Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids.

BACKGROUND: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. METHODS: In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. FINDINGS: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. INTERPRETATION: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. FUNDING: This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

Characterization of Proteus mirabilis and associated plasmids isolated from anaerobic dairy cattle manure digesters.

Proteus mirabilis is an opportunistic pathogen associated with a variety of human infections, including urinary tract infections. The prevalence of P. mirabilis in foods of animal origin and in the manure by-products created in animal production is not well documented. Further, the prevalence and persistence of extended-spectrum cephalosporin (ESC) resistant P. mirabilis is largely unknown. In this study, we characterized ESC-resistant P. mirabilis recovered from various stages of dairy manure anaerobic digestion. Isolates were screened by PCR for blaCTX-M, blaCMY and blaSHV, and antimicrobial susceptibility testing was performed. Fifty-six P. mirabilis carrying CTX-M were sequenced with short and long read sequencing technologies, and the assembled chromosomes and plasmids were compared. ESC-resistant Proteus was found in four of the six manure digesters, an indication that not all digesters were colonized with resistant strains. Both CTX-M-1 and CTX-M-15 plasmids were found in P. mirabilis isolates. Transfer of plasmid DNA by conjugation was also explored, with ESC-resistance plasmids able to transfer to Escherichia coli at high frequency. We concluded that P. mirabilis can harbour and transfer ESC-resistance genes and plasmids, and may be an overlooked reservoir of antimicrobial resistance.

Characterization of Escherichia coli and Other Enterobacterales Resistant to Extended-Spectrum Cephalosporins Isolated from Dairy Manure in Ontario, Canada.

Extended-spectrum cephalosporins (ESCs) resistance genes, such as blaCTX-M, blaCMY, and blaSHV, have been found regularly in bacteria from livestock. However, information on their distribution in dairy cattle in Canada and on the associated genome sequences of ESC-resistant Enterobacterales is sparse. In this study, the diversity and distribution of ESC-resistant Escherichia coli throughout manure treatments in six farms in Southern Ontario were assessed over a one-year period, and their ESC-resistance plasmids were characterized. The manure samples were enriched using selective media. The resulting isolates were screened via polymerase chain reaction for blaCTX-M, blaCMY, and blaSHV. No E. coli carrying blaSHV were detected. Escherichia coli (n = 248) carrying blaCTX-M or blaCMY underwent whole-genome sequencing using an Illumina MiSeq/NextSeq. These isolates were typed using multilocus sequence typing (MLST) and their resistance gene profiles. A subset of E. coli (n = 28) were sequenced using Oxford Nanopore Technologies. Plasmids were assembled using Unicycler and characterized via the resistance genes pattern, replicon type, plasmid MLST, phylogenetic analysis, and Mauve alignments. The recovery of ESC-resistant Enterobacterales (18 species, 8 genera) was drastically reduced in manure outputs. However, multiple treatment stages were needed to attain a significant reduction. 62 sequence types were identified, with ST10, ST46, ST58, ST155, ST190, ST398, ST685, and ST8761 being detected throughout the treatment pipeline. These STs overlapped with those found on multiple farms. The ESC-resistance determinants included CTX-M-1, -14, -15, -17, -24, -32, -55, and CMY-2. The plasmids carrying blaCTX-M were more diverse than were the plasmids carrying blaCMY. Known "epidemic plasmids" were detected for both blaCTX-M and blaCMY. IMPORTANCE The increase in antimicrobial resistance is of concern for human and animal health, especially when resistance is conferred to extended-spectrum cephalosporins, which are used to treat serious infections in both human and veterinary medicine. Bacteria carrying extended-spectrum cephalosporin resistance genes, including blaCTX-M and blaCMY, are frequently found in dairy manure. Manure treatment influences the loads and diversity of bacteria, including those carrying antimicrobial resistance genes, such as Enterobacterales and Escherichia coli. Any bacteria that survive the treatment process are subsequently applied to the environment. Enterobacterales carrying blaCTX-M or blaCMY can contaminate soil and crops consumed by humans and animals, thereby increasing the potential for antimicrobial resistance genes to integrate into the human gut microflora through horizontal gene transfer. This furthers the dissemination of resistance. Therefore, it is imperative to understand the effects manure treatments have on ESC-resistance in environmentally applied manure.

