Leakage of emerging clinically relevant multidrug-resistant Salmonella clones from pig farms

Abstract

Objectives

To assess the presence of multidrug-resistant (MDR) Salmonella with human clinical relevance in pig farms from different regions of Portugal and to analyse their mobile genetic elements associated with antibiotic resistance.

Methods

Seventy-nine samples were collected from six piggeries and analysed for the presence of Salmonella. All isolates were examined for susceptibility to antimicrobial agents and representative isolates for resistance genes and class 1 integrons (PCR/restriction fragment length polymorphism). Clonality was determined by PFGE and multilocus sequence typing (MLST). Plasmid analysis included determination of size, content and characterization of the incompatibility group (rep-PCR and I-CeuI/S1-hybridization).

Results

Sixty Salmonella isolates were recovered from five samples (two manure, two waste lagoons and one animal feed) in half of the piggeries studied. All isolates were resistant to at least one antibiotic (tetracycline) and 97% to at least four antibiotics from different families. In 10 isolates representing different serogroup and resistance phenotype combinations a diversity of resistance genes and integrons was detected. These isolates belonged to the internationally widespread Salmonella Rissen (ST469) and Salmonella Typhimurium DT104 (ST19) clones, as well as to the emerging Salmonella Typhimurium monophasic variant with examples of Spanish (carrying a sul3-atypical integron within IncA/C plasmids, here assigned to ST19) and European (ASSuT phenotype, assigned to ST34) clones.

Conclusions

This is one of the few studies reporting emerging MDR Salmonella clones and the first one detecting Salmonella Typhimurium monophasic variant in the pig production setting. The survival of these strains in manure and waste lagoons is of concern, since these environments might allow spread of MDR bacteria beyond pig farms’ boundaries.

antimicrobial agents, piggeries, PFGE clones, MLST

Introduction

In recent years, changing trends in salmonellosis and associated serotypes have been observed, with a marked increase in certain multidrug-resistant (MDR) clones of Salmonella Typhimurium and its monophasic variant in different countries.^1,2 Among these, Salmonella Typhimurium DT104, OXA-30-producing Salmonella Typhimurium clone and Salmonella Typhimurium monophasic variant (4,[5],12:i:-) are the most frequently reported in Europe.^2,3 The latter, which have recently emerged worldwide, range from pan-susceptible (USA, Brazil) to MDR (Europe) strains and seem to be largely distributed in animal hosts and their derived products (e.g. pork, poultry products, cattle).² Although there is evidence that the animal setting seems to be a reservoir of MDR strains, data concerning the spread of emerging Salmonella MDR clones with features of clinical interest within and off pig farms are still missing. Here, we assessed the presence of MDR Salmonella with human clinical relevance on pig farms from different regions of Portugal and analysed their mobile genetic elements associated with antibiotic resistance.

Materials and methods

Sample processing and Salmonella identification

Seventy-nine samples were collected within six geographically separated Portuguese piggeries (five with intensive and one with extensive production) during 2006 and 2007. They included samples from pigs (n = 21; faeces, nostril/surface swabs), feed/medicines (n = 22; feed, water, medicine, antiseptics), residues (n = 17; swine waste lagoons, residual waters, manure, septic tank) and piggery facilities (n = 19; water, walls/floors dust, soil). The presence of Salmonella was screened by the conventional method following ISO 6579, which includes two stages of enrichment and plating out in two selective solid media (four plates per sample).⁴ Suspected colonies (up to five from each of the four plates) were identified by slide agglutination (Salmonella O poly antisera and serogroup-specific antisera for serogroups B, C1 and D; BD, USA), biochemical tests (API 32 GN; bioMérieux, Marcy l'Étoile, France) and a PCR assay (targeting the invA gene and a DT104/U302 phage type-specific DNA sequence).⁵ The serotypes of representative isolates were determined at the National Centre of Salmonella. The Salmonella Typhimurium monophasic variant (4,[5],12:i:-) isolates were confirmed using PCR as previously described.²

