Étude de la virulence et de la résistance aux antibiotiques des Staphylococcus aureus résistants à la méthicilline chez le porc à l'abattoir au Québec

Depuis quelques années et dans plusieurs pays, un nouveau type de Staphylococcus aureus résistant à la méthicilline (SARM), le séquence type (ST) 398, a été fréquemment retrouvé chez les porcs et chez les fermiers en contact avec ces porcs. Au Canada, très peu d’informations sont disponibles concernant le SARM d’origine porcine. Une première étude dans notre laboratoire a permis de récolter 107 isolats de SARM provenant de deux abattoirs porcins du Québec. Le présent travail vise à caractériser les gènes de virulence et de résistance aux antibiotiques de ces SARM, d’étudier leur formation de biofilm en relation avec la spécificité du groupe agr et de vérifier la localisation plasmidique et la transférabilité de ces gènes à des souches de SARM d’origine humaine. Plusieurs souches ont démontré différents patrons phénotypiques de résistance aux antibiotiques. Vingt-quatre souches représentatives de ces isolats ont été soumises à une caractérisation plus approfondie par une étude génotypique en utilisant une biopuce à ADN et un grand nombre de gènes de virulence a été détecté codant pour des entérotoxines staphylococcales, des leucocidines, des hémolysines, des auréolysines, des facteurs d’immunoévasion, des superantigènes, des facteurs d’adhésion et des facteurs impliqués dans la formation de biofilm. Des gènes de résistance envers les aminoglycosides, les macrolides, les lincosamides, les tétracyclines et les biocides ont été également détectés par biopuce et leur localisation plasmidique a par la suite été déterminée. La transférabilité de ces gènes de souches porcines à des souches de SARM d’origine humaine a été démontrée par conjugaison bactérienne; ainsi le transfert horizontal de certains gènes de résistance aux antibiotiques et de virulence a été observé. Ces travaux de recherche apportent une meilleure connaissance de la résistance aux antibiotiques et de la virulence des SARM d’origine porcine et de leur potentiel de contribution à l’émergence de certaines résistances et facteurs de virulence chez le SARM d’origine humaine.

Étude de la résistance aux antibiotiques des entérocoques d'origine animale du Québec

Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence.

Role of the mar locus in virulence of Salmonella enterica serovar Typhimurium DT104 in chickens

The virulence of a Salmonella enterica serovar Typhimurium DT014 strain in which marA was insertionally inactivated was compared to its isogenic parent in vitro and in vivo. In vitro, the numbers of the marA mutant phagocytosed by porcine lung macrophages were significantly increased, while survival at 24 h inside macrophages and adherence to human gut cells were significantly reduced in comparison with the parent strain. In vivo, the marA inactivated strain, in competition with its parent strain, persisted for a shorter period in chickens, was present in the caeca at significantly lower levels and invaded the deeper organs to a significantly lesser extent. Therapeutic antibiotic treatment of one group of chickens with oxytetracycline favoured the persistence of both the parent strain and, to a lesser extent, the marA inactivated strain; but interestingly, increased tetracycline resistance of Salmonella isolates after treatment of birds with antibiotic was seen only for the parent strain. Further work is needed to elucidate how mar is involved in virulence and if its inactivation can minimise the ability of bacteria to become antibiotic-resistant in vivo.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

Aims: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracyclineresistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. Methods and Results: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l(-1) and a 10% increase in E. coli with an MIC > 50 mg l(-1) during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 mug g(-1) faeces during treatment. Conclusion: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. Significance and Impact of the Study: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

Multidrug-resistant urinary tract isolates of Escherichia coli from Ribeirão Preto, São Paulo, Brazil

Multiple resistances to antimicrobial drugs arising in Escherichia coli isolates may complicate therapeutic management of urinary tract infection (UTI) by this organism. In order to assess the multidrug resistance (MDR) among urinary E. coli isolates, we have tested 11 antimicrobial drugs against 67 isolates from outpatients attended in a tertiary-care teaching hospital and of 78 isolates from a municipal health unit, respectively in Ribeirão Preto, State of São Paulo, Brazil. Seventy-six percent and 22% of the isolates from the tertiary-care hospital and the municipal unit, respectively, were resistant to three or more different classes of agents, and were considered to present MDR. Among the isolates from the hospital patients, 73.0%, 65.0%, 58.0%, 58.0% and 31.0% were resistant to tetracycline, ampicillin, cephalothin, trimethoprim-sulfamethoxazole (TMP/SMX) and norfloxacin, respectively; resistance from the municipal unit patients were 31.0%, 37.0%, 8.0%, 29.0% and 12.0% respectively, to the same drugs. The predominant phenotype among the MDR isolates presented is ampicillin, TMP/SMX and tetracycline resistance. The high prevalence of drug resistance among UTI patients calls for continuous surveillance to assure effective control of this infection. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis. © 2013 American Dairy Science Association.

