Abstract

Despite using modern microbiological diagnostic approaches, the aetiological agents of pneumonia remain unidentified in about 50% of cases. Some bacteria that grow poorly or not at all in axenic media used in routine clinical bacteriology laboratory but which can develop inside amoebae may be the agents of these lower respiratory tract infections (RTIs) of unexplained aetiology. Such amoebae-resisting bacteria, which coevolved with amoebae to resist their microbicidal machinery, may have developed virulence traits that help them survive within human macrophages, i.e. the first line of innate immune defence in the lung. We review here the current evidence for the emerging pathogenic role of various amoebae-resisting microorganisms as agents of RTIs in humans. Specifically, we discuss the emerging pathogenic roles of Legionella-like amoebal pathogens, novel Chlamydiae (Parachlamydia acanthamoebae, Simkania negevensis), waterborne mycobacteria and Bradyrhizobiaceae (Bosea and Afipia spp.).

Introduction

Despite advances in antibiotic therapy, pneumonia remains one of the leading infectious causes of death in developed countries and a major cause of morbidity, especially in the elderly population and among patients with chronic underlying diseases (Mandell et al., 2007). Although a broad spectrum of microbial pathogens have been recognized as causal agents of respiratory tract infections (RTIs), the offending microorganism remains unknown in about half of the cases of community-acquired pneumonia (CAP) (Bochud et al., 2001; Echols et al., 2008), and three quarters of the cases of nosocomial pneumonia (Costa et al., 2001).

Microorganisms causing pneumonia may be acquired from respiratory droplets through human-to-human contact or from aerosolized particles from an animal or environmental reservoir. The epidemiology of RTI thus strongly depends on the interactions between humans and their ecosystem and evolves according to environmental changes due to human activities, climatic or ecological perturbations. Pandemics of influenza or the emergence of new respiratory diseases such as severe acute respiratory syndrome are dramatic illustrations of these phenomena. To some extent, agents of pneumonia have been identified in the context of outbreaks or case series occurring in a particular setting. For instance, psittacosis (parrot fever) was described for the first time in seven individuals exposed to pet birds in Switzerland in the 19th century (Ritter, 1880). More recently, the epidemics of severe pneumonia affecting war veterans in a hotel in Philadelphia in 1976 led to the discovery of the fastidious gram-negative rod Legionella pneumophila as a causal agent of respiratory diseases (Fraser et al., 1977; McDade et al., 1977). In this latter situation, water was found to be the source of contamination. Rowbotham (1980, 1983) then demonstrated that L. pneumophila may multiply within free-living amoebae and hypothesized that these protists may represent a reservoir for these intracellular bacteria.

Free-living amoebae live in water, soil and at the water–air interface. As they generally use bacteria as their main nutritional source, they are especially present in large quantities in sediments and biofilms (Rodriguez-Zaragoza, 1994). Thus, humans have been increasingly exposed to amoebae and to their related bacterial pathogens with the progressive development of various modern man-made water systems such as water-treatment plants, cooling towers, air conditioners, humidifiers, spas and swimming pools (Rodriguez-Zaragoza, 1994; Greub & Raoult, 2004; Pagnier et al., 2009a). Apart from L. pneumophila, which has mainly been recognized due to the dramatic importance of the Philadelphia outbreak, many other bacteria that resist the phagocytic amoebae may also use these protists as widespread reservoirs and may have acquired virulence traits promoting their resistance to macrophages. Interestingly, some of these amoebae-resisting bacteria have also been discovered during outbreaks of RTI (Herwaldt et al., 1984; Birtles et al., 1997). Indeed, given their intracellular lifestyle, they either do not grow or only poorly grow in conventional axenic media. It is important that the microbiology community be well aware of these new emerging human agents of pneumonia and develop new diagnostic tools for their identification. Amoebal coculture and amoebal enrichment coupled with detection of potential intra-amoebal bacteria have been demonstrated to be largely successful in identifying a large biodiversity of new pathogens from patients (Greub et al., 2004b; Thomas et al., 2006; Corsaro et al., 2009; Pagnier et al., 2009a, b).

In this review, we intend to present the current evidence of the role of various amoebae-resisting bacteria as agents of RTI in humans. More specifically, we will discuss the likely role of Legionella-like amoebal pathogens (LLAPs), novel Chlamydiae (Parachlamydia acanthamoebae, Simkania negevensis), waterborne mycobacteria and Bradyrhizobiaceae (Bosea and Afipia spp.).

Diagnostic tools for the identification of amoebae-resisting bacteria

For a better characterization of the microbial biodiversity, various molecular approaches are available, including a coupled cloning and sequencing approach, and metagenomics using new pyrosequencing techniques such as the 454 and Solexa/Illumina technologies. However, such sequence-based ecological studies do not provide the strains for subsequent studies, and consequently culture-based ecological studies are of equal if not greater importance. However, most culture-based studies are biased towards bacteria able to grow efficiently on different axenic media, broths and/or agar plates, and approaches that selectively amplify amoebae-resisting bacteria may be useful. Two main approaches, amoebal coculture and amoebal enrichment, have been applied so far to recover amoebae-resisting bacteria in culture (Fig. 1). Amoebal coculture is a cell culture method in which axenic amoebae are used as a host cell culture, whereas amoebal enrichment uses an enteric bacterium such as Escherichia coli as a food source for amoebae that are potentially present in the investigated sample. Once isolated by amoebal enrichment, the amoebae may then be studied for the possible presence of intra-amoebal microorganisms. Both approaches have been successfully used in recent years to uncover a variety of amoebae-resisting microorganisms from both environmental and clinical samples (Adekambi et al., 2004; Greub et al., 2004b; Thomas et al., 2006, 2008; Loret et al., 2008; Corsaro et al., 2009; Pagnier et al., 2009b).

Figure 1

Culture-based approaches that may be used to selectively grow amoebae-resisting microorganisms. (a) Amoebal coculture is a cell culture approach that uses amoebae as cell background and that can be used to isolate strict intracellular bacteria starting directly from clinical and/or environmental samples or by inoculation of free-living amoebae potentially containing amoebal pathogens or endosymbionts. Screening may then be achieved with various stainings and/or by PCRs. Subcultures on fresh amoebae in presence/absence of different antibiotics may help in isolating a given strain from heavily contaminated environmental samples. (b) Free-living amoebae may be isolated by amoebal enrichment, which consists in the inoculation of clinical and/or environmental samples on non-nutritive agar plates previously seeded with Escherichia coli and/or Enterobacter cloacae, which serve as a food source for the amoebae potentially present in the investigated sample. Once inoculated, agar plates can be screened daily for the presence of an amoebal migration front and, when positive, subcultured on new non-nutritive agar. Amoebae can then be screened for the presence of amoebae-resisting microorganisms by amoebal coculture and or molecular approaches.

Figure 1

Culture-based approaches that may be used to selectively grow amoebae-resisting microorganisms. (a) Amoebal coculture is a cell culture approach that uses amoebae as cell background and that can be used to isolate strict intracellular bacteria starting directly from clinical and/or environmental samples or by inoculation of free-living amoebae potentially containing amoebal pathogens or endosymbionts. Screening may then be achieved with various stainings and/or by PCRs. Subcultures on fresh amoebae in presence/absence of different antibiotics may help in isolating a given strain from heavily contaminated environmental samples. (b) Free-living amoebae may be isolated by amoebal enrichment, which consists in the inoculation of clinical and/or environmental samples on non-nutritive agar plates previously seeded with Escherichia coli and/or Enterobacter cloacae, which serve as a food source for the amoebae potentially present in the investigated sample. Once inoculated, agar plates can be screened daily for the presence of an amoebal migration front and, when positive, subcultured on new non-nutritive agar. Amoebae can then be screened for the presence of amoebae-resisting microorganisms by amoebal coculture and or molecular approaches.

Understanding the pathogenic role of amoebae-resisting bacteria: a comprehensive model approach

The availability of a bacterial strain growing in amoebae allows its pathogenic potential to be tested, for instance as an agent of pneumonia, using the comprehensive approach that has been applied to P. acanthamoebae (Greub, 2009). This global strategy includes (1) testing the permissiveness of lung fibroblasts, pneumocytes and alveolar macrophages to the new bacterial species; (2) developing diagnostic tools (serology, antigen-detection assays, PCR and immunohistochemistry) to study patients with and without lower RTIs; and (3) investigating the pathogenic role of some selected species in an animal model of pneumonia. For species that emerge as new pathogens or exhibit very peculiar interesting biological phenotypes, the availability of a given strain also allows better understanding of the biology of the species involved, using functional genomics, proteomics and cell biology.

Amoebal pathogens as causal agents of pneumonia: clinical evidence

Legionella species

Legionella pneumophila

The potential role of water systems as a reservoir of human respiratory diseases was recognized for the first time when L. pneumophila was identified as the causal agent of an outbreak of pneumonia in Philadelphia in 1976 (Fraser et al., 1977; McDade et al., 1977). This bacterium is widespread in our aquatic environment including man-made water systems and is one of the first examples of amoebae-resisting bacteria that has been described (Rowbotham et al., 1980, 1983). It is estimated to account for 2–7% of all cases of CAP affecting both immunocompromised and immunocompetent hosts (Doebbeling & Wenzel, 1987; Marrie et al., 1989; Fang et al., 1990a; Woodhead et al., 2002). Nosocomial outbreaks of Legionnaires' disease are also frequently reported and may result from bronchoaspiration of contaminated potable water rather than inhalation of aerosolized particules (Blatt et al., 1993; Sabria & Yu, 2002). In addition to potentially severe respiratory diseases, legionellosis may present as a flu-like syndrome called pontiac fever, which may be misdiagnosed as a viral infection and whose incidence is possibly underestimated (Doebbeling & Wenzel, 1987).

Legionella pneumophila is a fastidious gram-negative rod which is rarely detected by examination of gram stains of clinical samples and which needs buffered charcoal yeast extract (BCYE) agar to be grown. The availability of a urinary-specific antigen test for the detection of L. pneumophila serogroup 1, which accounts for about 85–90% of legionellosis in Europe and America, allows a rapid diagnosis of the disease. This test, in combination with cultures, exhibits an overall good sensitivity and specificity and its widespread use has improved the identification of Legionella pneumonia in hospitalized patients (Waterer et al., 2001). Molecular diagnostic tools have shown promising results but these methods lack standardization and their availability is limited (Waterer et al., 2001; Murdoch, 2003). The actual role of L. pneumophila in less severe pneumonia in the community as well as the incidence of respiratory diseases attributed to other Legionella species thus remain difficult to estimate (Waterer et al., 2001; Murdoch, 2003).

Legionella species other than L. pneumophila

Since the discovery of L. pneumophila, about 45 other Legionella species have been identified (Benson & Fields, 1998). These species share the same aquatic environment as L. pneumophila and about half of them have been associated with respiratory infections in humans (Table 1) (Muder & Yu, 2002; Roig et al., 2003). However, only a limited number of species seems to be relevant pathogens, whereas the others have been identified as the cause of pneumonia in anecdotal case reports. In a multinational survey, they have been estimated to account for 5–10% of legionellosis (Benin et al., 2002; Yu et al., 2002). The distribution of Legionella spp. is variable around the world: for instance, Legionella longbeachae is responsible for as many as 30% cases of Legionella diseases in Australia and New Zealand, whereas it accounts for only 3–4% of cases on the European and American continents (Yu et al., 2002). Legionella bozemanii, Legionella micdadei, Legionella dumoffii, Legionella anisa and Legionella feelei may account for most of the 1–5% remaining cases of Legionnaires' diseases (Fang et al., 1989; McNally et al., 2000; Benin et al., 2002; Muder & Yu, 2002; Yu et al., 2002). In contrast to L. pneumophila infections, pulmonary diseases attributed to other Legionella spp. have been mainly reported in a nosocomial context affecting immunocompromised patients such as haematopoietic or solid organ transplant recipients, patients under long-term corticoid therapy or splenectomized patients (Muder et al., 1983; Fang et al., 1989; Muder & Yu, 2002). With the exception of L. longbeachae, other Legionella spp. are rarely involved in CAP, although community outbreaks of pontiac fever have also been reported with L. anisa, L. micdadei, L. feelei and Legionella sainthelensi (Herwaldt et al., 1984; Goldberg et al., 1989; Fenstersheib et al., 1990; Loeb et al., 1999).