Antimicrobial susceptibility testing

All Salmonella isolates were tested for susceptibility to 10 antimicrobial agents (μg) [amoxicillin (10), gentamicin (10), kanamycin (30), streptomycin (10), ciprofloxacin (5), nalidixic acid (30), chloramphenicol (30), tetracycline (30), sulfamethoxazole (300) and trimethoprim (5)] by the disc diffusion method following CLSI standards.⁶Escherichia coli ATCC 25922 was used as the control strain. Amoxicillin-resistant isolates were further tested for susceptibility to several extended-spectrum β-lactams (ceftazidime, ceftriaxone, cefotaxime, cefepime, cefoxitin, aztreonam and imipenem) and the double disc synergy test (DDST) for ESBL detection was also conducted.⁶ From each sample, the selection of representative isolates for further studies was based on serogroup, presence of the invA gene and/or phage type DT104/U302 phage type-specific DNA sequence and antibiotic resistance phenotype.

Characterization of antimicrobial resistance genes, integrons and plasmids

Genes coding for resistance to sulfamethoxazole (sul1, sul2 and sul3), tetracycline [tet(A), tet(B) and tet(G)], chloramphenicol (floR, cmlA and catA), amoxicillin (bla_TEM, bla_PSE-1 and bla_OXA-30), gentamicin [aac(3)-IV], streptomycin (aadA and strA-strB) and trimethoprim (dfrA1 and dfrA12) were searched for by PCR using primers and conditions previously described.^7,8 The detection and characterization of class 1 integrons was performed by PCR and restriction fragment length polymorphism (RFLP) analysis with TaqI as previously reported.^7,8 Positive and negative controls were included in all PCRs. Plasmid content and the genetic localization of integrons and sul genes were investigated by S1 nuclease (Takara Bio Inc., Shiga, Japan) and I-CeuI (New England BioLabs, Ipswich, MA, USA) digestion of total genomic DNA followed by PFGE. Identification of plasmid incompatibility groups was determined by a rep-PCR typing method³ including three additional PCR assays for the IncU, IncR and ColE groups.⁹ Southern blot hybridization was performed by standard methods using intI1, sul1, sul2, sul3 and rep intragenic probes, following the manufacturer's instructions (Gene Images Alkphos Direct Labelling System Kit; Amersham GB/GE Healthcare Life Sciences UK Limited).

PFGE and MLST analysis

Clonal relatedness among isolates was assessed by PFGE following XbaI digestion of genomic DNA according to the standard 1 day protocol of the CDC.⁷Salmonella enterica serotype Braenderup H9812 (CDC) was used as a molecular size marker. Multilocus sequence typing (MLST) analysis was performed using specific primers to amplify a set of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA and thrA) and sequence type (ST) was assigned according to the MLST database (http://mlst.ucc.ie/mlst).

Results and discussion

Sixty Salmonella isolates were recovered from five positive samples (two manure, two waste lagoons and one animal feed) collected on three pig farms with intensive production (one located in the north and two located in the south of Portugal). Forty-seven isolates were serogroup B, of which 22 were positive for the DT104/U302 phage type-specific region, and 13 were serogroup C1. All isolates were resistant to at least one antibiotic (tetracycline) and 58 out of 60 were resistant to at least four antibiotics from different families (ranging from four to eight antibiotics). Resistances to tetracycline (n = 60 isolates), streptomycin (n = 58), sulfamethoxazole (n = 57) and amoxicillin (n = 56) were the most frequently detected and might reflect the high usage of these antibiotics in food-producing animals.⁷ Lesser rates of resistance were observed for the other antimicrobial agents tested; chloramphenicol (n = 22), trimethoprim (n = 19), nalidixic acid (n = 10), gentamicin (n = 8), ciprofloxacin (n = 0) and kanamycin (n = 0). Susceptibility to extended-spectrum β-lactams and absence of extended-spectrum β-lactamases (ESBLs) and AmpC were observed. Because enrichment steps allowed selection of more than one isolate of the same strain from the same sample, 10 isolates representing different combinations of serogroup and antibiotic resistance phenotype were selected for further studies (Table 1). The selected isolates belonged to three Salmonella serotypes (four Rissen, two Typhimurium and four from its monophasic variant 4,[5],12:i:-) and four PFGE clones spread among different farms and samples (Figure 1 and Table 1). Among them a diversity of antibiotic resistance genes were detected. These strains harboured common MDR genetic elements (e.g. integrons) and shared clonal relationships with previously nationally widespread/emerging clones in human and food products,^7,8 showing their potential transmission along the food chain and their ability to cause human infections (Figure 1).