Dynamics of extended-spectrum cephalosporin resistance genes in Escherichia coli from Europe and North America.

Extended-spectrum cephalosporins (ESCs) are critically important antimicrobial agents for human and veterinary medicine. ESC resistance (ESC-R) genes have spread worldwide through plasmids and clonal expansion, yet the distribution and dynamics of ESC-R genes in different ecological compartments are poorly understood. Here we use whole genome sequence data of Enterobacterales isolates of human and animal origin from Europe and North America and identify contrasting temporal dynamics. AmpC ß-lactamases were initially more dominant in North America in humans and farm animals, only later emerging in Europe. In contrast, specific extended-spectrum ß-lactamases (ESBLs) were initially common in animals from Europe and later emerged in North America. This study identifies differences in the relative importance of plasmids and clonal expansion across different compartments for the spread of different ESC-R genes. Understanding the mechanisms of transmission will be critical in the design of interventions to reduce the spread of antimicrobial resistance.

Diversity of blaCTX-M-1-carrying plasmids recovered from Escherichia coli isolated from Canadian domestic animals.

Conserved IncI1 and IncHI1 plasmids carrying blaCTX-M-1 have been found circulating in chickens and horses from continental Europe, respectively. In Canada, blaCTX-M-1 is overwhelmingly the most common blaCTX-M variant found in Escherichia coli from chicken and horses and can be recovered at lower frequencies in swine, cattle, and dogs. Whole-genome sequencing has identified a large genetic diversity of isolates carrying this variant, warranting further investigations into the plasmids carrying this gene. Therefore, the objective of this study was to describe the genetic profiles of blaCTX-M-1 plasmids circulating in E. coli from Canadian domestic animals and compare them to those recovered in animals in Europe. Fifty-one blaCTX-M-1 positive E. coli isolates from chicken (n = 14), horses (racetrack horses n = 11; community horses n = 3), swine (n = 7), turkey (n = 6), dogs (n = 5), beef cattle (n = 3), and dairy cattle (n = 2) were selected for plasmid characterization. Sequences were obtained through both Illumina and Oxford Nanopore technologies. Genomes were assembled using either Unicycler hybrid assembly or Flye with polishing performed using Pilon. blaCTX-M-1 was found residing on a plasmid in 45 isolates and chromosomally located in six isolates. A conserved IncI1/ST3 plasmid was identified among chicken (n = 12), turkey (n = 4), swine (n = 6), dog (n = 2), and beef cattle (n = 2) isolates. When compared against publicly available data, these plasmids showed a high degree of similarity to those identified in isolates from poultry and swine in Europe. These results suggest that an epidemic IncI1/ST3 plasmid similar to the one found in Europe is contributing to the spread of blaCTX-M-1 in Canada. A conserved IncHI1/FIA(HI1)/ST2 plasmid was also recovered from nearly all racetrack horse isolates (n = 10). Although IncHI1/ST2 plasmids have been reported among European horse isolates, IncHI1/ST9 plasmids appear to be more widespread. Further studies are necessary to understand the factors contributing to these plasmids' success in their respective populations.

Genomic Changes within a Subset of IncI2 Plasmids Associated with Dissemination of mcr-1 Genes and Other Important Antimicrobial Resistance Determinants.

IncI2 plasmids appear to have only recently become associated with resistance genes; however, their tendency to carry resistance to the antibiotics of last resort and their widespread distribution increase their relative importance. In this study, we describe lineages within this plasmid family that have an increased likelihood of acquisition of antimicrobial resistance genes. Globally distributed mcr-1-carrying IncI2 plasmids were found to cluster with other IncI2 plasmids carrying extended-spectrum beta-lactamase genes, and separately from the non-resistant IncI2 plasmids. In addition, insertion sequence (IS) elements with no direct association with the acquired resistance genes also clustered with the resistance plasmids in the phylogenetic tree. In recognition of the biased sequencing of resistant plasmids globally, the analysis was also performed on resistant and non-resistant IncI2 plasmids sequenced in the USA through government surveillance efforts that do not rely on antibiotic selection. This analysis confirmed a distinct clustering associated with both resistance and mobile elements and identified possible genomic changes in core genes that correlate with increased acquisition of foreign DNA. This work highlights a potential genetic mechanism for increased uptake of foreign DNA within this prevalent family of plasmids.