Table 1

Pathogenic role of Legionella spp. in pneumonia

Legionella species Pathogenic role in pneumonia References 
L. pneumophila 2–7% of community-acquired pneumonia (8% of those requiring ICU), 5% of nosocomial pneumonia Doebbeling & Wenzel (1987), Marrie et al. (1989), Fang et al. (1990a), Woodhead et al. (2002) 
Most frequent cause of Legionella pneumonia (91.5%) Yu et al. (2002) 
L. longbeachae Second cause of Legionella pneumonia worldwide (3.9%) Yu et al. (2002) 
Frequent cause of potentially severe community-acquired pneumonia in Australia, New Zealand and South East Asia Grove et al. (2002), Yu et al. (2002), Phares et al. (2007) 
Few cases reported in Europe or United States McKinney et al. (1981), Yu et al. (2002), McClelland et al. (2004), Kumpers et al. (2008) 
L. bozemanii Third cause of community-acquired Legionella pneumonia (2.4%) Fang et al. (1989), McNally et al. (2000), Yu et al. (2002) 
Cause of severe pneumonia in immunocompromised patients (frequent complications: empyema, cavitation) Fang et al. (1989), Swinburn et al. (1988), Taylor & Albrecht (1995), Harris et al. (1998), Muder & Yu (2002) 
L. micdadei Cause of life-threatening pneumonia in immunocompromised patients Myerowitz et al. (1979), Muder et al. (1983), Fang et al. (1987), Doebbeling et al. (1989), Muder & Yu (2002) 
Purulent pneumonia or pulmonary abscesses in solid-organ transplant recipients Myerowitz et al. (1979), Rogers et al. (1979), Mehta et al. (1983), Ernst et al. (1998), Knirsch et al. (2000) 
L. anisa Rare cause of community-acquired or nosocomial pneumonia McNally et al. (2000), Yu et al. (2002), La Scola et al. (2003b), Doleans et al. (2004) 
Identified as the cause of an outbreak of pontiac fever in California Fenstersheib et al. (1990) 
L dumoffii Some cases of pneumonia reported mainly in immunocompromised patients Fang et al. (1990b), Murdoch & Chambers (2000), Muder & Yu (2002), Yu et al. (2002) 
L. feeleii About 10 cases of pneumonia reported in the literature (75% in immunocompromised patients) Lee et al. (2009) 
One outbreak of pontiac fever in an automobile plant Herwaldt et al. (1984) 
L. jordanis Subacute or chronic respiratory infection with constitutional symptoms (rare cases described) Thacker et al. (1988b), Vinh et al. (2007) 
L. sainthelensi Two outbreaks of respiratory infections in nursing homes (Canada) Loeb et al. (1999) 
L. maceachernii Case reports of pneumonia in immunocompromised patients Wilkinson et al. (1985a), Thomas et al. (1992), Dumoff et al. (2004), van Dam et al. (2006) 
L. gormanii Case reports of pneumonia in immunocompromised patients Griffith et al. (1988), Ephros et al. (1989), Towns et al. (1994) 
L. wadsworthii Case reports of pneumonia in immunocompromised patients Edelstein et al. (1982), Yu et al. (2002) 
L. cincinnatiensis Case reports of pneumonia in renal transplant recipients or haemodialysis patient Thacker et al. (1988a), Jernigan et al. (1994) 
L. tucsonensis Case reports of pneumonia in immunocompromised patients Thacker et al. (1989), Doleans et al. (2004) 
L. oakridgensis 3 cases of pneumonia observed in patients with connective tissue diseases Tang et al. (1985), Lo Presti et al. (2000) 
L. parisiensis One case report of pneumonia in a liver transplant recipient Lo Presti et al. (1997) 
L. lansingensis One case report of pneumonia in a patient with chronic lymphocytic leukaemia Thacker et al. (1992) 
L. hackeliae One case report of pneumonia immunocompromised patient Wilkinson et al. (1985b) 
L. lytica (LLAP 3, 7 and 9) Seroconversion observed in cases of community-acquired pneumonia Fry et al. (1991), McNally et al. (2000) 
L. drancourtii (LLAP 4 and 12) Seroprevalence study suggesting a pathogenic role Marrie et al. (2001) 
L. fallonii (LLAP 10) Seroprevalence study suggesting a pathogenic role McNally et al. (2000) 
L. rowbothamii (LLAP 6) Seroprevalence study suggesting a pathogenic role McNally et al. (2000) 
L. drozanskii (LLAP 1) Seroprevalence study suggesting a pathogenic role McNally et al. (2000), Marrie et al. (2001) 
Legionella species Pathogenic role in pneumonia References 
L. pneumophila 2–7% of community-acquired pneumonia (8% of those requiring ICU), 5% of nosocomial pneumonia Doebbeling & Wenzel (1987), Marrie et al. (1989), Fang et al. (1990a), Woodhead et al. (2002) 
Most frequent cause of Legionella pneumonia (91.5%) Yu et al. (2002) 
L. longbeachae Second cause of Legionella pneumonia worldwide (3.9%) Yu et al. (2002) 
Frequent cause of potentially severe community-acquired pneumonia in Australia, New Zealand and South East Asia Grove et al. (2002), Yu et al. (2002), Phares et al. (2007) 
Few cases reported in Europe or United States McKinney et al. (1981), Yu et al. (2002), McClelland et al. (2004), Kumpers et al. (2008) 
L. bozemanii Third cause of community-acquired Legionella pneumonia (2.4%) Fang et al. (1989), McNally et al. (2000), Yu et al. (2002) 
Cause of severe pneumonia in immunocompromised patients (frequent complications: empyema, cavitation) Fang et al. (1989), Swinburn et al. (1988), Taylor & Albrecht (1995), Harris et al. (1998), Muder & Yu (2002) 
L. micdadei Cause of life-threatening pneumonia in immunocompromised patients Myerowitz et al. (1979), Muder et al. (1983), Fang et al. (1987), Doebbeling et al. (1989), Muder & Yu (2002) 
Purulent pneumonia or pulmonary abscesses in solid-organ transplant recipients Myerowitz et al. (1979), Rogers et al. (1979), Mehta et al. (1983), Ernst et al. (1998), Knirsch et al. (2000) 
L. anisa Rare cause of community-acquired or nosocomial pneumonia McNally et al. (2000), Yu et al. (2002), La Scola et al. (2003b), Doleans et al. (2004) 
Identified as the cause of an outbreak of pontiac fever in California Fenstersheib et al. (1990) 
L dumoffii Some cases of pneumonia reported mainly in immunocompromised patients Fang et al. (1990b), Murdoch & Chambers (2000), Muder & Yu (2002), Yu et al. (2002) 
L. feeleii About 10 cases of pneumonia reported in the literature (75% in immunocompromised patients) Lee et al. (2009) 
One outbreak of pontiac fever in an automobile plant Herwaldt et al. (1984) 
L. jordanis Subacute or chronic respiratory infection with constitutional symptoms (rare cases described) Thacker et al. (1988b), Vinh et al. (2007) 
L. sainthelensi Two outbreaks of respiratory infections in nursing homes (Canada) Loeb et al. (1999) 
L. maceachernii Case reports of pneumonia in immunocompromised patients Wilkinson et al. (1985a), Thomas et al. (1992), Dumoff et al. (2004), van Dam et al. (2006) 
L. gormanii Case reports of pneumonia in immunocompromised patients Griffith et al. (1988), Ephros et al. (1989), Towns et al. (1994) 
L. wadsworthii Case reports of pneumonia in immunocompromised patients Edelstein et al. (1982), Yu et al. (2002) 
L. cincinnatiensis Case reports of pneumonia in renal transplant recipients or haemodialysis patient Thacker et al. (1988a), Jernigan et al. (1994) 
L. tucsonensis Case reports of pneumonia in immunocompromised patients Thacker et al. (1989), Doleans et al. (2004) 
L. oakridgensis 3 cases of pneumonia observed in patients with connective tissue diseases Tang et al. (1985), Lo Presti et al. (2000) 
L. parisiensis One case report of pneumonia in a liver transplant recipient Lo Presti et al. (1997) 
L. lansingensis One case report of pneumonia in a patient with chronic lymphocytic leukaemia Thacker et al. (1992) 
L. hackeliae One case report of pneumonia immunocompromised patient Wilkinson et al. (1985b) 
L. lytica (LLAP 3, 7 and 9) Seroconversion observed in cases of community-acquired pneumonia Fry et al. (1991), McNally et al. (2000) 
L. drancourtii (LLAP 4 and 12) Seroprevalence study suggesting a pathogenic role Marrie et al. (2001) 
L. fallonii (LLAP 10) Seroprevalence study suggesting a pathogenic role McNally et al. (2000) 
L. rowbothamii (LLAP 6) Seroprevalence study suggesting a pathogenic role McNally et al. (2000) 
L. drozanskii (LLAP 1) Seroprevalence study suggesting a pathogenic role McNally et al. (2000), Marrie et al. (2001) 

Most Legionella species grow on BCYE agar media, but specific cultures for Legionella are not routinely performed in cases of CAP. Moreover, these media usually contain antibiotics for the selection of L. pneumophila that may inhibit the growth of some other Legionella species. The urinary antigen test does not detect species other than L. pneumophila serogroup 1, and PCR methods are as yet not widely used. For these reasons, Legionella spp. other than L. pneumophila are rarely identified as causal agents of infections and their role in the epidemiology of community- and hospital-acquired pneumonia has not been assessed precisely. The fact that these species exhibit a variable ability to infect and proliferate within amoebae may partly explain why they are less frequently involved in RTIs compared with L. pneumophila (Neumeister et al., 1997; Gao et al., 1999).

LLAPs

Historically, the term Legionella-like amoebal pathogens was introduced to designate obligate intracellular parasites of free-living amoebae which were closely related to the legionellae (Fig. 2) and which, unlike other Legionella spp., exhibited little or no growth on conventional bacteriological media such as BCYE agar (Rowbotham et al., 1986; Greub & Raoult, 2004). Most strains were originally isolated from water supplies during investigations of individual cases or outbreaks of Legionnaires' disease (Adeleke et al., 1996; Birtles et al., 1996). Because of their limited ability to grow in culture, these bacteria could not be completely characterized and were initially designated by numbers (e.g. LLAP-1–14). The first isolation of an LLAP (LLAP-3) in a clinical specimen was reported in 1991 using amoebal enrichment of the sputum of a patient with pneumonia who exhibited seroconversion against this strain (Fry et al., 1991). Phylogenetic analyses subsequently allowed its classification in the species Legionella lytica (Birtles et al., 1996), whereas other LLAP strains were characterized and assigned to new species of Legionella (Legionella drozanskii, Legionella rowbothamii, Legionella fallonii, Legionella drancourtii) (Adeleke et al., 1996, 2001; La Scola et al., 2004). The term of LLAPs thus has been retained for historical reasons, as most of these species have now been recognized to belong phylogenetically to the Legionella genus. Moreover, most of them are currently able to grow on BCYE agar because of the improvement in the quality of media and possibly because of a progressive adaptation by successive subcultures on amoebae.

Figure 2

Legionella drancourtii, i.e. LLAP-12, within Acanthamoeba castellannii as seen by gram staining (a) and Diff-Quick staining (modified May–Grünwald Giemsa) (b), respectively. Infected amoebae, which contain numerous rod-shaped bacteria, are circled by a dotted line. Microscopy performed 24 h postinfection at a low MOI of about 1/10; × 1000 magnification.

Figure 2

Legionella drancourtii, i.e. LLAP-12, within Acanthamoeba castellannii as seen by gram staining (a) and Diff-Quick staining (modified May–Grünwald Giemsa) (b), respectively. Infected amoebae, which contain numerous rod-shaped bacteria, are circled by a dotted line. Microscopy performed 24 h postinfection at a low MOI of about 1/10; × 1000 magnification.

The pathogenic role of LLAPs has been investigated in two series of patients suggesting that these fastidious bacteria may be a cause of pneumonia in some cases (Table 1) (McNally et al., 2000; Marrie et al., 2001). A French study reported serological evidence for recent infections with LLAP in 1.4% of CAP (Marrie et al., 2001). Most of them were attributed to LLAP-4 (L. drancourtii). An alternative potential pathogen was, however, isolated in about half of these cases. Similarly, a significant rise in antibodies against LLAP-1, -3, -6, -9 and -10 was reported in 7% of patients with CAP of unknown aetiology in a North American series (McNally et al., 2000). Data from other parts of the world or other subsets of population are lacking, although the ubiquity and biodiversity of Legionella spp., including LLAPs, in water systems has been reported worldwide (Thomas et al., 2006; Diederen et al., 2007; Wery et al., 2008; Hsu et al., 2009). The role of LLAPs in human diseases is difficult to assess, as their pathogenic role was suggested only by serological tests in relatively few cases, and in the absence of direct microbiological documentation by other diagnostic tools such as culture or molecular methods. Moreover, in vitro studies or animal models supporting their pathogenicity are currently lacking.

Chlamydia-related bacteria

Molecular and phylogenetic studies have recently revealed a rich diversity of microorganisms within the order Chlamydiales and allowed the identification of new families distinct from the well-known Chlamydiaceae (Everett et al., 1999; Corsaro & Greub, 2006; Greub, 2009). The term ‘Chlamydia-like organisms’, ‘novel Chlamydiae’, ‘Chlamydia-related bacteria’ or ‘amoebae-resistant Chlamydiae’ have been used to designate these strict intracellular bacteria that may infect and survive within free-living amoebae. Growing evidence suggests that some of them may be the cause of RTIs in humans (Friedman et al., 2003; Corsaro & Greub, 2006; Greub, 2009).

Simkaniaceae

Simkania negevensis, formerly called microorganism ‘Z’ or ‘Simkania Z’, is the member of these Chlamydia-like organisms whose implication in RTIs has been most extensively studied. This bacterium may easily grow within the Acanthamoeba amoeba (Fig. 3) and may also use free-living amoebae as a widespread environmental reservoir. Simkania negevensis was first described in 1993 as a cell culture contaminant of unknown origin exhibiting a typical two-stage developmental cycle, with infectious elementary bodies and replicative reticulate bodies (Fig. 4), but differing significantly from Chlamydiaceae (Kahane et al., 1993). Its seroprevalence in the population displays important variations around the world, having been reported to be as high as 55–80% in Israel and only 4% in Japan (Friedman et al., 1999, 2006; Johnsen et al., 2005; Yamaguchi et al., 2005). These differences may be partially due to the respective sensitivity and specificity of the serological approaches used and to the cut-off defining positivity. The involvement of S. negevensis in RTIs has been investigated in several large cohorts of patients in Europe, the Middle East and America using serological or molecular diagnostic methods (Table 2) (Kahane et al., 1998; Lieberman et al., 2002, 1997; Greenberg et al., 2003; Kumar et al., 2005; Friedman et al., 2006; Fasoli et al., 2008; Heiskanen-Kosma et al., 2008; Nascimento-Carvalho et al., 2009). Some of these analyses reported an association with CAP, exacerbations of chronic obstructive pulmonary diseases or bronchiolitis in adults and children (Kahane et al., 1998; Lieberman et al., 2002, 1997; Friedman et al., 2003, 2006; Greenberg et al., 2003; Fasoli et al., 2008; Heiskanen-Kosma et al., 2008; Nascimento-Carvalho et al., 2009). The importance of this bacterium as a causal agent of CAP is, however, difficult to evaluate and seems relatively marginal (<2% of all aetiologies) in view of these results. Its incidence may be higher in some populations or ethnic groups where a high seroprevalence of S. negevensis has been documented, for example in the Middle East (Bedouins) (Kahane et al., 1998; Friedman et al., 1999) or Northern Canada (Inuits) (Greenberg et al., 2003), whereas it remains to be determined in many other parts of the world. A study carried out in Brooklyn (NY) deserves mention as it did not identify any association with respiratory diseases despite a high prevalence (23.5%) of antibody titers against S. negevensis among adults and children in this population, suggesting that these Chlamydia-related bacteria are simple colonizers (Kumar et al., 2005). Thus, thorough evaluation of the pathogenic role of S. negevensis is warranted, as most studies did not include a control group, and among the few studies with a control group, most failed to demonstrate a significant correlation with lower RTIs (Table 2).

Figure 3

Simkania negevensis within Acanthamoeba castellannii, as seen by electron microscopy 24 h postinfection. Please note (a) the presence of several dividing reticulate bodies (arrows). (b) An elementary body in the process of being phagocytized (arrow). Magnification × 7000 and × 10 000, respectively. Scale bar=2 μm.

Figure 3

Simkania negevensis within Acanthamoeba castellannii, as seen by electron microscopy 24 h postinfection. Please note (a) the presence of several dividing reticulate bodies (arrows). (b) An elementary body in the process of being phagocytized (arrow). Magnification × 7000 and × 10 000, respectively. Scale bar=2 μm.

Figure 4

The two developmental stages of Simkania negevensis: the reticulate body, i.e. the metabolically active dividing stage (a) and the elementary body, i.e. the infectious stage (b). Simkania negevensis may also infrequently exhibit a third stage, the crescent body (initially reported for Parachlamydia acanthamoebae), which may be seen on some electron microscopy preparations (c). Electron micrographs: magnification × 70 000; Scale bar=0.2 μm.

Figure 4

The two developmental stages of Simkania negevensis: the reticulate body, i.e. the metabolically active dividing stage (a) and the elementary body, i.e. the infectious stage (b). Simkania negevensis may also infrequently exhibit a third stage, the crescent body (initially reported for Parachlamydia acanthamoebae), which may be seen on some electron microscopy preparations (c). Electron micrographs: magnification × 70 000; Scale bar=0.2 μm.

