Abstract

Objectives

Gonorrhoea remains a global public health problem and the treatment options are diminishing through the emergence of gonococci resistant to most antimicrobials. Previous in vitro studies have indicated a role for Neisseria gonorrhoeae pilQ alterations in conferring resistance to antimicrobials, including penicillin. In this study, we investigated whether pilQ polymorphisms were associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs) in clinical gonococcal strains.

Methods

Full-length pilQ nucleotide and PilQ amino acid sequences from geographically and temporally diverse gonococcal clinical isolates (n = 63), including the 2008 WHO reference strains, representing a range of ceftriaxone and cefixime MICs (≤0.008–0.25 and <0.016–0.5 mg/L, respectively) and 38 N. gonorrhoeae multiantigen sequence types, were examined. Previously described alterations associated with decreased ESC susceptibility (mosaic penA, mtrR and penB alterations) were also examined.

Results

Fifteen different pilQ nucleotide sequence types and nine different PilQ amino acid sequence types were observed, with two PilQ types accounting for 53 (84%) of the isolates. Independent of other genetic resistance determinants (penA mosaic, mtrR promoter deletion and penB), only one pilQ alteration, a D526N substitution, provided a statistically significant association with ceftriaxone (P < 0.01) and cefixime (P < 0.05) MICs. However, the two isolates exhibiting D526N lacked all three previously described alterations associated with decreased ESC susceptibility, thereby providing an alternative basis for the low MICs (≤0.008 mg/L) observed for these strains. The previously described E666K (pilQ2) and F595L (pilQ1) mutations were absent in all 63 isolates.

Conclusions

pilQ polymorphisms are unlikely contributors to decreased susceptibility to ESCs in clinical gonococcal strains.

Introduction

Gonorrhoea remains a global public health problem. Effective treatment options have rapidly diminished through the emergence and spread of Neisseria gonorrhoeae harbouring resistance to multiple classes of antimicrobials, including penicillins, tetracyclines, macrolides and quinolones. Extended-spectrum cephalosporins (ESCs), including the oral cefixime and the injectable ceftriaxone, are the last remaining options and, currently, the mainstay of treatment in most settings. However, there are now renewed concerns regarding the control of gonorrhoea, arising from recent reports of gonococci harbouring decreased in vitro susceptibility to all ESCs, and documented treatment failures of urogenital gonorrhoea using oral ESCs in Japan and Hong Kong. Accordingly, gonorrhoea may become untreatable under certain circumstances.1

Decreased susceptibility to ESCs in N. gonorrhoeae is due to: the presence of mosaic penA, encoding an altered penicillin-binding protein (PBP) 2; alterations in the mtrR promoter, causing an overexpression of the MtrC-MtrD-MtrE efflux pump; and penB, encoding altered forms of the porin PorB1b, particularly G120K and A121D.2 However, it has become clear that the decreased susceptibility to ESCs in N. gonorrhoeae is more complex. For example, an A501V alteration in PBP2, together with mtrR promoter deletion and penB mutations, may significantly contribute to decreased ESC susceptibility in non-mosaic penA gonococci.3,4

Using transformation experiments, Zhao et al.5 showed that the gonococcal PilQ oligomer forms a pore in the outer membrane through which antimicrobials may diffuse into the periplasm, and that an E666K alteration (pilQ2) in PilQ, when present in combination with penA, mtrR and penB alterations, leads to increased resistance to penicillin and tetracycline. A previous study showed that an F595L mutation (pilQ1) in PilQ increased the susceptibility to several antimicrobials, including penicillin, providing additional evidence for the involvement of gonococcal PilQ as a route of antimicrobial entry.6 In this study, we examined full-length pilQ nucleotide and PilQ amino acid sequences from temporally, geographically and genetically diverse N. gonorrhoeae clinical isolates representing a wide range of ceftriaxone and cefixime MICs.