Extended-spectrum cephalosporin resistance in Escherichia coli from broiler chickens raised with or without antibiotics in Ontario, Canada.

Extended-spectrum cephalosporin (ESC)-resistant Escherichia coli were isolated from fecal samples enriched in ESC-containing broth over two years from broiler chickens raised with and without therapeutic antimicrobials. Pooled samples were obtained from 13 different farms in Canada over two rearing cycles each. Resistant isolates were screened for blaCMY, blaCTX-M, and blaSHV by PCR, and several isolates were sequenced and assembled using Oxford Nanopore MinION technology. Flocks raised with or without antimicrobials contained similar ESC-resistant E. coli. Some plasmids were found in isolates from both farm types and many shared replicon types (IncI1, F, C, and B/O/K/Z) and resistance determinants. Although the use of cephalosporins has stopped in poultry production in Canada, the prevalence of ESC-resistant E. coli found in this study remained high; therefore, further studies are required to determine routes of transmission and persistence.

Resistance to extended-spectrum cephalosporins in Escherichia coli and other Enterobacterales from Canadian turkeys.

The goal of this study was to determine the frequency of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and other Enterobacterales from turkeys in Canada and characterize the associated resistance determinants. Pooled fecal samples were collected in 77 turkey farms across British Columbia, Québec, and Ontario. Isolates were obtained with and without selective enrichment cultures and compared to isolates from diagnostic submissions of suspected colibacillosis cases in Ontario. Isolates were identified using MALDI-TOF and susceptibility to ESCs was assessed by disk diffusion. The presence of blaCMY, blaCTX-M, blaTEM, and blaSHV was tested by PCR. Transformation experiments were used to characterize blaCMY plasmids. Genome sequencing with short and long reads was performed on a representative sample of blaCTX-M-positive isolates to assess isolates relatedness and characterize blaCTX-M plasmids. For the positive enrichment cultures (67% of total samples), 93% (587/610) were identified as E. coli, with only a few other Enterobacterales species identified. The frequency of ESC resistance was low in E. coli isolates from diagnostic submission (4%) and fecal samples without selective enrichment (5%). Of the ESC-resistant Enterobacterales isolates from selective enrichments, 71%, 18%, 14%, and 8% were positive for blaCMY, blaTEM, blaCTX-M, and blaSHV, respectively. IncI1 followed by IncK were the main incompatibility groups identified for blaCMY plasmids. The blaCTX-M-1 gene was found repeatedly on IncI1 plasmids of the pMLST type 3, while blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were associated with a variety of IncF plasmids. Clonal spread of strains carrying blaCTX-M genes between turkey farms was observed, as well as the presence of an epidemic blaCTX-M-1 plasmid in unrelated E. coli strains. In conclusion, Enterobacterales resistant to ESCs were still widespread at low concentration in turkey feces two years after the cessation of ceftiofur use. Although blaCMY-2 is the main ESC resistance determinant in E. coli from Canadian turkeys, blaCTX-M genes also occur which are often carried by multidrug resistance plasmids. Both clonal spread and horizontal gene transfer are involved in parallel in the spread of blaCTX-M genes in Enterobacterales from Canadian turkeys.

Presence and Diversity of Extended-Spectrum Cephalosporin Resistance Among Escherichia coli from Urban Wastewater and Feedlot Cattle in Alberta, Canada.

A recent preliminary study from our group found that extended-spectrum cephalosporin-resistance determinants can be detected in the majority of composite fecal samples collected from Alberta feedlot cattle. Most notably, blaCTX-M genes were detected in 46.5% of samples. Further isolate characterization identified blaCTX-M-15 and blaCTX-M-27, which are widespread in bacteria from humans. We hypothesized that Escherichia coli of human and beef cattle origins share the same pool of blaCTX-M genes. In this study, we aimed to assess and compare the genomic profiles of a larger collection of blaCTX-M-positive E. coli recovered from fecal composite samples from Canadian beef feedlot cattle and human wastewater through whole-genome sequencing. The variants blaCTX-M-55, blaCTX-M-32, blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14 were found in both urban wastewater and cattle fecal isolates. Core genome multilocus sequence typing showed little similarity between the fecal and wastewater isolates. Thus, if the dissemination of genes between urban wastewater and feedlot cattle occurs, it does not appear to be related to the expansion of specific clonal lineages. Further investigations are warranted to assemble and compare plasmids carrying these genes to better understand the modalities and directionality of transfer.