Table 2

Simkania negevensis as causal agents of pneumonia: review of the literature

Population (country) and number of patients Disease Diagnostic method Positive results P value (if controls) Recent infection and no alternative pathogen References 
Adults (Israel) 308 CAP Serology (IgG, IgA) 112 (37%) No control 4 (1.3%) Lieberman et al. (1997) 
Infants (Israel) 239 Bronchiolitis Culture and/or PCR (NP swabs) 60 (25%) P<0.001 38 (16%) Kahane et al. (1998) 
Adults (Israel) 190 COPD exacerbation Serology (IgG, IgA) 120 (63%) Not significant 1 (0.5%) Lieberman et al. (2002) 
Infants (Canada) 22 Bronchiolitis PCR (NP swabs) 14 (64%) No control 2 (9%) Greenberg et al. (2003) 
Adults/children (USA) 188 Bronchiolitis, Pneumonia Serology (IgG) (n=69) 14 (18%) Not significant NA Kumar et al. (2005) 
Asthma PCR (NPl swabs) (n=169) 29 (17%) Not significant NA  
Adults (UK) 29 RTI Serology (IgG) 18 (62%) Not significant NA Friedman et al. (2006) 
 Serology (IgA) 5 (17%) P=0.004 NA  
Children (UK) 222 Bronchiolitis Culture and/or PCR (NP swabs) 111 (50%) No control NA Friedman et al. (2006) 
Children (Italy) 101 CAP Serology (IgM, IgG) 20–30% No control 2 (2%) Fasoli et al. (2008) 
Children (Finland) 174 CAP Serology (IgM) 18 (10%) No control 6 (3.4%) Heiskanen-Kosma et al. (2008) 
Children (Brazil) 184 CAP Serology (IgM, IgG) 3 (1.6%) No control 1 (0.5%) Nascimento-Carvalho et al. (2009) 
Population (country) and number of patients Disease Diagnostic method Positive results P value (if controls) Recent infection and no alternative pathogen References 
Adults (Israel) 308 CAP Serology (IgG, IgA) 112 (37%) No control 4 (1.3%) Lieberman et al. (1997) 
Infants (Israel) 239 Bronchiolitis Culture and/or PCR (NP swabs) 60 (25%) P<0.001 38 (16%) Kahane et al. (1998) 
Adults (Israel) 190 COPD exacerbation Serology (IgG, IgA) 120 (63%) Not significant 1 (0.5%) Lieberman et al. (2002) 
Infants (Canada) 22 Bronchiolitis PCR (NP swabs) 14 (64%) No control 2 (9%) Greenberg et al. (2003) 
Adults/children (USA) 188 Bronchiolitis, Pneumonia Serology (IgG) (n=69) 14 (18%) Not significant NA Kumar et al. (2005) 
Asthma PCR (NPl swabs) (n=169) 29 (17%) Not significant NA  
Adults (UK) 29 RTI Serology (IgG) 18 (62%) Not significant NA Friedman et al. (2006) 
 Serology (IgA) 5 (17%) P=0.004 NA  
Children (UK) 222 Bronchiolitis Culture and/or PCR (NP swabs) 111 (50%) No control NA Friedman et al. (2006) 
Children (Italy) 101 CAP Serology (IgM, IgG) 20–30% No control 2 (2%) Fasoli et al. (2008) 
Children (Finland) 174 CAP Serology (IgM) 18 (10%) No control 6 (3.4%) Heiskanen-Kosma et al. (2008) 
Children (Brazil) 184 CAP Serology (IgM, IgG) 3 (1.6%) No control 1 (0.5%) Nascimento-Carvalho et al. (2009) 
*

Recent infection was defined as: serological evidence for recent infection (positive IgM or significant increase between initial and convalescent IgG titres) or positive PCR result during the course of infection.

COPD, chronic obstructive pulmonary disease; NA, data not available.

The implication of S. negevensis in other respiratory diseases, such as chronic cough or asthma, has also been investigated and could not be demonstrated conclusively (Johnsen et al., 2005; Kumar et al., 2005; Korppi et al., 2006). In one study, S. negevensis was detected by a PCR method in bronchoalveolar lavage samples of lung transplant recipients with a surprisingly high prevalence (97.5%, when compared with only 14.1% in other solid-organ transplant recipients; P<0.0001) (Husain et al., 2007). Many of these patients did not have documented pneumonia and the pathogenic role of S. negevensis in this context thus remains unclear. The authors of this study postulated a possible role in acute graft rejection, although the analysis was underpowered to reach statistical significance. Like other Chlamydia-related organisms, S. negevensis may infect free-living amoebae such as Acanthamoeba which are widespread in water, including hospital water supplies (La Scola et al., 2002; Thomas et al., 2006). Hospitalized patients exposed to aerosolized particles, such as those undergoing mechanical ventilation, may thus be colonized or infected by these intracellular bacteria, whose pathogenic role in this setting has been poorly investigated (La Scola et al., 2002). However, it is worth noting that in vitro studies of the pathogenesis of S. negevensis have demonstrated its ability to infect human macrophages and to induce a host cell inflammatory response, supporting its potential ability to cause human infections and the need for further clinical investigations (Kahane et al., 2008, 2007).

Parachlamydiaceae

This family has drawn increasing attention in the last decade because of the potential pathogenicity of its first recognized member, P. acanthamoebae, which was identified as the cause of an outbreak of fever of undetermined origin occurring in Vermont in 1989 (Birtles et al., 1997). An amoeba of the Acanthamoeba genus was isolated from the water of a humidifier and was subsequently shown to be infected with a gram-negative bacterium termed Hall's coccus. This organism was characterized by comparative sequence analyses (Birtles et al., 1997) and was found to be similar to a Chlamydia-like endoparasite of Acanthamoeba (strain Bn9) previously identified in the nasal mucosa of healthy subjects (Amann et al., 1997). Hall's coccus and strain Bn9 were subsequently assigned to the species P. acanthamoebae within the Chlamydiales order (Everett et al., 1999). The ability of P. acanthamoebae to replicate within free-living amoebae of the genus Acanthamoeba has been well described and is shown in Fig. 5.

Figure 5

Parachlamydia acanthamoebae within Acanthamoeba castellannii, as seen by gram staining (a), Diff-Quick staining (modified May–Grünwald Giemsa) (b), and immunofluorescence (c), respectively. Microscopy performed 24 h postinfection at an MOI of about × 10. (a) Note that elementary bodies are generally gram-positive, whereas the reticulate bodies are gram-negative. (c) The cell wall of P. acanthamoebae (in green) was stained with mice polyclonal anti-Parachlamydia antibodies whereas the amoeba was stained with concanavalin A. Magnification × 1000.

Figure 5

Parachlamydia acanthamoebae within Acanthamoeba castellannii, as seen by gram staining (a), Diff-Quick staining (modified May–Grünwald Giemsa) (b), and immunofluorescence (c), respectively. Microscopy performed 24 h postinfection at an MOI of about × 10. (a) Note that elementary bodies are generally gram-positive, whereas the reticulate bodies are gram-negative. (c) The cell wall of P. acanthamoebae (in green) was stained with mice polyclonal anti-Parachlamydia antibodies whereas the amoeba was stained with concanavalin A. Magnification × 1000.

Further studies reported a significantly higher rate of seropositivity for P. acanthamoebae among patients with RTIs when compared with healthy controls (Marrie et al., 2001; Greub et al., 2003b; Greub et al., 2009). However, serological evidence for a recent infection in the absence of other potential documented pathogens could be assessed in only a few cases, suggesting that these bacteria may account for <1% of CAP (Marrie et al., 2001; Greub et al., 2003a) and about 8% of ventilator-associated pneumonia (VAP) (Greub et al., 2003b) (Table 3). More recently, the use of a specific real-time PCR allowed the identification of P. acanthamoebae or a related species as the only potential pathogen in as many as 13% of children with bronchiolitis, suggesting that the pathogenic role of Parachlamydiaceae may be underestimated in some clinical settings (Casson et al., 2008c). Moreover, experimental models have demonstrated the ability of P. acanthamoebae to enter and replicate within human macrophages (Greub et al., 2003c, 2005) and pneumocytes (Casson et al., 2006) and to cause pneumonia in mice (Casson et al., 2008a). Molecular analyses also allowed the identification of other members of the family Parachlamydiaceae in respiratory samples of some cases of pneumonia (Corsaro et al., 2002; Casson et al., 2008b; Haider et al., 2008). Protochlamydia naegleriophila was detected as the unique potential pathogen in an immunocompromised patient with lung infiltrate (Casson et al., 2008b) and one case of CAP possibly attributed to Protochlamydia amoebophila has been reported (Haider et al., 2008) (Table 3). Further clinical studies are warranted to better define the pathogenicity and the epidemiology of P. acanthamoebae and other Parachlamydiaceae among the causal agents of RTIs.

Table 3

Parachlamydiaceae as causal agents of pneumonia: review of the literature

Genus/species Population (country) and number of patients Disease Diagnostic method Positive results P value (if controls) Recent infection and no alternative pathogen References 
Parachlamydia acanthamoebae Adults (Canada) 371 CAP Serology (IgM, IgG) 8 (2.2%) P< 0.01 1 (0.3%) Marrie et al. (2001) 
Adults/children (France) 1200 Pneumonia PCR (BAL) 1 (0.1%) No control 1 (0.1%) Greub et al. (2003a) 
Adults (France) 37 VAP Serology (IgM, IgG) 5 (13.5%) P< 0.001 3 (8.1%) Greub et al. (2003b) 
ICU adults (France) 210 Pneumonia PCR and culture (BAL), serology (IgM, IgG) 3 (1.4%) No control 1 (0.5%) Berger et al. (2006) 
Children (Switzerland) 39 Bronchiolitis PCR (NP swabs) 6 (15%) No control 5 (13%) Casson et al. (2008c) 
Protochlamydia amoebophila Adults (Austria) 387 CAP PCR (respiratory samples) 1 (0.3%) No control 1 (0.3%) Haider et al. (2008) 
Protochlamydia naegleriophila Adults/children (Switzerland) 65 Pneumonia PCR (BAL) 1 (1.5%) Not significant 1 (1.5%) Casson et al. (2008b) 
Other Parachlamydia spp. (unclassified) Adults/children (France/Italy) 170 Pneumonia PCR (respiratory samples) 2 (1.2%) No control NA Corsaro et al. (2002) 
Genus/species Population (country) and number of patients Disease Diagnostic method Positive results P value (if controls) Recent infection and no alternative pathogen References 
Parachlamydia acanthamoebae Adults (Canada) 371 CAP Serology (IgM, IgG) 8 (2.2%) P< 0.01 1 (0.3%) Marrie et al. (2001) 
Adults/children (France) 1200 Pneumonia PCR (BAL) 1 (0.1%) No control 1 (0.1%) Greub et al. (2003a) 
Adults (France) 37 VAP Serology (IgM, IgG) 5 (13.5%) P< 0.001 3 (8.1%) Greub et al. (2003b) 
ICU adults (France) 210 Pneumonia PCR and culture (BAL), serology (IgM, IgG) 3 (1.4%) No control 1 (0.5%) Berger et al. (2006) 
Children (Switzerland) 39 Bronchiolitis PCR (NP swabs) 6 (15%) No control 5 (13%) Casson et al. (2008c) 
Protochlamydia amoebophila Adults (Austria) 387 CAP PCR (respiratory samples) 1 (0.3%) No control 1 (0.3%) Haider et al. (2008) 
Protochlamydia naegleriophila Adults/children (Switzerland) 65 Pneumonia PCR (BAL) 1 (1.5%) Not significant 1 (1.5%) Casson et al. (2008b) 
Other Parachlamydia spp. (unclassified) Adults/children (France/Italy) 170 Pneumonia PCR (respiratory samples) 2 (1.2%) No control NA Corsaro et al. (2002) 
*

Recent infection was defined as: serological evidence for recent infection (positive IgM or significant increase between initial and convalescent IgG titres) or positive PCR result during the course of infection.

BAL, bronchoalveolar lavage fluid; NA, data not available.

Other Chlamydia-related organisms

Culture- and molecular-based studies on human, animal or environmental samples have led to the identification of many new Chlamydia-related organisms, attesting to the rich biodiversity within the order Chlamydiales (Greub et al., 2009). New strains have been identified and assigned to different families according to phylogenetic analyses, whereas others failed to grow in culture and could only be described by comparative sequence analyses. Some of them have been documented by PCR methods in human respiratory samples, but their pathogenic role remains unknown (Ossewaarde & Meijer, 1999; Corsaro et al., 2001; Haider et al., 2008). Using a molecular approach targeting the 16S rRNA gene (a conserved gene in the genome of all bacteria), Haider et al. (2008) recently found DNA of Chlamydia-related organisms in about 1% of adult patients with CAP, including Waddlia chondrophila (Family: Waddliaceae) and Rhabdochlamydia porcellionis (Family: Rhabdochlamydiaceae) (Haider et al., 2008). No sequence related to S. negevensis or P. acanthamoebae was detected in this series. The presence of Rhabdochlamydia spp. in respiratory samples of premature neonates has been recently reported, although their role as a causal agent of pneumonia or other systemic infections could not be assessed (Lamoth et al., 2009). It is thus difficult to estimate which proportion of RTIs in humans are caused by these Chlamydia-like organisms.

Waterborne mycobacteria

Nontuberculous mycobacteria are associated with disseminated or pulmonary infection in immunocompromised patients and may be the cause of various infectious diseases such as lymphadenitis, cutaneous infections or, rarely, pneumonia in the immunocompetent host (Falkinham et al., 1996). Among them, the Mycobacterium avium complex and Mycobacterium kansasii are the species most frequently associated with respiratory tract colonization or true infection in both immunocompromised and immunocompetent patients (Falkinham et al., 1996; Field & Cowie, 2006). Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium simae, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium gordonae and Mycobacterium malmoense are other recognized agents of pneumonia, occurring mainly in immunocompromised hosts or patients with underlying pulmonary diseases (Table 4) (Field & Cowie, 2006).

