Constança Pomba

Staphylococcus aureus CC398: Host Adaptation and Emergence of Methicillin Resistance in Livestock

ABSTRACT Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production. Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

Detection of the Pandemic O25-ST131 Human Virulent Escherichia coli CTX-M-15-Producing Clone Harboring the qnrB2 and aac(6′)-Ib-cr Genes in a Dog

The recent worldwide emergence of plasmid-mediated quinolone resistance due to qnr and aac(6′)-Ib-cr genes is a concerning fact among human and animal gram-negative pathogens (9, 14). The association of plasmid-mediated quinolone resistance with the production of extended-spectrum β-lactamases (ESBLs), including the widespread human CTX-M-15 enzyme, has been reported (8, 9). Very recently, an internationally disseminated Escherichia coli clone (O25:H4-ST131) with the CTX-M-15 ESBL was described (12). Furthermore, representative isolates of this globally diffusing clone were characterized as highly virulent and belonging to the B2 phylogenetic group (3). The aim of this study was to determine the prevalence of plasmid-mediated qnr genes among 61 consecutive nonrepetitive clinical strains of E. coli and to subsequently characterize positive isolates. These strains were isolated from dogs (n = 41) and cats (n = 20) with urinary tract infections at the Faculty of Veterinary Medicine in Lisbon, Portugal, from 2004 to 2006. Thirty-four percent and 26% of the strains were resistant to nalidixic acid and ciprofloxacin, respectively. Two were CMY-2 producers, and only one was an ESBL-producing strain. The presence of the qnrA, qnrB, and qnrS genes was investigated by dot blot DNA hybridization assays (ECL direct nucleic acid labeling and detection system 3000). The qnrA and qnrS genes were not found. The qnrB gene was detected in one (1.6%) of 61 E. coli isolates. The full qnrB2 gene nucleotide sequence was then determined. The QnrB2-positive strain, FMV5825, was isolated in Portugal from a 15-year-old female dog with chronic cystitis, after prolonged antimicrobial therapy, and was considered resistant to different antibiotics. These included ampicillin and cephalosporins (except 7-α-methoxy-cephalosporins), aztreonam, piperacillin, ticarcillin, trimethoprim-sulfamethoxazole, aminoglycosides (amikacin, gentamicin, and tobramycin), nalidixic acid, and fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) (Table ​(Table1).1). The MICs were determined by Dade MicroScan or by standard broth microdilution assays, performed and interpreted according to a Clinical and Laboratory Standards Institute guideline (5). Quinolone chromosome-encoded resistance of the FMV5825 strain was attributed to the Ser83→Ile and Asp87→Asn substitutions in GyrA and the Ser80→Ile and Glu84→Val substitutions in ParC, both previously associated with high-level fluoroquinolone resistance in E. coli (7). TABLE 1. Antimicrobial susceptibilities of the E. coli FMV5825 uropathogenic canine clinical isolate and E. coli J53 transconjugant and recipient strains The transferability of the qnrB2 gene was initially assessed by transformation. Plasmid DNA, extracted with a Qiagen plasmid mini kit, was transferred by electroporation into E. coli DH5α cells. Transformants were selected with 10 μg/ml ceftazidime. To avoid the nalidixic acid chromosome-encoded resistance of DH5α cells, further conjugation experiments were performed at 35°C using the E. coli DH5α-5825 transformant as the donor and E. coli J53 Azir as the recipient (11). Resistance to β-lactams, amikacin, tobramycin, ciprofloxacin, and norfloxacin but not to levofloxacin was cotransferred (Table ​(Table1).1). This phenotype raised the possibility of the association of the aminoglycoside acetyltransferase variant, AAC(6′)-Ib-cr, capable of acetylating ciprofloxacin and norfloxacin, as well as the presence of an ESBL enzyme. Indeed, we identified the following cotransferred genes: qnrB2, aac(6′)-Ib-cr, blaCTX-M-15, blaTEM-1B, and blaOXA-1. The insertion sequence ISEcp1 was found upstream of the blaCTX-M-15 gene. These genes and ISEcp1, except qnrB2, have also been detected in E. coli strains from humans with urinary tract infections in Portugal (10). To our knowledge, this is the first description of a canine uropathogenic E. coli CTX-M-15-producing strain containing the qnrB2 and aac(6′)-Ib-cr fluoroquinolone resistance genes. In this study, the plasmid encoding the multidrug resistance of the FMV5825 strain belonged to the narrow-host-range incompatibility group IncFII and had the FII replicon and the FIA replicon, according to a PCR-based replicon-typing scheme (2). The association of the blaCTX-M-15 gene with IncFII plasmids also containing blaTEM-1B, blaOXA-1, and aac(6′)-Ib-cr from human E. coli strains has been reported for different continents, suggesting a common origin for all of them (1, 6). A comparable multidrug resistance region was found in our animal IncFII plasmid except for the presence of qnrB2. Our E. coli strain of animal origin shares features identical to those of an intercontinental human clone (12), also belonging to the B2 phylogenetic group, O25 serogroup, and ST131 (determined by phylogenetic group multiplex PCR assay [4], O typing by the traditional antiserum method, and the MLST scheme [www.mlst.net] [15], respectively). Representative isolates of this globally diffusing clone constitute the B2 phylogenetic subgroup I, characterized by the abilities of its members to produce biofilm and to be highly virulent in mice despite lacking classical extraintestinal pathogenicity islands and their adhesion factors (3). The absence of pap, sfa, afa fimbriae, and hly operons was also noted for our E. coli strain. The presence of the iutD gene (representative of the siderophore aerobactin operon), the biofilm-producing ability (evaluated using a fluorescent in situ hybridization method [13]), and the similarity with the O25:H4-ST131 TNN (TE2) French E. coli isolate (3, 17) raise the possibility that the E. coli strain isolated from our canine patient belongs to the same phylogenetic B2 subgroup. This is the first report of the presence of the O25-ST131 human virulent E. coli CTX-M-15-producing clone harboring the disseminated incompatibility group IncFII plasmid with a multidrug resistance region containing the blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(6′)-Ib-cr, and qnrB2 genes in animals. It is possible that human-to-animal or animal-to-human transmission of the O25-ST131 clone has occurred.