Materials and methods

N. gonorrhoeae strains

A total of 63 gonococci were retrospectively selected on the basis of ceftriaxone MIC, and temporal and geographical diversity. Ceftriaxone MICs and cefixime MICs were in the range ≤0.008–0.25 and <0.016–0.5 mg/L (Table 1), respectively, as determined by using agar dilution (ceftriaxone) as previously described7 and Etest (cefixime) according to the manufacturer's instructions (AB bioMérieux, Solna, Sweden). The clinical isolates examined included 13 isolates from Australia, 7 from Asia (China, 1; Hong Kong, 1; Japan, 1; Korea, 1; Mongolia, 1; Thailand, 1; and the Philippines, 1), 25 from Sweden, 4 from the UK and 5 from the USA. The years of isolation were 1988 (n = 1), 1999 (n = 1), 2000 (n = 2), 2001 (n = 1), 2002 (n = 6), 2003 (n = 9), 2004 (n = 7), 2005 (n = 11), 2007 (n = 1), 2008 (n = 14) and 2009 (n = 1). A further nine gonococci (WHO C, F, G, K, L, M, N, O and P) used as WHO reference strains8 were included in the study. The isolates represented 38 different N. gonorrhoeae multiantigen sequence types (NG-MASTs).

Table 1.

The PilQ amino acid sequence types, mtrR promoter adenine deletion, penB and mosaic penA in 63 temporally, geographically and genetically diverse gonococci representing a wide range of ceftriaxone and cefixime MICs

Alleles
 
Ceftriaxone (cefixime) MIC (mg/L) for isolates
 
pilQ mtrRa penBb mosaic penA ≤0.008 0.016 0.030 0.060 0.125 0.25 0.5 
WTc WTc — 2 (2) — — — — — — 
II A-del KD — — — — — 1 (1) — — 
III A-del DA — — (1) (1) — — 
III WTc DA — — (1) — — — — 
IV WTc KD — — — — — (1) — 
WTc WTc — 1 (1) — — — — — — 
VI A-del KD — 1 (1) — (1) — — — 
VI A-del KD yes — — — (5) (2) 
VII A-del DG — 1 (1) — — — — — — 
VII A-del GS — — (1) (1) — — — 
VII A-del KD — (1) 1 (1) 3 (1) 5 (8) 8 (5) (1) — 
VII A-del KD yes — — — — (2) (3) 
VII A-del KG yes — — — — (1) — 
VII A-del KN yes — — — — 6 (5) 1 (2) — 
VII A to C GD — 1 (1) — — — — — — 
VII WTc DA — — — — 1 (2) — — 
VII WTc WTc — — (1) — 2 (1) — —  
VII WTc KD — — — — 2 (2) 1 (1) — — 
VII WTc NA — 1 (1) — — — — — — 
VII WTc PIAd — 1 (1) — — 1 (1) — — — 
VIII A-del DA — 1 (1) — — — — —  
IX WTc GG — 1 (1) — — — — —  
Total    13 (11) 2 (2) 4 (4) 12 (17) 31 (12) 1 (12) (5) 
Alleles
 
Ceftriaxone (cefixime) MIC (mg/L) for isolates
 
pilQ mtrRa penBb mosaic penA ≤0.008 0.016 0.030 0.060 0.125 0.25 0.5 
WTc WTc — 2 (2) — — — — — — 
II A-del KD — — — — — 1 (1) — — 
III A-del DA — — (1) (1) — — 
III WTc DA — — (1) — — — — 
IV WTc KD — — — — — (1) — 
WTc WTc — 1 (1) — — — — — — 
VI A-del KD — 1 (1) — (1) — — — 
VI A-del KD yes — — — (5) (2) 
VII A-del DG — 1 (1) — — — — — — 
VII A-del GS — — (1) (1) — — — 
VII A-del KD — (1) 1 (1) 3 (1) 5 (8) 8 (5) (1) — 
VII A-del KD yes — — — — (2) (3) 
VII A-del KG yes — — — — (1) — 
VII A-del KN yes — — — — 6 (5) 1 (2) — 
VII A to C GD — 1 (1) — — — — — — 
VII WTc DA — — — — 1 (2) — — 
VII WTc WTc — — (1) — 2 (1) — —  
VII WTc KD — — — — 2 (2) 1 (1) — — 
VII WTc NA — 1 (1) — — — — — — 
VII WTc PIAd — 1 (1) — — 1 (1) — — — 
VIII A-del DA — 1 (1) — — — — —  
IX WTc GG — 1 (1) — — — — —  
Total    13 (11) 2 (2) 4 (4) 12 (17) 31 (12) 1 (12) (5) 

aAlterations in the inverted repeat in the mtrR promoter region causing an overexpression of the MtrC-MtrD-MtrE efflux pump. The most common alteration is a deletion of a single nucleotide (‘A-del’).