Table 4

Mycobacteria other than tuberculosis (MOTT): review of the most frequent agents of pneumonia

Mycobacterium species Pathogenic role in pneumonia References 
M. avium intracellulare complex Most frequent cause of MOTT-associated respiratory infection (variable presentation) Olivier et al. (1998), Field & Cowie (2006), Kim et al. (2008), Parrish et al. (2008) 
More frequent in HIV and immunocompromised patients Olivier et al. (1998), Field & Cowie (2006), Parrish et al. (2008) 
Cause of hypersensitivity pneumonia after exposure to hot tubes Embil et al. (1997), Field & Cowie (2006) 
M. kansasii Pulmonary disease similar to M. tuberculosis. One of the MOTT most frequently associated with pneumonia in both immunocompetent and immunocompromised hosts Evans et al. (1996a, b), Campo & Campo (1997), Taillard et al. (2003), Maliwan & Zvetina (2005), Kim et al. (2008) 
M. xenopi One of the MOTT most frequently associated with pneumonia in both immunocompetent and immunocompromised hosts Juffermans et al. (1998), Faress et al. (2003), Andrejak et al. (2007), Kim et al. (2008), van Ingen et al. (2008), Marusic et al. (2009) 
M. malmoense Most frequent cause of MOTT-associated respiratory infection in the United Kingdom and Sweden, especially in patients with underlying lung diseases. Cavitations are often present Research Committee of the British Thoracic Society (2001), Henriques et al. (1994), Field & Cowie (2006) 
M. chelonae Rare cause of fever and pneumonia in neutropenic cancer patients McWhinney et al. (1992), Levendoglu-Tugal et al. (1998), Peres et al. (2009) 
Rare cause of pneumonia in patients with oesophageal or swallowing disorders Burke & Ullian (1977), Hadjiliadis et al. (1999) 
M. fortuitum Rare cause of pneumonia in patients with oesophageal or swallowing disorders Howard et al. (1991), Hadjiliadis et al. (1999) 
Some cases of pneumonia reported mainly in immunocompromised patients Marchevsky et al. (1982), Ellis & Qadri (1993), Al Shaalan et al. (1997), Abe et al. (1999), Miguez-Burbano et al. (2006) 
M. simae One of the MOTT most frequently isolated from respiratory samples Valero et al. (1995), El Sahly et al. (2002), Samra et al. (2005) 
Cause of pneumonia in patients with underlying pulmonary diseases, rarely in AIDS patients Bell et al. (1983), Huminer et al. (1993), Valero et al. (1995), Maoz et al. (2008) 
M. gordonae Frequently isolated from sputum, but rare cause of respiratory infection Eckburg et al. (2000), Thomsen et al. (2002), Field & Cowie (2006) 
Mycobacterium species Pathogenic role in pneumonia References 
M. avium intracellulare complex Most frequent cause of MOTT-associated respiratory infection (variable presentation) Olivier et al. (1998), Field & Cowie (2006), Kim et al. (2008), Parrish et al. (2008) 
More frequent in HIV and immunocompromised patients Olivier et al. (1998), Field & Cowie (2006), Parrish et al. (2008) 
Cause of hypersensitivity pneumonia after exposure to hot tubes Embil et al. (1997), Field & Cowie (2006) 
M. kansasii Pulmonary disease similar to M. tuberculosis. One of the MOTT most frequently associated with pneumonia in both immunocompetent and immunocompromised hosts Evans et al. (1996a, b), Campo & Campo (1997), Taillard et al. (2003), Maliwan & Zvetina (2005), Kim et al. (2008) 
M. xenopi One of the MOTT most frequently associated with pneumonia in both immunocompetent and immunocompromised hosts Juffermans et al. (1998), Faress et al. (2003), Andrejak et al. (2007), Kim et al. (2008), van Ingen et al. (2008), Marusic et al. (2009) 
M. malmoense Most frequent cause of MOTT-associated respiratory infection in the United Kingdom and Sweden, especially in patients with underlying lung diseases. Cavitations are often present Research Committee of the British Thoracic Society (2001), Henriques et al. (1994), Field & Cowie (2006) 
M. chelonae Rare cause of fever and pneumonia in neutropenic cancer patients McWhinney et al. (1992), Levendoglu-Tugal et al. (1998), Peres et al. (2009) 
Rare cause of pneumonia in patients with oesophageal or swallowing disorders Burke & Ullian (1977), Hadjiliadis et al. (1999) 
M. fortuitum Rare cause of pneumonia in patients with oesophageal or swallowing disorders Howard et al. (1991), Hadjiliadis et al. (1999) 
Some cases of pneumonia reported mainly in immunocompromised patients Marchevsky et al. (1982), Ellis & Qadri (1993), Al Shaalan et al. (1997), Abe et al. (1999), Miguez-Burbano et al. (2006) 
M. simae One of the MOTT most frequently isolated from respiratory samples Valero et al. (1995), El Sahly et al. (2002), Samra et al. (2005) 
Cause of pneumonia in patients with underlying pulmonary diseases, rarely in AIDS patients Bell et al. (1983), Huminer et al. (1993), Valero et al. (1995), Maoz et al. (2008) 
M. gordonae Frequently isolated from sputum, but rare cause of respiratory infection Eckburg et al. (2000), Thomsen et al. (2002), Field & Cowie (2006) 

The genus Mycobacterium has been considerably enriched with the discovery of a large number of new species during the last decades (Primm et al., 2004). These mycobacterial species have been isolated from water sources, soil, air, human or animal reservoirs, reflecting their ubiquity in the environment (Falkinham et al., 2002; Primm et al., 2004). Natural fresh or salt waters, such as lakes, rivers, swamps, estuaries or marine streams, are common habitats of opportunistic mycobacteria. Their resistance to chlorine and most disinfectants used for water treatment as well as their ability to survive despite low nutrient levels, low oxygen content or extreme temperatures allow them to colonize drinking water supplies, cooling towers, swimming pools and other recreational water systems (Falkinham et al., 2002; Black & Berk, 2003; Pagnier et al., 2009b). Their selection by surface disinfectants may also promote their widespread occurrence in the hospital environment. Moreover, these pathogens are able to form biofilms and to colonize medical devices such as bronchoscopes (Wallace et al., 1998; Falkinham et al., 2002). Nontuberculous waterborne mycobacteria are frequently isolated from clinical specimens (Martin-Casabona et al., 2004) and nosocomial outbreaks have been reported (Wallace et al., 1998; Phillips & von Reyn, 2001). In most cases, the presence of such microorganisms in the respiratory tract reflects transient colonization. However, several cases or small outbreaks of hospital-acquired pneumonia have been ascribed to M. xenopi, M. chelonae and M. simae (Wallace et al., 1998; Phillips & von Reyn, 2001; Conger et al., 2004). Community-acquired respiratory diseases such as hypersensitivity pneumonitis and even cases of true pneumonia have been reported in healthy individuals exposed to aerosols from different water sources (spas, swimming pools, hot tubs, metalworking fluid) (Embil et al., 1997; Primm et al., 2004).

Mycobacteria and free-living amoebae thus share the same ecosystem and their close interaction has been suspected since the presence of mycobacteria within an amoebal host was first reported in 1973 (Jadin et al., 1973). Further in vitro studies not only demonstrated that mycobacteria were able to enter and replicate within the trophozoites and cysts of amoebae or other protozoa (Krishna Prasad & Gupta, 1978; Cirillo et al., 1997; Steinert et al., 1998; Strahl et al., 2001; Taylor et al., 2003; Adekambi et al., 2006; Mura et al., 2006; Whan et al., 2006; Thomas & McDonnell, 2007), but also that amoeba-grown mycobacteria displayed increased virulence in macrophage and mouse models of infection (Cirillo et al., 1997). Their recovery in hospital water networks has been strongly associated with the presence of amoebae (Thomas et al., 2006). Moreover, pathogenic strains of M. kansasii exhibit a better ability to grow in Acanthamoeba castellanii than the nonpathogenic strains colonizing the respiratory tract (Goy et al., 2007). The mechanisms involved in the pathogenicity of M. avium with respect to macrophages and amoebae have been found to be very similar (Danelishvili et al., 2007). These findings suggest that mycobacteria, like legionellae, may take advantage of amoebae, using them as a reservoir and as an evolutionary niche for the development of virulence factors. In contrast to legionellae and to some Chlamydia-like organisms residing within the cytosol of the amoebal cysts, mycobacteria are located within the double layers of the cysts (Steinert et al., 1998). This evolutionary adaptation may allow them to survive in a hostile environment such as phagocytic human cells and to resist to high chlorine concentrations or to the action of antibiotics (Miltner & Bermudez, 2000; Adekambi et al., 2006; Thomas & McDonnell, 2007).

Bradyrhizobiaceae

Afipia spp. and Bosea spp. are recently discovered gram-negative bacteria belonging to the class of Alphaproteobacteria (family: Bradyrhizobiaceae) and somehow related to Brucella spp. and Bartonella spp. (Brenner et al., 1991; Das et al., 1996). These Bradyrhizobiaceae are able to resist to amoebal microbicidal effectors (Fig. 6). The original isolation of Afipia spp. in clinical specimens such as lymph nodes (Afipia felis) or bone biopsy (Afipia clevelandensis) raised the question about their pathogenic role in human diseases, particularly in cat scratch disease (English et al., 1988; Brenner et al., 1991; Hall et al., 1991). However, subsequent analyses identified Bartonella henselae as the causal agent of this latter disease and the precise pathogenic role of Afipia remained undetermined (Jerris & Regnery, 1996). These bacteria were first detected in human respiratory samples by Brenner et al. (1991), who described the new genus Afipia. Drancourt et al. (1997) detected the presence of antibodies against A. clevelandensis in 1.5% of sera tested in the French national centre for rickettsial diseases and postulated a cross-reactivity between this bacterium and Brucella spp. or Yersinia spp., as about half of the cases had a diagnosis of certain or probable brucellosis or yersiniosis. However, about 15% of patients from this series were diagnosed as having pneumonia (Drancourt et al., 1997). The subsequent isolation of Afipia spp. and Bosea spp. in hospital water supplies suggests a possible role in nosocomial pneumonia (La Scola et al., 2000, 2002, 2003b, c; Thomas et al., 2006, 2007). Sero-epidemiological analyses revealed evidence of exposure to Afipia spp. and closely related Alphaproteobacteria in 13% of patients with hospital-acquired pneumonia, whereas no specific antibodies were detected in healthy blood donors (P<0.01) (La Scola et al., 2002). The same strains were simultaneously detected in the water supplies of the intensive care units where these patients were staying. In another series of 30 patients in a single intensive care unit in France, seroconversion to Afipia spp. and Bosea spp. was documented in 17% and 20% patients with VAP, respectively (La Scola et al., 2003b). No alternative potential pathogen was documented in about half of these cases. This study also reported a case of pneumonia with detection of Bosea massiliensis by PCR in bronchoalveolar lavage fluid associated with seroconversion to the same microorganism (La Scola et al., 2003b). In another analysis of 210 ICU patients with pneumonia, Bosea spp. were detected in eight (3.8%) patients by culture, PCR or serological testing (B. massiliensis and Bosea thiooxidans) (Berger et al., 2006). However, evidence for recent infection was documented in only one case and potential alternative pathogens of pneumonia were isolated in all cases. All patients but one had hospital-acquired pneumonia. Afipia spp. were not detected in this population (Berger et al., 2006). A Bosea and some other Alphaproteobacteria have also been isolated from nasal swabs of hospitalized patients by amoebal coculture, although their pathogenic role was unclear (Greub et al., 2004b). Interestingly, the possible implication of the Bradyrhizobiaceae in CAP has not yet been investigated. Bosea strains have also been isolated from environmental sources of water other than hospital networks such as river water or drinking water plants (Rapala et al., 2006; Thomas et al., 2007) and Afipia spp. were the most common bacterial species found in biofilms from a dental unit water system in Baltimore (Singh et al., 2003). The actual role of Bradyrhizobiaceae in RTIs thus remains difficult to assess on the basis of serological diagnostic tools, which lack sensitivity and specificity, and in the absence of microbiological documentation in clinical specimens in most cases.

Figure 6

Bosea sequanensis (arrows) within Acanthamoeba castellannii, as seen by electron microscopy. Magnification × 7000. Scale bar=2 μm. The inset is a drawing showing the limits of the amoeba (discontinuous line) that contains about 19 bacteria (light grey); three bacteria that are localized outside of the amoeba are highlighted in dark grey.

Figure 6

Bosea sequanensis (arrows) within Acanthamoeba castellannii, as seen by electron microscopy. Magnification × 7000. Scale bar=2 μm. The inset is a drawing showing the limits of the amoeba (discontinuous line) that contains about 19 bacteria (light grey); three bacteria that are localized outside of the amoeba are highlighted in dark grey.

Biodiversity of amoebae-resisting microorganisms and perspectives for further investigations

Free-living amoebae represent a widespread evolutionary niche that may favour the selection of virulence traits in intra-amoebal bacteria, enabling them to survive in other phagocytic cells, including alveolar macrophages, which are one a major line of immune defence against invading pathogens. The examples of emerging pathogens provided in this review probably represent only the tip of the iceberg, and there is still a largely underestimated biodiversity of amoebae-resisting bacteria, which may have acquired their ability to cause diseases in humans by the development of virulence traits during their intra-amoebal life. This huge and so far unexplored biodiversity not only includes members of the clade presented in this review, i.e. Legionella spp., Chlamydiae, Bradyrhizobiaceae and mycobacteria, but also many other bacterial clades and giant viruses (Greub & Raoult, 2004). At least one of the amoebae-resisting viruses discovered so far, the mimivirus, is also resistant to human macrophages and may be involved in lower RTIs, as suggested by clinical studies and by a well documented laboratory-acquired infection (see Box 1).

Box 1.