Small plasmids carrying vga(A) or vga(C) genes mediate resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-resistant Staphylococcus aureus ST398 from swine

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) isolates of the sequence type ST398 have been identified recently from cases of exudative epidermitis in swine, but also as colonizers of apparently healthy swine in Portugal. A considerable number of these isolates displayed an unusual resistance phenotype, namely resistance to the lincosamide clindamycin, but susceptibility to the macrolide erythromycin. Since the genetic basis of this resistance phenotype is unknown, we selected five representative isolates to identify the respective resistance genes, to determine whether they are transferable and to investigate their genetic environment. Of the five MRSA ST398 isolates, three (E32, E33 and E49) were obtained from dust samples in three different holdings of breeding pigs during the European Community baseline study, one (E8) was from the nasal swab of an apparently healthy sow and the remaining one (E18) was from a skin lesion of a piglet suffering from exudative epidermitis. PCR screening for the presence of the lincosamide resistance gene lnu(A) yielded negative results in repeated experiments using plasmid DNA or chromosomal DNA as targets. A full antibiogram of these isolates, as conducted by broth microdilution according to CLSI document M31-A3, revealed that besides the previously reported resistance to oxacillin, clindamycin and tetracycline, all five isolates displayed high MIC values of ≥128 mg/L for the pleuromutilin tiamulin and ≥32 mg/L for the streptogramin A antibiotic virginiamycin M1. Combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics has recently been described to be associated with the resistance genes vga(A) and vga(C), both of which are located on plasmids. Plasmid profiling revealed 1–3 plasmids in each of the five MRSA ST398 isolates. The plasmids were subjected to transformation into the plasmid-free recipient strain S. aureus RN4220, with subsequent selection of the transformants on medium containing clindamycin (2 mg/L). All five isolates yielded transformants that harboured a single plasmid of either 5.3 (E49) or 5.7 kb (E8, E18, E32 and E33). Susceptibility testing confirmed that all transformants displayed resistance or high MICs (in cases where no applicable breakpoints are available to classify an isolate as resistant) to lincosamides, pleuromutilins and streptogramin A antibiotics. Restriction analysis with the enzymes PvuII, DraI, PstI and BglII showed that the 5.3 kb plasmid, designated pCPS49, differed distinctly from the 5.7 kb plasmids, which were indistinguishable based on restriction analysis with the aforementioned endonucleases, but also with HindIII, PstI, ClaI, EcoRI, XhoI and EcoRV. The plasmid obtained from the E32 transformant, designated pCPS32, was chosen as representative of this group of plasmids for further analysis. HindIII fragments of plasmids pCPS32 and pCPS49 were cloned into pBluescript II SK+, and sequenced completely on both strands. Plasmid pCPS32 was 5718 bp in size (accession no. FN806791). It showed 99.9% sequence identity to the 5713 bp plasmid pVGA obtained from a human clinical S. aureus in Portugal. Sequence analysis revealed the presence of three open reading frames (Figure 1). The first reading frame coded for a Rep protein of 312 amino acids involved in replication of plasmid pCPS32. The second reading frame encoded a Vga(A) ABC transporter protein of 522 amino acids that is responsible for combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The third reading frame coded for a Mob protein of 325 amino acids involved in plasmid mobilization. While the Rep and Vga(A) proteins of pCPS32 were indistinguishable from those of pVGA, the Mob protein showed a single amino acid exchange, Thr50 (pCPS32) versus Ala50 (pVGA). Plasmid pCPS49 was 5292 bp in size (accession no. FN806792). It also comprised three reading frames for proteins involved in plasmid replication, antimicrobial resistance and mobilization. Plasmid pCPS49 harboured a vga(C) gene encoding a 522 amino acid ABC transporter identical to that described in the revised sequence of plasmid pKKS825 (accession no. FN377602). The Vga(C) protein also conferred combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics. The pre/mob gene for plasmid recombination and

Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows

The role of Escherichia coli in the pathogenesis of the puerperal uterine infection of the cow is largely unknown. It is proposed that E. coli favors the persistence of Arcanobacterium pyogenes and gram-negative bacteria that are pivotal to the establishment of the infection. Here, we report the genomic and phenotypic characteristics of 72 E. coli isolates recovered from the uterus of dairy cows with normal puerperium (n = 12; 35 isolates) or clinical metritis (n = 18; 37 isolates), in an attempt to identify characteristics that are related to the establishment of uterine infection. We evaluated DNA fingerprints generated by repetitive element sequence-based PCR, phylogenetic grouping, the presence of 15 virulence factor genes, in vitro biofilm formation and its relationship to curli fimbriae expression, and cellulose production. We found a wide genetic diversity (40 clonal types), including types common to normal puerperium and clinical metritis cows (n = 6), as well as types specific to normal puerperium (n = 14) or clinical metritis (n = 20) cows. Isolates were assigned to phylogenetic groups B1 (58%), A (31%), and D (11%). Only 4 virulence factor genes were detected (hlyE, hlyA, iuc, and eaeA). In vitro biofilm formation was significantly affected by culture medium and incubation temperature. Curli fimbriae expression and cellulose production, although related to biofilm formation, were not required for it. None of the evaluated E. coli characteristics were significantly related to the establishment of the uterine infection. In conclusion, data presented in this paper indicate that E. coli isolates recovered from the uterus of puerperal cows present a wide genetic diversity, do not belong to a known pathogenic group, and have a low potential of virulence and persistence. This corroborates the putative role of the bacterium in the pathogenesis of the puerperal uterine infection of the cow.

Prevalence of meticillin-resistant staphylococci among dogs and cats at a veterinary teaching hospital in Portugal

THE emergence of meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-resitant Staphylococcus pseudintermedius (MRSP) in pet animals is a public health concern due to the high zoonotic potential of these multidrug-resistant bacteria (Perreten and others 2010, Ruscher and others 2010, Weese and van Duijkeren 2010). Major human epidemic clones of MRSA, mainly sequence type (ST) 22 (EMRSA-15), have been isolated from dogs and cats in various countries (Weese and van Duijkeren 2010). Although rare, human infections with MRSP of canine origin have recently been reported (Van Hoovels and others 2006, Chuang and others 2010) and high carriage rates have been shown among small animal practitioners in the USA (Morris and others 2010). As data on MRSA and MRSP carriage rates in dogs and cats are scarce, particularly in Europe, this study investigated the prevalence of MRSA and MRSP carriers among animals presenting at the teaching hospital of the Faculty of Veterinary Medicine, Technical University of Lisbon, Portugal, thus representing a sample from a tertiary referral centre population. From April 2008 to May 2010, nasal swabs were obtained from 141 cats and 146 dogs within two hours after admission to the hospital. The swabs were enriched in 3 ml broth containing 10 g/l mannitol, 65 g/l sodium chloride, 2.5 g/l yeast extract and 10 g/l tryptone. After overnight incubation, 10 μl bacterial suspension was plated on to a selective medium (ChromID MRSA; bioMerieux) and suspected MRSA/MRSP colonies were confirmed by PCR detection of mecA (Technical University of Denmark [DTU] 2008). Meticillin-resistant staphylococci were isolated …

Involvement, and dissemination, of the enterococcal small multidrug resistance transporter QacZ in resistance to quaternary ammonium compounds