bAlterations of the outer membrane porin PorB1b resulting in decreased intake of antimicrobials. The most frequent penB alterations are G120K and A121D. K, lysine; D, aspartic acid; A, alanine; G, glycine; S, serine; N, asparagine.

cWT, wild-type.

dPossessed a PIA (PorB1a) porin.

PCR and DNA sequencing

DNA sequencing was performed at the Queensland Paediatric Infectious Diseases Laboratory, Brisbane, Australia (QPID-Australia) and Örebro University Hospital, Örebro, Sweden (OUH-Sweden). At QPID-Australia, pilQ sequences were amplified by PCR, as previously described,6 and then sent for sequencing at the Australian Genome Research Facility (http://www.agrf.org.au/). NG-MASTs and penA, mtrR and penB sequence data were available for the Australian and Asian isolates and eight out of the nine WHO reference strains (n = 28) as part of previous studies.3,8,9 At OUH-Sweden (examining 34 clinical isolates and WHO C), pilQ was PCR amplified using previously described primers.6,10 NG-MAST determination and penA, mtrR and penB sequencing were performed as previously described.8

Sequence analysis

Alignments of the pilQ nucleotide sequences and PilQ amino acid sequences were performed with BioEdit (version 5.0.9) software and manual adjustment using N. gonorrhoeae strain FA1090 (www.genome.ou.edu/gono.html; GenBank accession number AE004969) as the reference sequence. The assignment of PilQ amino acid sequence types was based on similarities to PilQ in N. gonorrhoeae FA1090.

Statistical analysis

Data analysis was performed using the Mann–Whitney U-test. Significance was set at a P value of <0.05.

Results

A total of 15 different pilQ nucleotide sequence types and nine different PilQ amino acid sequences (PilQ types I–IX; Figure 1) were observed (Table 1). In the alignment of the pilQ nucleotide sequences (2196 unambiguously aligned nucleotides), 62 polymorphic nucleotide sites (a ratio of 2.8 per 100 sites) were identified compared with the pilQ sequence of FA1090, comprising a 24 nucleotide deletion, a 24 nucleotide insertion and 14 single nucleotide substitutions. PilQ alterations included a combination of a 147AKQQAAAP deletion (in PilQ type IX), an 180QAATPAKQ insertion (all PilQ types, but lacking in FA1090) and seven substitutions [Q172E (PilQ type II), S341N (PilQ types V–IX), N432S (PilQ type IV), D494N (PilQ type V), G500S (PilQ type VIII), D526N (PilQ type I) and N648S (PilQ types III–VI)] (Figure 1). The most common PilQ type was VII (n = 44 isolates), followed by type VI (n = 9) (Table 1). All other PilQ types were represented by one to three isolates only (Table 1). By geographical origin, PilQ types V (n = 1), VI (n = 2), VII (n = 9) and VIII (n = 1) were observed in Australian gonococci, and types I (n = 2), VI (n = 6) and VII (n = 17) were observed in the Swedish isolates. The seven Asian isolates comprised types II (China), III (Hong Kong and Thailand) and VII (Japan, Korea, Mongolia and the Philippines). The four isolates from the UK and the five isolates from the USA were all of type VII. The WHO reference strains were represented by types III (WHO C), IV (WHO L), VI (WHO K), VII (WHO G, M, N, O and P) and IX (WHO F).

Figure 1.

Full-length PilQ amino acid sequences from 63 temporally, geographically and genetically diverse N. gonorrhoeae isolates. The sequences are classified into different amino acid types (I–IX) and are aligned with the N. gonorrhoeae FA1090 sequence (GenBank accession number AE004969). The numbers of isolates of each amino acid type are indicated in parentheses.