Mimivirus, a giant virus likely involved in lower respiratory tract infections

Mimivirus (Mimiviridae) is a double-stranded DNA virus belonging to the nucleocytoplasmic large DNA viruses (NCLDV). Initially discovered within free-living amoebae recovered by amoebal enrichment from a cooling tower during the investigation of an outbreak of community-acquired pneumonia in Bradford (UK), this microorganism was first considered to be a gram-positive bacterial coccus (La Scola et al., 2003a). However, electron microscopy suggested that it was a giant virus of about 400 nm in diameter and subsequent studies unequivocally confirmed its affiliation within the NCLDV, which also includes the Iridoviridae, the Phycodnaviridae, the Asfarviridae and the Poxviridae (Koonin et al., 2005). Its genome of about 1.18 Mb encodes for about 911 ORFs and six tRNA genes (Raoult et al., 2004). 
As this virus was shown to grow well within Acanthamoeba polyphaga (La Scola et al., 2003a), its possible resistance to destruction by human macrophages was readily suspected and confirmed by Ghigo et al. (2008). Thus, mimivirus was shown to selectively enter within human macrophages and not into epithelial cells (Ghigo et al., 2008). More importantly, entry occurred by phagocytosis (as demonstrated by its inhibition by overexpression of a dominant-negative form of a regulator of phagocytosis, dynamin-II), and was followed by efficient exponential replication (Ghigo et al., 2008). This further supported the paradigm that intra-amoebal pathogens may also be resistant to macrophages and suggested its possible pathogenic role towards humans. 
Some clinical studies have further supported the possible role of mimivirus in lower respiratory tract infections. Thus, 36 (9.7%) patients with community-acquired pneumonia exhibited antibody reactivity against mimivirus as compared with 12 (2.3%) of 511 healthy controls (P<0.01) (La Scola et al., 2005). In addition, serologic evidence of mimivirus infection was also observed in five (19.2%) of 26 intensive care unit patients, whereas none of the 50 control patients were seropositive for mimivirus (P<0.01). More importantly, mimivirus DNA was detected in a bronchoalveolar lavage sample from a 60-year-old comatose patient who presented two episodes of hospital-acquired pneumonia during hospitalization in the intensive care unit (La Scola et al., 2005). Moreover, in another study, patients exhibiting antimimivirus antibodies had longer durations of mechanical ventilation and intensive-care unit stay, with median excesses of 7 and 10 days, respectively (Vincent et al., 2009). However, when testing 496 different pneumonia patients with two different real-time PCRs, other investigators failed to identify any case of mimivirus (Dare et al., 2008). This discrepancy might potentially be due to the possible occurrence of mimivirus-like strains, which cross-react with mimivirus but exhibit some differences in the region of the viral helicase and thiol-oxidoreductase genes used as PCR targets, preventing their detection using this method (Dare et al., 2008). It is noteworthy that the pathogenic role of mimivirus has been clearly demonstrated by an accidental exposure of a laboratory technician to mimivirus (Raoult et al., 2006). Besides strongly supporting the pathogenicity of mimivirus, this laboratory-acquired infection highlights the importance of cautious manipulation of such emerging potential pathogens, especially given the current absence of antivirals that are efficient against such giant viruses. 
Finally, the pathogenic role of mimivirus was further supported by a mouse model of infection (Khan et al., 2007). Thus, mice inoculated through the intracardiac route presented pneumonia similar to viral pneumonia due to measles, smallpox and rubella. Interestingly, a virophage called Sputnik was found closely associated with a new giant virus also belonging to the Mimiviridae (La Scola et al., 2008). Both the giant virus and the virophage may replicate efficiently within Acanthamoeba amoebae (Fig. 7). Whether this small icosahedral virus may modify the pathogenicity of the Mimiviridae remains to be investigated. 
In conclusion, humans are commonly exposed to mimivirus or cross-reacting agents and mimivirus was pathogenic at least towards humans following accidental laboratory exposure and to mice following intravenous challenge. Moreover, this amoebae-resisting virus (or a related cross-reactive species) should be considered as an emerging agent of both nosocomial and community-acquired pneumonia and clearly merits further study. 
Mimivirus (Mimiviridae) is a double-stranded DNA virus belonging to the nucleocytoplasmic large DNA viruses (NCLDV). Initially discovered within free-living amoebae recovered by amoebal enrichment from a cooling tower during the investigation of an outbreak of community-acquired pneumonia in Bradford (UK), this microorganism was first considered to be a gram-positive bacterial coccus (La Scola et al., 2003a). However, electron microscopy suggested that it was a giant virus of about 400 nm in diameter and subsequent studies unequivocally confirmed its affiliation within the NCLDV, which also includes the Iridoviridae, the Phycodnaviridae, the Asfarviridae and the Poxviridae (Koonin et al., 2005). Its genome of about 1.18 Mb encodes for about 911 ORFs and six tRNA genes (Raoult et al., 2004). 
As this virus was shown to grow well within Acanthamoeba polyphaga (La Scola et al., 2003a), its possible resistance to destruction by human macrophages was readily suspected and confirmed by Ghigo et al. (2008). Thus, mimivirus was shown to selectively enter within human macrophages and not into epithelial cells (Ghigo et al., 2008). More importantly, entry occurred by phagocytosis (as demonstrated by its inhibition by overexpression of a dominant-negative form of a regulator of phagocytosis, dynamin-II), and was followed by efficient exponential replication (Ghigo et al., 2008). This further supported the paradigm that intra-amoebal pathogens may also be resistant to macrophages and suggested its possible pathogenic role towards humans. 
Some clinical studies have further supported the possible role of mimivirus in lower respiratory tract infections. Thus, 36 (9.7%) patients with community-acquired pneumonia exhibited antibody reactivity against mimivirus as compared with 12 (2.3%) of 511 healthy controls (P<0.01) (La Scola et al., 2005). In addition, serologic evidence of mimivirus infection was also observed in five (19.2%) of 26 intensive care unit patients, whereas none of the 50 control patients were seropositive for mimivirus (P<0.01). More importantly, mimivirus DNA was detected in a bronchoalveolar lavage sample from a 60-year-old comatose patient who presented two episodes of hospital-acquired pneumonia during hospitalization in the intensive care unit (La Scola et al., 2005). Moreover, in another study, patients exhibiting antimimivirus antibodies had longer durations of mechanical ventilation and intensive-care unit stay, with median excesses of 7 and 10 days, respectively (Vincent et al., 2009). However, when testing 496 different pneumonia patients with two different real-time PCRs, other investigators failed to identify any case of mimivirus (Dare et al., 2008). This discrepancy might potentially be due to the possible occurrence of mimivirus-like strains, which cross-react with mimivirus but exhibit some differences in the region of the viral helicase and thiol-oxidoreductase genes used as PCR targets, preventing their detection using this method (Dare et al., 2008). It is noteworthy that the pathogenic role of mimivirus has been clearly demonstrated by an accidental exposure of a laboratory technician to mimivirus (Raoult et al., 2006). Besides strongly supporting the pathogenicity of mimivirus, this laboratory-acquired infection highlights the importance of cautious manipulation of such emerging potential pathogens, especially given the current absence of antivirals that are efficient against such giant viruses. 
Finally, the pathogenic role of mimivirus was further supported by a mouse model of infection (Khan et al., 2007). Thus, mice inoculated through the intracardiac route presented pneumonia similar to viral pneumonia due to measles, smallpox and rubella. Interestingly, a virophage called Sputnik was found closely associated with a new giant virus also belonging to the Mimiviridae (La Scola et al., 2008). Both the giant virus and the virophage may replicate efficiently within Acanthamoeba amoebae (Fig. 7). Whether this small icosahedral virus may modify the pathogenicity of the Mimiviridae remains to be investigated. 
In conclusion, humans are commonly exposed to mimivirus or cross-reacting agents and mimivirus was pathogenic at least towards humans following accidental laboratory exposure and to mice following intravenous challenge. Moreover, this amoebae-resisting virus (or a related cross-reactive species) should be considered as an emerging agent of both nosocomial and community-acquired pneumonia and clearly merits further study. 

The research community should thus be aware of the wide biodiversity of amoebae-resisting microorganisms including novel Chlamydiae, new LLAP, some Bradyrhizobiaceae, the recently described novel waterborne mycobacterial species and giant viruses. Although Parachlamydia and some other amoebae-resisting Chlamydiae have already been investigated for their pathogenic potential (Corsaro & Greub, 2006; Greub, 2009), there is still an infinite and exciting perspective for further investigations with regard to the development of new diagnostic tools and the comprehension of the pathogenic roles and the cell biology of such microorganisms.

Further investigations may be especially important given the fact that the intra-amoebal environment may select strains that are preferentially resistant to antibiotics and to biocides. Such resistance may be due to the partial protection conferred by amoebal trophozoites and cysts, or may result from the acquisition of efflux mechanisms following exposition to heavy metals and other toxic compounds when inside the amoebal host. The concomitant resistance to biocides and chemical compounds that was first described in mycobacteria (Miltner & Bermudez, 2000), as well as the unexpected resistance of Chlamydiae to quinolones (Maurin et al., 2002; Casson & Greub, 2006; Goy & Greub, 2009), are illustrations of the potential of amoebae-resisting bacteria to select new virulence traits. Moreover, the intra-amoebal environment likely represents an important niche for gene exchange between intracellular pathogens, as exemplified by the occurrence in Rickettsia bellii (Ogata et al., 2006) and in P. amoebophila (Greub et al., 2004a) of similar genes encoding a putative F-like conjugative DNA transfer system. Such gene exchanges not only occur between different bacterial clades, but also likely take place with giant amoebae-resisting viruses and with the genomic content of the amoebal host itself, explaining the relatively large genomes of amoebae-resisting microorganisms.

Conclusion

Amoebae and intra-amoebal microorganisms have coevolved for millions of years and have generated a wide biodiversity of microorganisms that are likely to be able to resist both the phagocytic machinery of amoebae and human macrophages. Thus, the amoebal evolutionary crib may have produced a widespread biodiversity of potential pathogenic species that remain to be discovered. The isolation of these fastidious bacteria and their species identification by culture-based methods, such as amoebal coculture and the amoebal enrichment, as well as the development of molecular methods for their detection in clinical samples, are warranted for a better assessment of their actual role in human diseases such as pneumonia, which remains a major cause of morbidity and mortality in the world. A better comprehensive approach of the interactions between free-living amoebae and amoebae-resisting organisms may give further insights into the mechanisms of pathogenicity of microorganisms and their mode of acquisition of resistance to environmental aggressions, such as phagocytosis by amoebae and macrophages, biocides or chemical compounds, with potential implications for therapeutic approaches.

Acknowledgements

Research performed in the Greub group is currently supported by the Swiss National Science Foundation (grants nos 310030-124843, 3200BO-116445 and PDFMP3-127302), by the American National Institutes of Health (NIH subcontract for grant no. 7R03AI067412-02), by the Swiss HIV cohort study (projects SHCS421 & 515), by SUEZ-ONDEO (Paris, France), by the 3R Foundation (no. 99/05), by the Infectigen foundation (In010) and by the University of Lausanne. We warmly thank A. Croxatto for providing (panel c) of Fig. 4, Bernard La Scola for providing Fig. 7 and Dieter Haas for his constructive review of this manuscript.

Figure 7

Presence of numerous viral particles (mimivirus) within the Acanthamoeba polyphaga amoeba. Note the presence in the middle of the picture of very small viral particles corresponding to the Sputnik virus. Scale bar=2 μm.

Figure 7

Presence of numerous viral particles (mimivirus) within the Acanthamoeba polyphaga amoeba. Note the presence in the middle of the picture of very small viral particles corresponding to the Sputnik virus. Scale bar=2 μm.

References

Abe
Y
Nakamura
M
Suzuki
K
Hashizume
T
Tanigaki
T
Saito
T
Fujino
T
Kikuchi
K
(
1999
)
Massive hemoptysis due to Mycobacterium fortuitum infection controlled with bronchial artery embolization – a case report
.
Clin Imag
 
23
:
361
363
.
Adekambi
T
Reynaud-Gaubert
M
Greub
G
Gevaudan
MJ
La Scola
B
Raoult
D
Drancourt
M
(
2004
)
Amoebal coculture of ‘Mycobacterium massiliense’ sp. nov. from the sputum of a patient with hemoptoic pneumonia
.
J Clin Microbiol
 
42
:
5493
5501
.
Adekambi
T
Ben Salah
S
Khlif
M
Raoult
D
Drancourt
M
(
2006
)
Survival of environmental mycobacteria in Acanthamoeba polyphaga
.
Appl Environ Microb
 
72
:
5974
5981
.
Adeleke
A
Pruckler
J
Benson
R
Rowbotham
T
Halablab
M
Fields
B
(
1996
)
Legionella-like amebal pathogens – phylogenetic status and possible role in respiratory disease
.
Emerg Infect Dis
 
2
:
225
230
.
Adeleke
AA
Fields
BS
Benson
RF
et al. (
2001
)
Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species
.
Int J Syst Evol Micr
 
51
:
1151
1160
.
Al Shaalan
M
Law
BJ
Israels
SJ
Pianosi
P
Lacson
AG
Higgins
R
(
1997
)
Mycobacterium fortuitum interstitial pneumonia with vasculitis in a child with Wilms' tumor
.
Pediatr Infect Dis J
 
16
:
996
1000
.
Amann
R
Springer
N
Schonhuber
W
Ludwig
W
Schmid
EN
Muller
KD
Michel
R
(
1997
)
Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp
.
Appl Environ Microb
 
63
:
115
121
.
Andrejak
C
Lescure
FX
Douadi
Y
Laurans
G
Smail
A
Duhaut
P
Jounieaux
V
Schmit
JL
(
2007
)
Non-tuberculous mycobacteria pulmonary infection: management and follow-up of 31 infected patients
.
J Infection
 
55
:
34
40
.
Bell
RC
Higuchi
JH
Donovan
WN
Krasnow
I
Johanson
WG
Jr
(
1983
)
Mycobacterium simiae. Clinical features and follow-up of twenty-four patients
.
Am Rev Respir Dis
 
127
:
35
38
.
Benin
AL
Benson
RF
Besser
RE
(
2002
)
Trends in Legionnaire's disease, 1980–1998: declining mortality and new patterns of diagnosis
.
Clin Infect Dis
 
35
:
1039
1046
.
Benson
RF
Fields
BS
(
1998
)
Classification of the genus Legionella
.
Semin Respir Infect
 
13
:
90
99
.
Berger
P
Papazian
L
Drancourt
M
La Scola
B
Auffray
JP
Raoult
D
(
2006
)
Ameba-associated microorganisms and diagnosis of nosocomial pneumonia
.
Emerg Infect Dis
 
12
:
248
255
.
Birtles
RJ
Rowbotham
TJ
Raoult
D
Harrison
TG
(
1996
)
Phylogenetic diversity of intra-amoebal legionellae as revealed by 16S rRNA gene sequence comparison
.
Microbiology
 
142
(Part 12)
:
3525
3530
.
Birtles
RJ
Rowbotham
TJ
Storey
C
Marrie
TJ
Raoult
D
(
1997
)
Chlamydia-like obligate parasite of free-living amoebae
.
Lancet
 
349
:
925
926
.
Black
WC
Berk
SG
(
2003
)
Cooling towers – a potential environmental source of slow-growing mycobacterial species
.
AIHA J
 
64
:
238
242
.
Blatt
SP
Parkinson
MD
Pace
E
Hoffman
P
Dolan
D
Lauderdale
P
Zajac
RA
Melcher
GP
(
1993
)
Nosocomial Legionnaires' disease: aspiration as a primary mode of disease acquisition
.
Am J Med
 
95
:
16
22
.
Bochud
PY
Moser
F
Erard
P
et al. (
2001
)
Community-acquired pneumonia. A prospective outpatient study
.
Medicine
 
80
:
75
87
.
Brenner
DJ
Hollis
DG
Moss
CW
English
CK
Hall
GS
Vincent
J
Radosevic
J
Birkness
KA
Bibb
WF
Quinn
FD
(
1991
)
Proposal of Afipia gen. nov., with Afipia felis sp. nov. (formerly the cat scratch disease bacillus), Afipia clevelandensis sp. nov. (formerly the Cleveland Clinic Foundation strain), Afipia broomeae sp. nov., and three unnamed genospecies
.
J Clin Microbiol
 
29
:
2450
2460
.
Burke
DS
Ullian
RB
(
1977
)
Megaesophagus and pneumonia associated with Mycobacterium chelonei. A case report and a literature review
.
Am Rev Respir Dis
 
116
:
1101
1107
.
Campo
RE
Campo
CE
(
1997
)
Mycobacterium kansasii disease in patients infected with human immunodeficiency virus
.
Clin Infect Dis
 