Diversity of CTX-M-positive Escherichia coli recovered from animals in Canada.

Historically, extended-spectrum cephalosporin resistance in bacteria from animals in Canada has been attributed to the SHV and CMY ß-lactamase families. This pattern is beginning to change with the emergence of the blaCTX-M gene family among Escherichia coli recovered from various animal species. Here we analyze and compare whole genome sequences of blaCTX-M-positive E. coli isolates (n = 173) from dogs, chicken, swine, horses and beef cattle in Canada. Ten blaCTX-M variants were identified with blaCTX-M-1,-14, -15, -27 and blaCTX-M-55 being identified in most animal species. These variants occurred across many sequence types, suggesting that mobile genetic elements mediate the spread of blaCTX-M. The variants blaCTX-M-14, -15, -27 and blaCTX-M-55 are associated with the global spread of blaCTX-M in human clinical isolates and their presence could be indicative of transfer between humans and animals. These variants were also the principal variants identified among sequence type 131 isolates, which were not associated with any other species than dogs. These isolates carried the same blaCTX-M variants as E. coli isolates found in humans. Close contact may promote the transmission of these isolates between humans and companion animals.

Distribution of the pco Gene Cluster and Associated Genetic Determinants among Swine Escherichia coli from a Controlled Feeding Trial.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonella enterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.

A method to detect Escherichia coli carrying the colistin-resistance genes mcr-1 and mcr-2 using a single real-time polymerase chain reaction and its application to chicken cecal and porcine fecal samples.

Colistin is one of the last-resort antibiotics for the treatment of multidrug-resistant infections in humans, but transmissible colistin-resistance genes have emerged in bacteria from animals. The rapid and sensitive detection among animals of colonization with bacteria carrying these genes is critical in helping to control further spread. Here we describe a method for broth enrichment of colistin-resistant Escherichia coli from animal fecal and cecal samples followed by real-time polymerase chain reaction (PCR) for the simultaneous detection of two of the main colistin-resistance genes, mcr-1 and mcr-2. The PCR uses a single set of nondegenerative primers, and mcr variants can be differentiated by melt-curve analysis. Overnight culture enrichment was effective for amplifying colistin-resistant E. coli, even when initially present in numbers as low as 10 bacteria per gram of sample. The mcr-1 and mcr-2 genes were not found in any of the Ontario swine and poultry samples investigated.

La colistine est un des antibiotiques de dernier recours pour le traitement d'infections causées par des bactéries multi-résistantes chez l'humain, mais des gènes transmissibles de résistance à la colistine ont émergé chez des bactéries provenant d'animaux. La détection rapide et sensible parmi les animaux de la colonisation par ces bactéries porteuses de ces gènes est critique afin d'aider à limiter la propagation. Nous décrivons ici une méthode pour un enrichissement en bouillon des souches de Escherichia coli résistantes à la colistine provenant d'échantillons de fèces animales et de caecum suivi d'une réaction d'amplification en chaine par la polymérase (ACP) en temps réel pour la détection simultanée des deux gènes principaux de résistance à la colistine, mcr-1 et mcr-2. La réaction d'ACP utilise une seule série d'amorces non-dégénératives, et les variants de mcr peuvent être différenciés par l'analyse de la courbe de fusion. Une culture d'enrichissement d'une nuit était efficace pour amplifier les E. coli résistants à la colistine, même si présents initialement en quantité aussi faible que 10 bactéries par gramme d'échantillon. Les gènes mcr-1 et mcr-2 n'ont pas été trouvés dans aucun des échantillons porcins ou aviaires étudiés.(Traduit par Docteur Serge Messier).

Prevalence and mechanisms of extended-spectrum cephalosporin resistance in clinical and fecal Enterobacteriaceae isolates from dogs in Ontario, Canada.