Estudo de vigilância bacteriológica: isolamento, fatores de virulência e resistência antimicrobiana de cepas de Escherichia coli isoladas de gatos domésticos na região de Ribeirão Preto

Pós-graduação em Microbiologia Agropecuária - FCAV

Streptococcus agalactiae in Brazil: serotype distribution, virulence determinants and antimicrobial susceptibility

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Resistência antimicrobiana e prevalência de sorovares de salmonella spp. isolados de fezes e linfonodos de suinos

Pós-graduação em Medicina Veterinária - FMVZ

Conjugal Transfer of the Tn916-like Transposon TnFO1 from Enterococcus faecalis Isolated from Cheese to Other Gram-positive Bacteria

A Tn916-like transposon (TnFO1) was found in the multiple antibiotic resistant Enterococcus faecalis strain FO1 isolated from a raw milk cheese. In this strain, the tetracycline determinant was localized by DNA-DNA hybridization with a tetM nucleotide probe on the chromosome and on a 30-kb plasmid. The transposon TnFO1 was identified and characterized by DNA-DNA hybridization experiments with the five internal HincII fragments of Tn916. The tetracycline resistance determinant was identified by its complete nucleotide sequence as TetM. Transposon TnFO1 was also detected in its circular form by DNA-DNA hybridization and PCR amplification. Both ends including the joining region of the closed circular transposon TnFO1 were sequenced. TnFO1 could be transferred by conjugation from Enterococcus faecalis into Enterococcus faecalis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Listeria innocua, Leuconostoc mesenteroides and Staphylococcus aureus, and from Lactococcus lactis subsp. lactis biovar. diacetylactis into Listeria innocua. Pulsed-field electrophoresis of genomic DNA from E. faecalis FO1 transconjugants showed that transposon TnFO1 integrated at different sites.

Prevalence, genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.

New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Genomic diversity of Lactobacillus salivarius

Lactobacillus salivarius is unusual among the lactobacilli due to its multireplicon genome architecture. The circular megaplasmids harboured by L. salivarius strains encode strain-specific traits for intestinal survival and probiotic activity. L. salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. In terms of probiotic strain selection, it is important to have an understanding of the level of genomic diversity present in this species. Comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) were employed to assess the level of genomic diversity in L. salivarius. The wellcharacterised probiotic strains L. salivarius UCC118 was employed as a genetic reference strain. The group of test strains were chosen to reflect the range of habitats from which L. salivarius strains are frequently recovered, including human, animal, and environmental sources. Strains of L. salivarius were found to be genetically diverse when compared to the UCC118 genome. The most conserved strains were human GIT isolates, while the greatest level of divergence were identified in animal associated isolates. MLST produced a better separation of the test strains according to their isolation origins, than that produced by CGHbased strain clustering. The exopolysaccharide (EPS) associated genes of L. salivarius strains were found to be highly divergent. The EPS-producing phenotype was found to be carbonsource dependent and inversely related to a strain's ability to produce a biofilm. The genome of the porcine isolate L. salivarius JCM1046 was shown by sequencing to harbour four extrachromosomal replicons, a circular megaplasmid (pMP1046A), a putative chromid (pMP1046B), a linear megaplasmid (pLMP1046) and a smaller circular plasmid (pCTN1046) which contains an integrated Tn916-like element (Tn6224), which carries the tetracycline resistance gene tetM. pLMP1046 represents the first sequence of a linear plasmid in a Lactobacillus species. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable, and the identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. In summary, this thesis used a comparative genomics approach to examine the level of genotypic diversity in L. salivarius, a species which contains probiotic strains. The genome sequence of strain JCM1046 provides additional insight into the spectrum of extrachromosomal replicons present in this species.