24
:
1233
1238
.
Casson
N
Greub
G
(
2006
)
Resistance of different Chlamydia-like organisms to quinolones and mutations in the quinoline resistance-determining region of the DNA gyrase A- and topoisomerase-encoding genes
.
Int J Antimicrob Ag
 
27
:
541
544
.
Casson
N
Medico
N
Bille
J
Greub
G
(
2006
)
Parachlamydia acanthamoebae enters and multiplies within pneumocytes and lung fibroblasts
.
Microbes Infect
 
8
:
1294
1300
.
Casson
N
Entenza
JM
Borel
N
Pospischil
A
Greub
G
(
2008a
)
Murine model of pneumonia caused by Parachlamydia acanthamoebae
.
Microb Pathogenesis
 
45
:
92
97
.
Casson
N
Michel
R
Muller
KD
Aubert
JD
Greub
G
(
2008b
)
Protochlamydia naegleriophila as aetiologic agent of pneumonia
.
Emerg Infect Dis
 
14
:
168
172
.
Casson
N
Posfay-Barbe
KM
Gervaix
A
Greub
G
(
2008c
)
New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples
.
J Clin Microbiol
 
46
:
1491
1493
.
Cirillo
JD
Falkow
S
Tompkins
LS
Bermudez
LE
(
1997
)
Interaction of Mycobacterium avium with environmental amoebae enhances virulence
.
Infect Immun
 
65
:
3759
3767
.
Conger
NG
O'Connell
RJ
Laurel
VL
Olivier
KN
Graviss
EA
Williams-Bouyer
N
Zhang
Y
Brown-Elliott
BA
Wallace
RJ
Jr
(
2004
)
Mycobacterium simae outbreak associated with a hospital water supply
.
Infect Cont Hosp Ep
 
25
:
1050
1055
.
Corsaro
D
Greub
G
(
2006
)
Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria
.
Clin Microbiol Rev
 
19
:
283
297
.
Corsaro
D
Venditti
D
Le Faou
A
Guglielmetti
P
Valassina
M
(
2001
)
A new Chlamydia-like 16S rDNA sequence from a clinical sample
.
Microbiology
 
147
:
515
516
.
Corsaro
D
Venditti
D
Valassina
M
(
2002
)
New parachlamydial 16S rDNA phylotypes detected in human clinical samples
.
Res Microbiol
 
153
:
563
567
.
Corsaro
D
Feroldi
V
Saucedo
G
Ribas
F
Loret
JF
Greub
G
(
2009
)
Novel Chlamydiales strains isolated from a water treatment plant
.
Environ Microbiol
 
11
:
188
200
.
Costa
SF
Newbaer
M
Santos
CR
Basso
M
Soares
I
Levin
AS
(
2001
)
Nosocomial pneumonia: importance of recognition of aetiological agents to define an appropriate initial empirical therapy
.
Int J Antimicrob Ag
 
17
:
147
150
.
Danelishvili
L
Wu
M
Stang
B
Harriff
M
Cirillo
SL
Cirillo
JD
Bildfell
R
Arbogast
B
Bermudez
LE
(
2007
)
Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection
.
P Natl Acad Sci USA
 
104
:
11038
11043
.
Dare
RK
Chittaganpitch
M
Erdman
DD
(
2008
)
Screening pneumonia patients for mimivirus
.
Emerg Infect Dis
 
14
:
465
467
.
Das
SK
Mishra
AK
Tindall
BJ
Rainey
FA
Stackebrandt
E
(
1996
)
Oxidation of thiosulfate by a new bacterium, Bosea thiooxidans (strain BI-42) gen. nov., sp. nov.: analysis of phylogeny based on chemotaxonomy and 16S ribosomal DNA sequencing
.
Int J Syst Bacteriol
 
46
:
981
987
.
Diederen
BM
De Jong
CM
Aarts
I
Peeters
MF
Van Der Zee
A
(
2007
)
Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations
.
J Water Health
 
5
:
375
383
.
Doebbeling
BN
Wenzel
RP
(
1987
)
The epidemiology of Legionella pneumophila infections
.
Semin Respir Infect
 
2
:
206
221
.
Doebbeling
BN
Ishak
MA
Wade
BH
Pasquale
MA
Gerszten
RE
Groschel
DH
Kadner
RJ
Wenzel
RP
(
1989
)
Nosocomial Legionella micdadei pneumonia: 10 years experience and a case–control study
.
J Hosp Infect
 
13
:
289
298
.
Doleans
A
Aurell
H
Reyrolle
M
Lina
G
Freney
J
Vandenesch
F
Etienne
J
Jarraud
S
(
2004
)
Clinical and environmental distributions of Legionella strains in France are different
.
J Clin Microbiol
 
42
:
458
460
.
Drancourt
M
Brouqui
P
Raoult
D
(
1997
)
Afipia clevelandensis antibodies and cross-reactivity with Brucella spp. and Yersinia enterocolitica O:9
.
Clin Diagn Lab Immun
 
4
:
748
752
.
Dumoff
K
McGovern
PC
Edelstein
PH
Nachamkin
I
(
2004
)
Legionella maceachernii pneumonia in a patient with HIV infection
.
Diagn Micr Infec Dis
 
50
:
141
145
.
Echols
RM
Tillotson
GS
Song
JX
Tosiello
RL
(
2008
)
Clinical trial design for mild-to-moderate community-acquired pneumonia – an industry perspective
.
Clin Infect Dis
 
47
(suppl 3)
:
S166
S175
.
Eckburg
PB
Buadu
EO
Stark
P
Sarinas
PS
Chitkara
RK
Kuschner
WG
(
2000
)
Clinical and chest radiographic findings among persons with sputum culture positive for Mycobacterium gordonae: a review of 19 cases
.
Chest
 
117
:
96
102
.
Edelstein
PH
Brenner
DJ
Moss
CW
Steigerwalt
AG
Francis
EM
George
WL
(
1982
)
Legionella wadsworthii species nova: a cause of human pneumonia
.
Ann Intern Med
 
97
:
809
813
.
Ellis
ME
Qadri
SM
(
1993
)
Mycobacteria other than tuberculosis producing disease in a tertiary referral hospital
.
Ann Saudi Med
 
13
:
508
515
.
El Sahly
HM
Septimus
E
Soini
H
Septimus
J
Wallace
RJ
Pan
X
Williams-Bouyer
N
Musser
JM
Graviss
EA
(
2002
)
Mycobacterium simiae pseudo-outbreak resulting from a contaminated hospital water supply in Houston, Texas
.
Clin Infect Dis
 
35
:
802
807
.
Embil
J
Warren
P
Yakrus
M
Stark
R
Corne
S
Forrest
D
Hershfield
E
(
1997
)
Pulmonary illness associated with exposure to Mycobacterium avium complex in hot tub water. Hypersensitivity pneumonitis or infection?
Chest
 
111
:
813
816
.
English
CK
Wear
DJ
Margileth
AM
Lissner
CR
Walsh
GP
(
1988
)
Cat-scratch disease. Isolation and culture of the bacterial agent
.
J Am Med Assoc
 
259
:
1347
1352
.
Ephros
M
Engelhard
D
Maayan
S
Bercovier
H
Avital
A
Yatsiv
I
(
1989
)
Legionella gormanii pneumonia in a child with chronic granulomatous disease
.
Pediatr Infect Dis J
 
8
:
726
727
.
Ernst
A
Gordon
FD
Hayek
J
Silvestri
RC
Koziel
H
(
1998
)
Lung abscess complicating Legionella micdadei pneumonia in an adult liver transplant recipient: case report and review
.
Transplantation
 
65
:
130
134
.
Evans
AJ
Crisp
AJ
Hubbard
RB
Colville
A
Evans
SA
Johnston
ID
(
1996a
)
Pulmonary Mycobacterium kansasii infection: comparison of radiological appearances with pulmonary tuberculosis
.
Thorax
 
51
:
1243
1247
.
Evans
SA
Colville
A
Evans
AJ
Crisp
AJ
Johnston
ID
(
1996b
)
Pulmonary Mycobacterium kansasii infection: comparison of the clinical features, treatment and outcome with pulmonary tuberculosis
.
Thorax
 
51
:
1248
1252
.
Everett
KD
Bush
RM
Andersen
AA
(
1999
)
Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms
.
Int J Syst Bacteriol
 
49
(Part 2)
:
415
440
.
Falkinham
JO
III
(
1996
)
Epidemiology of infection by nontuberculous mycobacteria
.
Clin Microbiol Rev
 
9
:
177
215
.
Falkinham
JO
III
(
2002
)
Nontuberculous mycobacteria in the environment
.
Clin Chest Med
 
23
:
529
551
.
Fang
GD
Yu
VL
Vickers
RM
(
1987
)
Infections caused by the Pittsburgh pneumonia agent
.
Semin Respir Infect
 
2
:
262
266
.
Fang
GD
Yu
VL
Vickers
RM
(
1989
)
Disease due to the Legionellaceae (other than Legionella pneumophila). Historical, microbiological, clinical, and epidemiological review
.
Medicine
 
68
:
116
132
.
Fang
GD
Fine
M
Orloff
J
Arisumi
D
Yu
VL
Kapoor
W
Grayston
JT
Wang
SP
Kohler
R
Muder
RR
(
1990a
)
New and emerging aetiologies for community-acquired pneumonia with implications for therapy. A prospective multicenter study of 359 cases
.
Medicine
 
69
:
307
316
.
Fang
GD
Stout
JE
Yu
VL
Goetz
A
Rihs
JD
Vickers
RM
(
1990b
)
Community-acquired pneumonia caused by Legionella dumoffii in a patient with hairy cell leukemia
.
Infection
 
18
:
383
385
.
Faress
JA
McKinney
LA
Semaan
MT
Byrd
RP
Jr
Mehta
JB
Roy
TM
(
2003
)
Mycobacterium xenopi pneumonia in the southeastern United States
.
South Med J
 
96
:
596
599
.
Fasoli
L
Paldanius
M
Don
M
Valent
F
Vetrugno
L
Korppi
M
Canciani
M
(
2008
)
Simkania negevensis in community-acquired pneumonia in Italian children
.
Scand J Infect Dis
 
40
:
269
272
.
Fenstersheib
MD
Miller
M
Diggins
C
Liska
S
Detwiler
L
Werner
SB
Lindquist
D
Thacker
WL
Benson
RF
(
1990
)
Outbreak of pontiac fever due to Legionella anisa
.
Lancet
 
336
:
35
37
.
Field
SK
Cowie
RL
(
2006
)
Lung disease due to the more common nontuberculous mycobacteria
.
Chest
 
129
:
1653
1672
.
Fraser
DW
Tsai
TR
Orenstein
W
et al. (
1977
)
‘Legionnaires’ disease: description of an epidemic of pneumonia
.
New Engl J Med
 
297
:
1189
1197
.
Friedman
MG
Galil
A
Greenberg
S
Kahane
S
(
1999
)
Seroprevalence of IgG antibodies to the Chlamydia-like microorganism ‘Simkania Z’ by ELISA
.
Epidemiol Infect
 
122
:
117
123
.
Friedman
MG
Dvoskin
B
Kahane
S
(
2003
)
Infections with the Chlamydia-like microorganism Simkania negevensis, a possible emerging pathogen
.
Microbes Infect
 
5
:
1013
1021
.
Friedman
MG
Kahane
S
Dvoskin
B
Hartley
JW
(
2006
)
Detection of Simkania negevensis by culture, PCR, and serology in respiratory tract infection in Cornwall, UK
.
J Clin Pathol
 
59
:
331
333
.
Fry
NK
Rowbotham
TJ
Saunders
NA
Embley
TM
(
1991
)
Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia
.
FEMS Microbiol Lett
 
67
:
165
168
.
Gao
LY
Susa
M
Ticac
B
Abu Kwaik
Y
(
1999
)
Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells
.
Microb Pathog
 
27
:
273
287
.
Ghigo
E
Kartenbeck
J
Lien
P
Pelkmans
L
Capo
C
Mege
JL
Raoult
D
(
2008
)
Amoebal pathogen mimivirus infects macrophages through phagocytosis
.
PLoS Pathog
 
4
:
e1000087
.
Goldberg
DJ
Wrench
JG
Collier
PW
Emslie
JA
Fallon
RJ
Forbes
GI
McKay
TM
Macpherson
AC
Markwick
TA
Reid
D
(
1989
)
Lochgoilhead fever: outbreak of non-pneumonic legionellosis due to Legionella micdadei
.
Lancet
 
i
:
316
318
.
Goy
G
Greub
G
(
2009
)
Antibiotic susceptibility of Waddlia chondrophila in Acanthamoeba castellanii amoebae
.
Antimicrob Agents Ch
 
53
:
2663
2666
.
Goy
G
Thomas
V
Rimann
K
Jaton
K
Prod'hom
G
Greub
G
(
2007
)
The Neff strain of Acanthamoeba castellanii, a tool for testing the virulence of Mycobacterium kansasii
.
Res Microbiol
 
158
:
393
397
.
Greenberg
D
Banerji
A
Friedman
MG
Chiu
CH
Kahane
S
(
2003
)
High rate of Simkania negevensis among Canadian inuit infants hospitalized with lower respiratory tract infections
.
Scand J Infect Dis
 
35
:
506
508
.
Greub
G
(
2009
)
Parachlamydia acanthamoebae, an emerging agent of pneumonia
.
Clin Microbiol Infec
 
15
:
18
28
.
Greub
G
Raoult
D
(
2004
)
Microorganisms resistant to free-living amoebae
.
Clin Microbiol Rev
 
17
:
413
433
.
Greub
G
Berger
P
Papazian
L
Raoult
D
(
2003a
)
Parachlamydiaceae as rare agents of pneumonia
.
Emerg Infect Dis
 
9
:
755
756
.
Greub
G
Boyadjiev
I
La Scola
B
Raoult
D
Martin
C
(
2003b
)
Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive care patients
.
Ann NY Acad Sci
 
990
:
311
319
.
Greub
G
Mege
JL
Raoult
D
(
2003c
)
Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis
.
Infect Immun
 
71
:
5979
5985
.
Greub
G
Collyn
F
Guy
L
Roten
CA
(
2004a
)
A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system
.
BMC Microbiol
 
4
:
48
.
Greub
G
La Scola
B
Raoult
D
(
2004b
)
Amoebae-resisting bacteria isolated from human nasal swabs by amoebal coculture
.
Emerg Infect Dis
 
10
:
470
477
.
Greub
G
Mege
JL
Gorvel
JP
Raoult
D
Meresse
S
(
2005
)
Intracellular trafficking of Parachlamydia acanthamoebae
.
Cell Microbiol
 
7
:
581
589
.
Griffith
ME
Lindquist
DS
Benson
RF
Thacker
WL
Brenner
DJ
Wilkinson
HW
(
1988
)
First isolation of Legionella gormanii from human disease
.
J Clin Microbiol
 