There is little information on the genetic basis of resistance to the critically important extended-spectrum cephalosporins (ESCs) in Enterobacteriaceae from dogs in Canada. This study assessed the frequency of ESC resistance in Enterobacteriaceae isolated from dogs in Ontario and the distribution of major ESC resistance genes in these bacteria. A total of 542 Enterobacteriaceae were isolated from 506 clinical samples from two diagnostic laboratories in Ontario. Eighty-eight ESC-resistant Enterobacteriaceae and 217 Escherichia coli were isolated from 234 fecal samples from dogs collected at leash-free dog parks. These fecal isolates were tested for ESC resistance along with the clinical isolates. Isolates with reduced ESC susceptibility were screened for blaCMY, blaCTX-M, and blaSHV, and all CTX-M-positive isolates underwent whole-genome sequencing. The prevalence of ESC resistance in clinical Enterobacteriaceae was 10.4%. The average frequency of fecal carriage of ESC-resistant Enterobacteriaceae in healthy dogs was 26.5%. The majority of ESC-resistant isolates were E. coli and the other major Enterobacteriaceae carrying ESC resistance genes were Klebsiella pneumoniae and Proteus mirabilis. The results show that the same ESC resistance genes can be found in clinical and fecal Enterobacteriaceae in dogs. The identified E. coli sequence types (including ST131 and ST648) and CTX-M variants (including CTX-M-14, -15, and -27) support the hypothesis of transfer of resistant bacteria between humans and dogs. CTX-M-1 was frequently found in canine fecal Enterobacteriaceae, while it is still rare in human Enterobacteriaceae in Canada, thus suggesting transfer of resistant bacteria to dogs from food animals or other sources.

Determinants of virulence and of resistance to ceftiofur, gentamicin, and spectinomycin in clinical Escherichia coli from broiler chickens in Québec, Canada.

Antimicrobials are frequently used for the prevention of avian colibacillosis, with gentamicin used for this purpose in Québec until 2003. Ceftiofur was also used similarly, but voluntarily withdrawn in 2005 due to increasing resistance. Spectinomycin-lincomycin was employed as a replacement, but ceftiofur use was partially reinstated in 2007 until its definitive ban by the poultry industry in 2014. Gentamicin resistance frequency increased during the past decade in clinical Escherichia coli isolates from broiler chickens in Québec, despite this antimicrobial no longer being used. Since this increase coincided with the use of spectinomycin-lincomycin, co-selection of gentamicin resistance through spectinomycin was suspected. Therefore, relationships between spectinomycin, gentamicin, and ceftiofur resistance determinants were investigated here. The distribution of 13 avian pathogenic E. coli virulence-associated genes and their association with spectinomycin resistance were also assessed. A sample of 586 E. coli isolates from chickens with colibacillosis in Québec between 2009 and 2013 was used. The major genes identified for resistance to ceftiofur, gentamicin, and spectinomycin were blaCMY, aac(3)-VI, and aadA, respectively. The aadA and aac(3)-VI genes were strongly associated and shown to be located on a modified class 1 integron. The aadA and blaCMY genes were negatively associated, but when present together, were generally located on the same plasmids. No statistical positive association was observed between aadA and virulence genes, and virulence genes were only rarely detected on plasmids encoding spectinomycin resistance. Thus, the use of spectinomycin-lincomycin may likely select for gentamicin but not ceftiofur resistance, nor for any of the virulence-associated genes investigated.

Staphylococcus spp., Streptococcus canis, and Arcanobacterium phocae of healthy Canadian farmed mink and mink with pododermatitis.

Pododermatitis is a disease of concern for mink breeders in Canada and worldwide, as it causes discomfort and lowers the breeding rates on farms affected by the disease. Unfortunately, the etiology and pathogenesis of pododermatitis are still unknown. In this study, we compared Staphylococcus spp. and Streptococcus canis isolates from healthy mink with isolates from animals with pododermatitis on 2 farms in Ontario. Almost all hemolytic Staphylococcus spp. isolated were shown to be Staphylococcus delphini Group A by 16S ribosomal ribonucleic acid (rRNA) sequence analysis and polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) did not reveal any S. delphini or S. canis clonal lineages specifically associated with pododermatitis, which suggests that these bacteria do not act as primary pathogens, but does not dismiss their potential roles as opportunistic pathogens. While S. delphini and S. canis were the most prevalent bacterial pathogens in mink pododermatitis, they were also present in samples from healthy mink. Arcanobacterium phocae is occasionally isolated from pododermatitis cases, but is difficult to recover with conventional culture methods due to its slow growth. A quantitative real-time PCR was developed for the detection of A. phocae and was tested on 138 samples of footpad tissues from 14 farms. The bacterium was detected only in pododermatitis-endemic farms in Canada and was at higher concentrations in tissues from infected footpads than in healthy tissues. This finding suggests that A. phocae is involved in the pathogenesis of pododermatitis.