Table 1.

Characterization of the Salmonella clones from Portuguese piggeries

Serotype (phage type)/PFGE typea	No. of isolates	MLST – ST (no. of isolates)b	Date of isolation	Piggery	Sample	Resistance phenotypec/resistance genes profile (no. of isolates; sample)	Class 1 integron genes (bp)	Class 1 integron and/or sul2 location Chr or PL (kb, Inc)d
Rissen/N	4	ST469 (n = 2)	2006	B, C	manure B1;	AMX, STR, SUL, TET, TMP	intI1, sul1	PL (35, R)
					manure C7	blaTEM, aadA2, sul1, tet(A), dfrA12	dfrA12, orfF, aadA2 (2000)
						(n = 2; B1 and C7)
						AMX, STR, TET, TMP	intI1	PL (30, R)
						blaTEM, tet(A)
						(n = 1; B1)
						TET	—	—
						tet(A)
						(n = 1; C7)
Typhimurium (DT104/U302)/A	2	ST19 (n = 1)	2007	E	waste lagoon E32; manure E34	AMX, CHL, STR, SUL, TET	intI1, sul1	Chr
						blaPSE-1, floR, aadA2, sul1, tet(G)	aadA2 (1000)
						(n = 2; E32 and E34)	blaPSE-1 (1200)
Typhimurium (DT104/U302) 4,[5],12:i:-/O	2	ST19 (n = 1)	2006	C	manure C7	AMX, CHL, GEN, NAL, STR, SUL, TET, TMP	intI1, sul1dfrA12, orfF,	PL (170, A/C)
						blaTEM, cmlA1, aac(3)-IV, aadA2, sul1-sul2-sul3, tet(A), dfrA12	aadA2 (2000)type III-sul3e
						(n = 1; C7)
						CHL, NAL, STR, TET, SUL	intI1, sul1	PL (130, A/C)
						cmlA1, aadA, tet(A), sul1-sul2-sul3	type III-sul3e
						(n = 1; C7)
Typhimurium 4,[5],12:i:-/Y	2	ST34 (n = 2)	2007	E	feed E2;	AMX, STR, SUL, TET	—	Chr (sul2)
					manure E34	blaTEM, strA-strB, sul2, tet(B)
						(n = 2; E2 and E34)

^aClones are designated by capital letters, as previously published.^7,8

^bNumber of isolates submitted to MLST database.

^cAMX, amoxicillin; CHL, chloramphenicol; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfamethoxazole; TET, tetracycline; TMP, trimethoprim.

^dChromosomal (Chr) and/or plasmid (PL) location of integrons and the sul2 gene was assessed by hybridization of I-CeuI/S1-digested genomic DNA using int1, sul1, sul2, sul3 and rep probes.

^eStructure of the type III sul3 integron: 5′CS-estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3.⁸

Figure 1.

PFGE patterns of Salmonella isolates from piggeries and others previously characterized.^7,8 Lanes: M, Salmonella Braenderup H9812 (CDC); 1, clone O, human isolate (Salmonella Typhimurium 4,[5],12:i:-, 459/02); 2, clone O, pork isolate (Salmonella Typhimurium 4,[5],12:i:-, A30); 3, C7.5 isolate (Salmonella Typhimurium 4,[5],12:i:-, piggery C; clone O); 4, C7.17 isolate (Salmonella Typhimurium 4,[5],12:i:-, piggery C; clone O); 5, clone O, pork isolate (Salmonella Typhimurium 4,[5],12:i:-, B296); 6, C7.7 isolate (Salmonella Rissen, piggery C; clone N); 7, B1.1 isolate (Salmonella Rissen, piggery B; clone N); 8, clone N, pork isolate (Salmonella Rissen, A27); 9, C7.16 isolate (Salmonella Rissen, piggery C; clone N); 10, B1.3 isolate (Salmonella Rissen, piggery B; clone N); 11, E32.2 isolate (Salmonella Typhimurium DT104, piggery E; clone A); 12, E2.5 isolate (Salmonella Typhimurium 4,[5],12:i:-, piggery E; clone Y); 13, E34.1 isolate (Salmonella Typhimurium 4,[5],12:i:-, piggery E; clone Y); and 14, E34.11 isolate (Salmonella Typhimurium DT104, piggery E; clone A).