26
:
380
381
.
Grove
DI
Lawson
PJ
Burgess
JS
Moran
JL
O'Fathartaigh
MS
Winslow
WE
(
2002
)
An outbreak of Legionella longbeachae infection in an intensive care unit?
J Hosp Infect
 
52
:
250
258
.
Hadjiliadis
D
Adlakha
A
Prakash
UB
(
1999
)
Rapidly growing mycobacterial lung infection in association with esophageal disorders
.
Mayo Clin Proc
 
74
:
45
51
.
Haider
S
Collingro
A
Walochnik
J
Wagner
M
Horn
M
(
2008
)
Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients
.
FEMS Microbiol Lett
 
281
:
198
202
.
Hall
GS
Pratt-Rippin
K
Washington
JA
(
1991
)
Isolation of agent associated with cat scratch disease bacillus from pretibial biopsy
.
Diagn Micr Infec Dis
 
14
:
511
513
.
Harris
A
Lally
M
Albrecht
M
(
1998
)
Legionella bozemanii pneumonia in three patients with AIDS
.
Clin Infect Dis
 
27
:
97
99
.
Heiskanen-Kosma
T
Paldanius
M
Korppi
M
(
2008
)
Simkania negevensis may be a true cause of community acquired pneumonia in children
.
Scand J Infect Dis
 
40
:
127
130
.
Henriques
B
Hoffner
SE
Petrini
B
Juhlin
I
Wahlen
P
Kallenius
G
(
1994
)
Infection with Mycobacterium malmoense in Sweden: report of 221 cases
.
Clin Infect Dis
 
18
:
596
600
.
Herwaldt
LA
Gorman
GW
McGrath
T
Toma
S
Brake
B
Hightower
AW
Jones
J
Reingold
AL
Boxer
PA
Tang
PW
(
1984
)
A new Legionella species, Legionella feeleii species nova, causes pontiac fever in an automobile plant
.
Ann Intern Med
 
100
:
333
338
.
Howard
RS
Woodring
JH
Vandiviere
HM
Dillon
ML
(
1991
)
Mycobacterium fortuitum pulmonary infection complicating achalasia
.
South Med J
 
84
:
1391
1393
.
Hsu
BM
Lin
CL
Shih
FC
(
2009
)
Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area
.
Water Res
 
43
:
2817
2828
.
Huminer
D
Dux
S
Samra
Z
Kaufman
L
Lavy
A
Block
CS
Pitlik
SD
(
1993
)
Mycobacterium simiae infection in Israeli patients with AIDS
.
Clin Infect Dis
 
17
:
508
509
.
Husain
S
Kahane
S
Friedman
MG
Paterson
DL
Studer
S
McCurry
KR
Wolf
DG
Zeevi
A
Pilewski
J
Greenberg
D
(
2007
)
Simkania negevensis in bronchoalveolar lavage of lung transplant recipients: a possible association with acute rejection
.
Transplantation
 
83
:
138
143
.
Jadin
JB
(
1973
)
Hypotheses on the adaptation of amoebas of the limax group to man and animals
.
Ann Parasit Hum Comp
 
48
:
199
204
.
Jernigan
DB
Sanders
LI
Waites
KB
Brookings
ES
Benson
RF
Pappas
PG
(
1994
)
Pulmonary infection due to Legionella cincinnatiensis in renal transplant recipients: two cases and implications for laboratory diagnosis
.
Clin Infect Dis
 
18
:
385
389
.
Jerris
RC
Regnery
RL
(
1996
)
Will the real agent of cat-scratch disease please stand up?
Annu Rev Microbiol
 
50
:
707
725
.
Johnsen
S
Birkebaek
N
Andersen
PL
Emil
C
Jensen
JS
Ostergaard
L
(
2005
)
Indirect immunofluorescence and real time PCR for detection of Simkania negevensis infection in Danish adults with persistent cough and in healthy controls
.
Scand J Infect Dis
 
37
:
251
255
.
Juffermans
NP
Verbon
A
Danner
SA
Kuijper
EJ
Speelman
P
(
1998
)
Mycobacterium xenopi in HIV-infected patients: an emerging pathogen
.
AIDS
 
12
:
1661
1666
.
Kahane
S
Gonen
R
Sayada
C
Elion
J
Friedman
MG
(
1993
)
Description and partial characterization of a new Chlamydia-like microorganism
.
FEMS Microbiol Lett
 
109
:
329
333
.
Kahane
S
Greenberg
D
Friedman
MG
Haikin
H
Dagan
R
(
1998
)
High prevalence of ‘Simkania Z,’ a novel Chlamydia-like bacterium, in infants with acute bronchiolitis
.
J Infect Dis
 
177
:
1425
1429
.
Kahane
S
Fruchter
D
Dvoskin
B
Friedman
MG
(
2007
)
Versatility of Simkania negevensis infection in vitro and induction of host cell inflammatory cytokine response
.
J Infection
 
55
:
e13
e21
.
Kahane
S
Dvoskin
B
Friedman
MG
(
2008
)
The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model
.
FEMS Immunol Med Mic
 
52
:
219
227
.
Khan
M
La Scola
B
Lepidi
H
Raoult
D
(
2007
)
Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus
.
Microb Pathogenesis
 
42
:
56
61
.
Kim
RD
Greenberg
DE
Ehrmantraut
ME
et al. (
2008
)
Pulmonary nontuberculous mycobacterial disease: prospective study of a distinct preexisting syndrome
.
Am J Resp Crit Care
 
178
:
1066
1074
.
Knirsch
CA
Jakob
K
Schoonmaker
D
Kiehlbauch
JA
Wong
SJ
Della-Latta
P
Whittier
S
Layton
M
Scully
B
(
2000
)
An outbreak of Legionella micdadei pneumonia in transplant patients: evaluation, molecular epidemiology, and control
.
Am J Med
 
108
:
290
295
.
Koonin
EV
(
2005
)
Virology: Gulliver among the Lilliputians
.
Curr Biol
 
15
:
R167
R169
.
Korppi
M
Paldanius
M
Hyvarinen
A
Nevalainen
A
(
2006
)
Simkania negevensis and newly diagnosed asthma: a case–control study in 1- to 6-year-old children
.
Respirology
 
11
:
80
83
.
Krishna Prasad
BN
Gupta
SK
(
1978
)
Preliminary report on engulfment and retention of mycobacteria by trophozoites of axenically grow Acanthamoeba castellanii Douglas
.
Curr Sci
 
47
:
245
247
.
Kumar
S
Kohlhoff
SA
Gelling
M
Roblin
PM
Kutlin
A
Kahane
S
Friedman
MG
Hammerschlag
MR
(
2005
)
Infection with Simkania negevensis in Brooklyn, New York
.
Pediatr Infect Dis J
 
24
:
989
992
.
Kumpers
P
Tiede
A
Kirschner
P
Girke
J
Ganser
A
Peest
D
(
2008
)
‘Legionnaires’ disease in immunocompromised patients: a case report of Legionella longbeachae pneumonia and review of the literature
.
J Med Microbiol
 
57
:
384
387
.
Lamoth
F
Aeby
S
Schneider
A
Jaton-Ogay
K
Vaudaux
B
Greub
G
(
2009
)
Parachlamydia and Rhabdochlamydia in respiratory secretions of premature neonates: prevalence and clinical impact
.
Emerg Infect Dis
 
15
:
2072
2075
.
La Scola
B
Barrassi
L
Raoult
D
(
2000
)
Isolation of new fastidious alpha proteobacteria and Afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures
.
FEMS Microbiol Ecol
 
34
:
129
137
.
La Scola
B
Mezi
L
Auffray
JP
Berland
Y
Raoult
D
(
2002
)
Patients in the intensive care unit are exposed to amoeba-associated pathogens
.
Infect Cont Hosp Ep
 
23
:
462
465
.
La Scola
B
Audic
S
Robert
C
Jungang
L
De Lamballerie
X
Drancourt
M
Birtles
R
Claverie
JM
Raoult
D
(
2003a
)
A giant virus in amoebae
.
Science
 
299
:
2033
.
La Scola
B
Boyadjiev
I
Greub
G
Khamis
A
Martin
C
Raoult
D
(
2003b
)
Amoeba-resisting bacteria and ventilator-associated pneumonia
.
Emerg Infect Dis
 
9
:
815
821
.
La Scola
B
Mallet
MN
Grimont
PA
Raoult
D
(
2003c
)
Bosea eneae sp. nov., Bosea massiliensis sp. nov. and Bosea vestrisii sp. nov., isolated from hospital water supplies, and emendation of the genus Bosea (Das et al. 1996)
.
Int J Syst Evol Micr
 
53
:
15
20
.
La Scola
B
Birtles
RJ
Greub
G
Harrison
TJ
Ratcliff
RM
Raoult
D
(
2004
)
Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen
.
Int J Syst Evol Micr
 
54
:
699
703
.
La Scola
B
Marrie
TJ
Auffray
JP
Raoult
D
(
2005
)
Mimivirus in pneumonia patients
.
Emerg Infect Dis
 
11
:
449
452
.
La Scola
B
Desnues
C
Pagnier
I
et al. (
2008
)
The virophage as a unique parasite of the giant mimivirus
.
Nature
 
455
:
100
104
.
Lee
J
Caplivski
D
Wu
M
Huprikar
S
(
2009
)
Pneumonia due to Legionella feeleii: case report and review of the literature
.
Transpl Infect Dis
 
11
:
337
340
.
Levendoglu-Tugal
O
Munoz
J
Brudnicki
A
Fevzi
OM
Sandoval
C
Jayabose
S
(
1998
)
Infections due to nontuberculous mycobacteria in children with leukemia
.
Clin Infect Dis
 
27
:
1227
1230
.
Lieberman
D
Kahane
S
Lieberman
D
Friedman
MG
(
1997
)
Pneumonia with serological evidence of acute infection with the Chlamydia-like microorganism ‘Z’
.
Am J Resp Crit Care
 
156
:
578
582
.
Lieberman
D
Dvoskin
B
Lieberman
DV
Kahane
S
Friedman
MG
(
2002
)
Serological evidence of acute infection with the Chlamydia-like microorganism Simkania negevensis (Z) in acute exacerbation of chronic obstructive pulmonary disease
.
Eur J Clin Microbiol
 
21
:
307
309
.
Loeb
M
Simor
AE
Mandell
L
et al. (
1999
)
Two nursing home outbreaks of respiratory infection with Legionella sainthelensi
.
J Am Geriatr Soc
 
47
:
547
552
.
Lo Presti
F
Riffard
S
Vandenesch
F
Reyrolle
M
Ronco
E
Ichai
P
Etienne
J
(
1997
)
The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia
.
J Clin Microbiol
 
35
:
1706
1709
.
Lo Presti
F
Riffard
S
Jarraud
S
Le
GF
Richet
H
Vandenesch
F
Etienne
J
(
2000
)
Isolation of Legionella oakridgensis from two patients with pleural effusion living in the same geographical area
.
J Clin Microbiol
 
38
:
3128
3130
.
Loret
JF
Jousset
M
Robert
S
Saucedo
G
Ribas
F
Thomas
V
Greub
G
(
2008
)
Amoebae-resisting bacteria in drinking water: risk assessment and management
.
Water Sci Technol
 
58
:
571
577
.
Maliwan
N
Zvetina
JR
(
2005
)
Clinical features and follow up of 302 patients with Mycobacterium kansasii pulmonary infection: a 50 year experience
.
Postgrad Med J
 
81
:
530
533
.
Mandell
LA
Wunderink
RG
Anzueto
A
et al. (
2007
)
Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults
.
Clin Infect Dis
 
44
(suppl 2)
:
S27
S72
.
Maoz
C
Shitrit
D
Samra
Z
Peled
N
Kaufman
L
Kramer
MR
Bishara
J
(
2008
)
Pulmonary Mycobacterium simiae infection: comparison with pulmonary tuberculosis
.
Eur J Clin Microbiol
 
27
:
945
950
.
Marchevsky
A
Damsker
B
Gribetz
A
Tepper
S
Geller
SA
(
1982
)
The spectrum of pathology of nontuberculous mycobacterial infections in open-lung biopsy specimens
.
Am J Clin Pathol
 
78
:
695
700
.
Marrie
TJ
Durant
H
Yates
L
(
1989
)
Community-acquired pneumonia requiring hospitalization: 5-year prospective study
.
Rev Infect Dis
 
11
:
586
599
.
Marrie
TJ
Raoult
D
La Scola
B
Birtles
RJ
De Carolis
E
(
2001
)
Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia
.
Emerg Infect Dis
 
7
:
1026
1029
.
Martin-Casabona
N
Bahrmand
AR
Bennedsen
J
et al. (
2004
)
Non-tuberculous mycobacteria: patterns of isolation. A multi-country retrospective survey
.
Int J Tuberc Lung D
 
8
:
1186
1193
.
Marusic
A
Katalinic-Jankovic
V
Popovic-Grle
S
Jankovic
M
Mazuranic
I
Puljic
I
Sertic
MH
(
2009
)
Mycobacterium xenopi pulmonary disease – epidemiology and clinical features in non-immunocompromised patients
.
J Infection
 
58
:
108
112
.
Maurin
M
Bryskier
A
Raoult
D
(
2002
)
Antibiotic susceptibilities of Parachlamydia acanthamoeba in amoebae
.
Antimicrob Agents Ch
 
46
:
3065
3067
.
McClelland
MR
Vaszar
LT
Kagawa
FT
(
2004
)
Pneumonia and osteomyelitis due to Legionella longbeachae in a woman with systemic lupus erythematosus
.
Clin Infect Dis
 
38
:
e102
e106
.
McDade
JE
Shepard
CC
Fraser
DW
Tsai
TR
Redus
MA
Dowdle
WR
(
1977
)
‘Legionnaires’ disease: isolation of a bacterium and demonstration of its role in other respiratory disease
.
New Engl J Med
 
297
:
1197
1203
.
McKinney
RM
Porschen
RK
Edelstein
PH
et al. (
1981
)
Legionella longbeachae species nova, another aetiologic agent of human pneumonia
.
Ann Intern Med
 
94
:
739
743
.
McNally
C
Hackman
B
Fields
BS
Plouffe
JF
(
2000
)
Potential importance of Legionella species as aetiologies in community acquired pneumonia (CAP)
.
Diagn Micr Infec Dis
 
38
:
79
82
.
McWhinney
PH
Yates
M
Prentice
HG
Thrussell
M
Gillespie
SH
Kibbler
CC
(
1992
)
Infection caused by Mycobacterium chelonae: a diagnostic and therapeutic problem in the neutropenic patient
.
Clin Infect Dis
 
14
:
1208
1212
.
Mehta
P
Patel
JD
Milder
JE
(
1983
)
Legionella micdadei (Pittsburgh pneumonia agent). Two infections with unusual clinical features
.
J Am Med Assoc
 