Figure 1.

Full-length PilQ amino acid sequences from 63 temporally, geographically and genetically diverse N. gonorrhoeae isolates. The sequences are classified into different amino acid types (I–IX) and are aligned with the N. gonorrhoeae FA1090 sequence (GenBank accession number AE004969). The numbers of isolates of each amino acid type are indicated in parentheses.

The previously described E666K (pilQ2) and F595L (pilQ1) mutations were not observed in these isolates. Independent of other genetic resistance determinants (penA mosaic, mtrR promoter deletion and penB), a statistically significant association with ceftriaxone and cefixime MICs was observed for the D526N substitution only (P < 0.01 and P < 0.05, respectively). However, the two isolates exhibiting the D526N alteration of PilQ (PilQ type I; Table 1 and Figure 1) lacked all the three previously described alterations associated with reduced ESC susceptibility, and displayed low ceftriaxone and cefixime MICs (≤0.008 and <0.016 mg/L, respectively) (Table 1). Consistent with previous studies, significant associations with ceftriaxone and cefixime MICs were observed for mosaic penA (P < 0.001), the adenine deletion in the mtrR promoter (P < 0.01 and P < 0.001, respectively) and the most common penB alterations, i.e. G120K and A121D (P < 0.01) (Table 1).

Discussion

The results of the present study showed that there were only eight different PilQ amino acid alterations (resulting in nine divergent sequence types) in 63 temporally and geographically diverse gonococci representing a wide range of ceftriaxone and cefixime MICs, with two PilQ types (VI and VII) accounting for the majority (84%) of isolates. Accordingly, the gonococcal pore-forming secretin PilQ is highly conserved for an outer membrane protein implicated in antimicrobial resistance, particularly when compared with the highly variable PorB porin. The only PilQ alteration that was significantly associated with ceftriaxone and cefixime MICs was D526N; however, both isolates harbouring this particular mutation lacked mosaic penA, as well as mtrR and penB alterations (Table 1; isolates with PilQ type I). Thus, the low MICs observed for these two isolates could be explained by the absence of these key alterations. The previously described E666K (pilQ25) and F595L (pilQ16) alterations were absent in all isolates investigated in this study.

It is becoming increasingly clear that emerging chromosomally mediated resistance to ESCs, similar to penicillin, in N. gonorrhoeae is exceedingly complex and multifaceted, and involves multiple alterations in various genes and that these act synergistically.2–4 In this respect, PilQ E666K (pilQ2) was previously shown to have little to no impact in the absence of penA, mtrR promoter and penB alterations.5 It is therefore conceivable that the lack of clear association between pilQ and ceftriaxone and cefixime susceptibility observed in our study arose from use of univariate analysis and that larger studies using multivariate analysis may be required to fully investigate the relative contributions of each mutation. However, based on our data there are no clear candidate pilQ alterations that may be implicated in decreased susceptibility to ESCs in clinical gonococcal isolates. Furthermore, the previously described pilQ2 polymorphism is also unlikely to contribute to clinical resistance, because this mutation interferes with the formation of the PilQ secretin complex and disrupts proper piliation. Accordingly, this PilQ mutant is not functional for pilus assembly and the type IV pili are essential in gonococcal mucosal pathogenesis, as they are involved in the adherence to the epithelium cells of the host.10,11

In conclusion, the present study showed that the N. gonorrhoeae pilQ nucleotide sequence and PilQ amino acid sequence are highly conserved in temporally, geographically and genetically diverse gonococci. Furthermore, alterations of pilQ are unlikely contributors to decreased susceptibility to ESCs, such as ceftriaxone and cefixime, in clinical gonococcal strains.

Funding

This study was conducted as part of the reference work of the Australian National Neisseria Network, and was supported by core funding of the Queensland Paediatric Infectious Diseases Laboratory provided by the Royal Children's Hospital Foundation, Australia, as well as by grants from the Örebro County Council Research Committee, Sweden.

Transparency declarations

None to declare.

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