More than one resistant genotype or clone was detected within the same piggery, suggesting enrichment of the local metagenome with a broad range of drug-resistant strains and genetic elements, such as integrons. Isolates belonging to the emerging Salmonella Rissen clone (n = 4), detected in the manure of two geographically separated piggeries, were assigned to ST469, only previously reported in the few European isolates of the same serotype allocated in the MLST database (http://mlst.ucc.ie/mlst). The human acquisition of Salmonella Rissen was previously associated with tourism or international trade outside the EU,¹⁰ but the data of this and a few other studies suggest a more local source of infection with this emerging serotype.⁷ Different resistance patterns and the presence of a specific class 1 integron (dfrA12-orfF-aadA2), located in IncR plasmids (35 kb), were observed, which may provide a selective advantage for this emergent serotype in the animal niche. Two isolates from the widely disseminated clone of Salmonella Typhimurium, DT104, showing the chromosomal located R-type ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline), were detected in environmental samples from one piggery (manure and waste lagoon), suggesting that, beside the classical dissemination routes (animals and foods of animal origin), environmental reservoirs might also play a role in the spread of such strains. Of particular interest was the detection of two MDR clones of the emerging Salmonella Typhimurium monophasic variant, related to the so-called European and Spanish clones.^11,12 The pattern ASSuT was observed in the isolates from one piggery associated with the chromosomal resistance genes [bla_TEM-1, strA-strB, sul2 and tet(B)] typical of the European monophasic strains,¹² here demonstrated to belong to ST34 (a single-locus variant of ST19). The other monophasic variant MDR clone, belonging to the worldwide-distributed ST19 and DT104/U302 phage type, presented an IncA/C plasmid (130–170 kb) with the recently characterized atypical sul3-carrying integron type III.⁸ Interestingly, isolates of this monophasic variant clone carrying similar MDR features have been recovered for more than 10 years in Spain¹¹ and from humans and food products in Portugal, and were initially identified as Salmonella Typhimurium.⁸ Successful adaptation of this pathogen and the intense commercial trade in live pigs and pig meat products between these countries might explain the maintenance and dissemination of the monophasic MDR so-called Spanish clone in the Iberian Peninsula.

This study indicates that clinically relevant MDR clones are prevalent and disseminated in piggeries. The success of these bacterial strains might be related to antimicrobial resistance features, but also to other properties, such as virulence factors, as recently described for the IncA/C plasmids in the Spanish clone.¹¹ Also, the detection of these widely frequent MDR genetic lineages of Salmonella in manure and swine waste lagoons raises concerns due to potential water, air and soil contamination, as well as of wild animals, indicating potentially high risks of transmission of the pathogen in the environment, animals and humans.^13–15 The frequent use of manure as a fertilizer and the long-term survival potential of this zoonotic pathogen seem to contribute to the increasing reports of salmonellosis outbreaks caused by consumption of fresh products (e.g. ready-to-eat salads).^13,14 This alerts us to the ability of the emergent clones, such as subtypes of Salmonella Typhimurium monophasic variant to be transmitted to humans by sources other than pigs and pork, until now considered sporadic.

In conclusion, this is one of the few studies reporting emerging MDR Salmonella clones carrying features with clinical interest and the first one identifying Salmonella Typhimurium monophasic variant in pig farms. The presence of isolates resistant to several antibiotic families and belonging to clonal lineages increasingly associated with human infections, particularly the emergence of Salmonella Typhimurium monophasic variants in piggeries, is a public health threat.

Funding

This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT), which belongs to the Ministry of Science, Technology and Innovation from Portugal (POCI/AMB/61814/2004).

Transparency declarations

None to declare.

Acknowledgements

We are deeply grateful to Carla Novais for critical review of this paper prior to submission. We are also grateful to Alessandra Carattoli for kindly providing the positive controls for the IncU, IncR and ColE plasmids, to Centro Nacional de Salmonella (Lisboa, Portugal) for serotyping the strains and CDC for the PFGE protocols and the control strain Salmonella Braenderup H9812.