249
:
1620
1623
.
Miguez-Burbano
MJ
Flores
M
Ashkin
D
Rodriguez
A
Granada
AM
Quintero
N
Pitchenik
A
(
2006
)
Non-tuberculous mycobacteria disease as a cause of hospitalization in HIV-infected subjects
.
Int J Infect Dis
 
10
:
47
55
.
Miltner
EC
Bermudez
LE
(
2000
)
Mycobacterium avium grown in Acanthamoeba castellanii is protected from the effects of antimicrobials
.
Antimicrob Agents Ch
 
44
:
1990
1994
.
Muder
RR
Yu
VL
(
2002
)
Infection due to Legionella species other than L. pneumophila
.
Clin Infect Dis
 
35
:
990
998
.
Muder
RR
Yu
VL
Zuravleff
JJ
(
1983
)
Pneumonia due to the Pittsburgh pneumonia agent: new clinical perspective with a review of the literature
.
Medicine
 
62
:
120
128
.
Mura
M
Bull
TJ
Evans
H
Sidi-Boumedine
K
McMinn
L
Rhodes
G
Pickup
R
Hermon-Taylor
J
(
2006
)
Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga
.
Appl Environ Microb
 
72
:
854
859
.
Murdoch
DR
(
2003
)
Diagnosis of Legionella infection
.
Clin Infect Dis
 
36
:
64
69
.
Murdoch
DR
Chambers
ST
(
2000
)
Detection of Legionella DNA in peripheral leukocytes, serum, and urine from a patient with pneumonia caused by Legionella dumoffii
.
Clin Infect Dis
 
30
:
382
383
.
Myerowitz
RL
Pasculle
AW
Dowling
JN
Pazin
GJ
Sr
Puerzer
M
Yee
RB
Rinaldo
CR
Jr
Hakala
TR
(
1979
)
Opportunistic lung infection due to ‘Pittsburgh Pneumonia Agent’
.
New Engl J Med
 
301
:
953
958
.
Nascimento-Carvalho
CM
Cardoso
MR
Paldanius
M
Barral
A
Araujo-Neto
CA
Saukkoriipi
A
Vainionpaa
R
Leinonen
M
Ruuskanen
O
(
2009
)
Simkania negevensis infection among Brazilian children hospitalized with community-acquired pneumonia
.
J Infection
 
58
:
250
253
.
Neumeister
B
Schöniger
S
Faigle
M
Eichner
M
Dietz
K
(
1997
)
Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii
.
Appl Environ Microbiol
 
63
:
1219
1224
.
Ogata
H
La Scola
B
Audic
S
Renesto
P
Blanc
G
Robert
C
Fournier
PE
Claverie
JM
Raoult
D
(
2006
)
Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens
.
PLoS Genet
 
2
:
e76
.
Olivier
KN
(
1998
)
Nontuberculous mycobacterial pulmonary disease
.
Curr Opin Pulm Med
 
4
:
148
153
.
Ossewaarde
JM
Meijer
A
(
1999
)
Molecular evidence for the existence of additional members of the order Chlamydiales
.
Microbiology
 
145
(Part 2)
:
411
417
.
Pagnier
I
Merchat
M
La Scola
B
(
2009a
)
Potentially pathogenic amoeba-associated microorganisms in cooling towers and their control
.
Future Microbiol
 
4
:
615
629
.
Pagnier
I
Merchat
M
Raoult
D
La Scola
B
(
2009b
)
Emerging mycobacteria spp. in cooling towers
.
Emerg Infect Dis
 
15
:
121
122
.
Parrish
SC
Myers
J
Lazarus
A
(
2008
)
Nontuberculous mycobacterial pulmonary infections in non-HIV patients
.
Postgrad Med
 
120
:
78
86
.
Peres
E
Khaled
Y
Krijanovski
OI
Mineishi
S
Levine
JE
Kaul
DR
Riddell
J
(
2009
)
Mycobacterium chelonae necrotizing pneumonia after allogeneic hematopoietic stem cell transplant: report of clinical response to treatment with tigecycline
.
Transpl Infect Dis
 
11
:
57
63
.
Phares
CR
Wangroongsarb
P
Chantra
S
et al. (
2007
)
Epidemiology of severe pneumonia caused by Legionella longbeachae, Mycoplasma pneumoniae, and Chlamydia pneumoniae: 1-year, population-based surveillance for severe pneumonia in Thailand
.
Clin Infect Dis
 
45
:
e147
e155
.
Phillips
MS
Von Reyn
CF
(
2001
)
Nosocomial infections due to nontuberculous mycobacteria
.
Clin Infect Dis
 
33
:
1363
1374
.
Primm
TP
Lucero
CA
Falkinham
JO
III
(
2004
)
Health impacts of environmental mycobacteria
.
Clin Microbiol Rev
 
17
:
98
106
.
Raoult
D
Audic
S
Robert
C
Abergel
C
Renesto
P
Ogata
H
La Scola
B
Suzan
M
Claverie
JM
(
2004
)
The 1.2-megabase genome sequence of mimivirus
.
Science
 
306
:
1344
1350
.
Raoult
D
Renesto
P
Brouqui
P
(
2006
)
Laboratory infection of a technician by mimivirus
.
Ann Intern Med
 
144
:
702
703
.
Rapala
J
Niemela
M
Berg
KA
Lepisto
L
Lahti
K
(
2006
)
Removal of cyanobacteria, cyanotoxins, heterotrophic bacteria and endotoxins at an operating surface water treatment plant
.
Water Sci Technol
 
54
:
23
28
.
Research Committee of the British Thoracic Society
(
2001
)
First randomised trial of treatments for pulmonary disease caused by M. avium intracellulare, M. malmoense, and M. xenopi in HIV negative patients: rifampicin, ethambutol and isoniazid versus rifampicin and ethambutol
.
Thorax
 
56
:
167
172
.
Ritter
J
(
1880
)
Beitrag zur Frage des Pneumotyphus [eine Hausepidemie in Uster (Schweiz) betreffend]
.
Deut Arch Klin Med
 
53
96
.
Rodriguez-Zaragoza
S
(
1994
)
Ecology of free-living amoebae
.
Crit Rev Microbiol
 
20
:
225
241
.
Rogers
BH
Donowitz
GR
Walker
GK
Harding
SA
Sande
MA
(
1979
)
Opportunistic pneumonia: a clinicopathological study of five cases caused by an unidentified acid-fast bacterium
.
New Engl J Med
 
301
:
959
961
.
Roig
J
Sabria
M
Pedro-Botet
ML
(
2003
)
Legionella spp.: community acquired and nosocomial infections
.
Curr Opin Infect Dis
 
16
:
145
151
.
Rowbotham
TJ
(
1980
)
Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae
.
J Clin Pathol
 
33
:
1179
1183
.
Rowbotham
TJ
(
1983
)
Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae
.
J Clin Pathol
 
36
:
978
986
.
Rowbotham
TJ
(
1986
)
Current views on the relationships between amoebae, legionellae and man
.
Israel J Med Sci
 
22
:
678
689
.
Sabria
M
Yu
VL
(
2002
)
Hospital-acquired legionellosis: solutions for a preventable infection
.
Lancet Infect Dis
 
2
:
368
373
.
Samra
Z
Kaufman
L
Pitlik
S
Shalit
I
Bishara
J
(
2005
)
Emergence of Mycobacterium simiae in respiratory specimens
.
Scand J Infect Dis
 
37
:
838
841
.
Singh
R
Stine
OC
Smith
DL
Spitznagel
JK
Jr
Labib
ME
Williams
HN
(
2003
)
Microbial diversity of biofilms in dental unit water systems
.
Appl Environ Microb
 
69
:
3412
3420
.
Steinert
M
Birkness
K
White
E
Fields
B
Quinn
F
(
1998
)
Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls
.
Appl Environ Microb
 
64
:
2256
2261
.
Strahl
ED
Gillaspy
GE
Falkinham
JO
III
(
2001
)
Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth
.
Appl Environ Microb
 
67
:
4432
4439
.
Swinburn
CR
Gould
FK
Corris
PA
Hooper
TL
Odom
NJ
Freeman
R
McGregor
CG
(
1988
)
Opportunistic pneumonia caused by Legionella bozemanii
.
Lancet
 
1
:
472
.
Taillard
C
Greub
G
Weber
R
et al. (
2003
)
Clinical implications of Mycobacterium kansasii species heterogeneity: Swiss National Survey
.
J Clin Microbiol
 
41
:
1240
1244
.
Tang
PW
Toma
S
MacMillan
LG
(
1985
)
Legionella oakridgensis: laboratory diagnosis of a human infection
.
J Clin Microbiol
 
21
:
462
463
.
Taylor
SJ
Ahonen
LJ
De Leij
FA
Dale
JW
(
2003
)
Infection of Acanthamoeba castellanii with Mycobacterium bovis and M. bovis BCG and survival of M. bovis within the amoebae
.
Appl Environ Microb
 
69
:
4316
4319
.
Taylor
TH
Albrecht
MA
(
1995
)
Legionella bozemanii cavitary pneumonia poorly responsive to erythromycin: case report and review
.
Clin Infect Dis
 
20
:
329
334
.
Thacker
WL
Benson
RF
Staneck
JL
Vincent
SR
Mayberry
WR
Brenner
DJ
Wilkinson
HW
(
1988a
)
Legionella cincinnatiensis sp. nov. isolated from a patient with pneumonia
.
J Clin Microbiol
 
26
:
418
420
.
Thacker
WL
Wilkinson
HW
Benson
RF
Edberg
SC
Brenner
DJ
(
1988b
)
Legionella jordanis isolated from a patient with fatal pneumonia
.
J Clin Microbiol
 
26
:
1400
1401
.
Thacker
WL
Benson
RF
Schifman
RB
Pugh
E
Steigerwalt
AG
Mayberry
WR
Brenner
DJ
Wilkinson
HW
(
1989
)
Legionella tucsonensis sp. nov. isolated from a renal transplant recipient
.
J Clin Microbiol
 
27
:
1831
1834
.
Thacker
WL
Dyke
JW
Benson
RF
Havlichek
DH
Jr
Robinson-Dunn
B
Stiefel
H
Schneider
W
Moss
CW
Mayberry
WR
Brenner
DJ
(
1992
)
Legionella lansingensis sp. nov. isolated from a patient with pneumonia and underlying chronic lymphocytic leukemia
.
J Clin Microbiol
 
30
:
2398
2401
.
Thomas
E
Gupta
NK
Van Der Westhuizen
NG
Chan
E
Bernard
K
(
1992
)
Fatal Legionella maceachernii pneumonia in Canada
.
J Clin Microbiol
 
30
:
1578
1579
.
Thomas
V
McDonnell
G
(
2007
)
Relationship between mycobacteria and amoebae: ecological and epidemiological concerns
.
Lett Appl Microbiol
 
45
:
349
357
.
Thomas
V
Herrera-Rimann
K
Blanc
DS
Greub
G
(
2006
)
Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network
.
Appl Environ Microb
 
72
:
2428
2438
.
Thomas
V
Casson
N
Greub
G
(
2007
)
New Afipia and Bosea strains isolated from various water sources by amoebal co-culture
.
Syst Appl Microbiol
 
30
:
572
579
.
Thomas
V
Loret
JF
Jousset
M
Greub
G
(
2008
)
Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant
.
Environ Microbiol
 
10
:
2728
2745
.
Thomsen
VO
Andersen
AB
Miorner
H
(
2002
)
Incidence and clinical significance of non-tuberculous mycobacteria isolated from clinical specimens during a 2-y nationwide survey
.
Scand J Infect Dis
 
34
:
648
653
.
Towns
ML
Fisher
D
Moore
J
(
1994
)
Community-acquired pneumonia due to Legionella gormanii
.
Clin Infect Dis
 
18
:
265
266
.
Valero
G
Peters
J
Jorgensen
JH
Graybill
JR
(
1995
)
Clinical isolates of Mycobacterium simiae in San Antonio, Texas. An 11-yr review
.
Am J Resp Crit Care
 
152
:
1555
1557
.
Van Dam
AP
Pronk
M
Van Hoek
B
Claas
EC
(
2006
)
Successful treatment of Legionella maceachernii pneumonia after diagnosis by polymerase chain reaction and culture
.
Clin Infect Dis
 
42
:
1057
1059
.
Van Ingen
J
Boeree
MJ
De Lange
WC
Hoefsloot
W
Bendien
SA
Magis-Escurra
C
Dekhuijzen
R
Van Soolingen
D
(
2008
)
Mycobacterium xenopi clinical relevance and determinants, the Netherlands
.
Emerg Infect Dis
 
14
:
385
389
.
Vincent
A
La Scola
B
Forel
JM
Pauly
V
Raoult
D
Papazian
L
(
2009
)
Clinical significance of a positive serology for mimivirus in patients presenting a suspicion of ventilator-associated pneumonia
.
Crit Care Med
 
37
:
111
118
.
Vinh
DC
Garceau
R
Martinez
G
Wiebe
D
Burdz
T
Reimer
A
Bernard
K
(
2007
)
Legionella jordanis lower respiratory tract infection: case report and review
.
J Clin Microbiol
 
45
:
2321
2323
.
Wallace
RJ
Jr
Brown
BA
Griffith
DE
(
1998
)
Nosocomial outbreaks/pseudo-outbreaks caused by nontuberculous mycobacteria
.
Annu Rev Microbiol
 
52
:
453
490
.
Waterer
GW
Baselski
VS
Wunderink
RG
(
2001
)
Legionella and community-acquired pneumonia: a review of current diagnostic tests from a clinician's viewpoint
.
Am J Med
 
110
:
41
48
.
Wery
N
Bru-Adan
V
Minervini
C
Delgenes
JP
Garrelly
L
Godon
JJ
(
2008
)
Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility
.
Appl Environ Microb
 
74
:
3030
3037
.
Whan
L
Grant
IR
Rowe
MT
(
2006
)
Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa
.
BMC Microbiol
 
6
:
63
.
Wilkinson
HW
Thacker
WL
Brenner
DJ
Ryan
KJ
(
1985a
)
Fatal Legionella maceachernii pneumonia
.
J Clin Microbiol
 
22
:
1055
.
Wilkinson
HW
Thacker
WL
Steigerwalt
AG
Brenner
DJ
Ampel
NM
Wing
EJ
(
1985b
)
Second serogroup of Legionella hackeliae isolated from a patient with pneumonia
.
J Clin Microbiol
 
22
:
488
489
.
Woodhead
M
(
2002
)
Community-acquired pneumonia in Europe: causative pathogens and resistance patterns
.
Eur Respir J
 
36
(suppl)
:
20s
27s
.
Yamaguchi
T
Yamazaki
T
Inoue
M
et al. (
2005
)
Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test
.
FEMS Immunol Med Mic
 
43
:
21
27
.
Yu
VL
Plouffe
JF
Pastoris
MC
et al. (
2002
)
Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey
.
J Infect Dis
 
186
:
127
128
.
Editor: Colin Berry