OBJECTIVES To investigate the role of a putative small multidrug resistance transporter, annotated in Enterococcus faecalis V583 genome as EFA0010 (we will refer to this gene as qacZ), in decreased susceptibility to biocides. METHODS A derivative strain of V583, susceptible to erythromycin (V583ErmS), was complemented with pORI23 carrying the qacZ gene (strain EF-SAVE1). MICs of benzalkonium chloride, chlorhexidine and ethidium bromide were determined for the complemented strain and wild-type. RT-PCR and ethidium bromide efflux assays were performed in order to fully understand the role and specificity of the qacZ gene. The presence of qacZ in 73 enterococcal strains from different origins was investigated by PCR, and MICs of benzalkonium chloride and chlorhexidine were determined for the same strains. RESULTS The complemented strain, EF-SAVE1, presented a higher MIC of benzalkonium chloride (8 mg/L) than V583ErmS (4 mg/L); the MICs of chlorhexidine and ethidium bromide were the same for both strains, 4 mg/L and 16 mg/L, respectively. Expression of qacZ was found to be higher in EF-SAVE1 and constitutive, i.e. not inducible by any of the three tested biocides. Overexpression of qacZ was not responsible for changes in ethidium bromide efflux. This gene was present in 52% of the enterococcal isolates studied and the MICs of benzalkonium chloride and chlorhexidine ranged between 2 and 8 mg/L. CONCLUSIONS We demonstrate the involvement of the qacZ gene in tolerance to the quaternary ammonium compound benzalkonium chloride, but not ethidium bromide. This work constitutes the first report of a biocide resistance mechanism in E. faecalis, and reveals its dissemination amongst the genus Enterococcus.

Use of herd information for predicting Salmonella status in pig herds.

Salmonella surveillance‐and‐control programs in pigs are highly resource demanding, so alternative cost‐effective approaches are desirable. The aim of this study was to develop and evaluate a tool for predicting the Salmonella test status in pig herds based on herd information collected from 108 industrial farrow‐to‐finish pig herds in Portugal. A questionnaire including known risk factors for Salmonella was used. A factor analysis model was developed to identify relevant factors that were then tested for association with Salmonella status. Three factors were identified and labelled: general biosecurity (factor 1), herd size (factor 2) and sanitary gap implementation (factor 3). Based on the loadings in factor 1 and factor 3, herds were classified according to their biosecurity practices. In total, 59% of the herds had a good level of biosecurity (interpreted as a loading below zero in factor 1) and 37% of the farms had good biosecurity and implemented sanitary gap (loading below zero in factor 1 and loading above zero in factor 3). This implied that they, among other things, implemented preventive measures for visitors and workers entering the herd, controlled biological vectors, had hygiene procedures in place, water quality assessment, and sanitary gap in the fattening and growing sections. In total, 50 herds were tested for Salmonella. Logistic regression analysis showed that factor 1 was significantly associated with Salmonella test status (P = 0.04). Herds with poor biosecurity had a higher probability of testing Salmonella positive compared with herds with good biosecurity. This study shows the potential for using herd information to classify herds according to their Salmonella status in the absence of good testing options. The method might be used as a potentially cost‐effective tool for future development of risk‐based approaches to surveillance, targeting interventions to high‐risk herds or differentiating sampling strategies in herds with different levels of infection.

Methicillin-resistant Staphylococcus aureus CC398 isolates with indistinguishable ApaI restriction patterns in colonized and infected pigs and humans

19A, p. 34. American Society for Microbiology, Washington, DC, USA. J Antimicrob Chemother 2010 doi:10.1093/jac/dkq343 Advance Access publication 16 September 2010 High frequency of b-lactam susceptibility in CTX-M-type extended- spectrum-b-lactamase-producing Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis according to the new CLSI recommendations Katia Ruschel Pilger de Oliveira1, Ana Lucia Peixoto de Freitas2, Denise Maria Cunha Willers1, Afonso Luis Barth1,2*† and Alexandre Prehn Zavascki3† Unidade de Microbiologia e Biologia Molecular, Servico de Patologia Clinica, Hospital de Clinicas de Porto Alegre, Avenida Ramiro Barcelos 2350, 90.035-903, Porto Alegre, RS, Brazil; Departamento de Analises, Faculdade de Farmacia da Universidade Federal do Rio Grande do Sul, Avenida Ramiro Barcelos 2350, 90.035-903, Porto Alegre, RS, Brazil; Servico de Infectologia Hospital de Clinicas de Porto Alegre, Avenida Ramiro Barcelos 2350, 90.035-903, Porto Alegre, RS, Brazil *Corresponding author. Servico de Patologia Clinica, Hospital de Clinicas de Porto Alegre, Avenida Ramiro Barcelos 2350, 90.035-903, Porto Alegre, RS, Brazil. Tel/Fax: +51-21018607; E-mail: albarth@hcpa.ufrgs.br †Both senior authors, who have contributed equally.

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period

OBJECTIVES The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. METHODS Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. RESULTS The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. CONCLUSIONS The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the dissemination of highly successful human clones. Urgent measures, such as determination of clinical breakpoints and guidelines for antimicrobial use, are needed.

Public health risk of antimicrobial resistance transfer from companion animals.

Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.