La pododermatite est une maladie qui préoccupe les éleveurs de visons au Canada et ailleurs dans le monde, étant donné qu'elle cause un inconfort et diminue les taux de reproduction dans les fermes où la maladie est retrouvée. Malheureusement, l'étiologie et la pathogénie de la pododermatite sont encore inconnues. Dans la présente étude nous avons comparé des isolats de Staphylococcus spp. et Streptococcus canis provenant de visons en santé à ceux provenant d'animaux avec pododermatite de deux fermes en Ontario. Presque tous les isolats hémolytiques de Staphylococcus spp. ont été identifiés comme étant des S. delphini Groupe A par analyse des séquences de l'ARN ribosomal (ARNr) 16S et par réaction d'amplification en chaîne par la polymérase (PCR). L'analyse par électrophorèse en gel à champs pulsés (PFGE) n'a pas permis de mettre en évidence pour S. delphini ou S. canis de lignées clonales associées spécifiquement à la pododermatite, ce qui suggère que ces bactéries n'agissent pas en tant qu'agents pathogènes primaires, mais ne diminue pas pour autant leur rôle potentiel comme agents pathogènes opportunistes. Malgré le fait que S. delphini et S. canis étaient les bactéries pathogènes les plus prévalentes dans les cas de pododermatite chez le vison, elles étaient également présentes dans des échantillons provenant de visons en santé. Arcanobacterium phocae est isolé occasionnellement de cas de pododermatite, mais il est difficile de l'isoler en utilisant des méthodes conventionnelles de culture étant donné sa croissance lente. Une épreuve PCR quantitative en temps réel fut développée pour la détection d'A. phocae et fut testée sur 138 échantillon de tissus des coussinets plantaires provenant de 14 fermes. La bactérie fut détectée seulement de fermes canadienne où la pododermatite était endémique et se retrouvait en plus grande concentrations dans les tissus de coussinets plantaires infectés comparativement à des tissus sains. Ces résultats suggèrent qu'A. phocae est impliqué dans la pathogénie de la pododermatite.(Traduit par Docteur Serge Messier).

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome.

The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2), bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins.

Genetic diversity of Haemophilus parasuis from sick and healthy pigs.

A collection of 94 Haemophilus parasuis isolates was used for this study. It consisted of isolates from organs of pigs with Glässer's disease and pneumonia (n=54), from nasal swabs of healthy pigs in farms without Glässer's disease problems (n=25), and 15 reference strains. These isolates were typed using a new multilocus variable number of tandem repeats analysis (MLVA) protocol and investigated for the presence of nine putative virulence genes. The new MLVA protocol was highly discriminatory (54 types identified and discrimination index of 97.4%) and reproducible. Similar to previous investigations done with other methods, two major genetic clusters were identified by MLVA, which partially correlated with serotype and virulence gene distributions. Gene linkage analysis suggested that lateral gene transfer occurs within each of these clusters, but rarely between them. Although one single MLVA type included more than 20% of the clinical isolates, no significant correlation was detected between a specific MLVA type, the major genetic clusters, or the presence of any of the virulence genes investigated or the source of the isolates (clinical infection vs. healthy pig). The MLVA typing protocol described in this study is a promising new tool for future investigations into the epidemiology of Glässer's disease and could help us to better understand interacting microbial, host and environmental factors that lead to the development of H. parasuis disease.

Occurrence of ß-lactamase genes among non-Typhi Salmonella enterica isolated from humans, food animals, and retail meats in the United States and Canada.

Non-Typhi Salmonella cause over 1.7 million cases of gastroenteritis in North America each year, and food-animal products are commonly implicated in human infections. For invasive infections, antimicrobial therapy is indicated. In North America, the antimicrobial susceptibility of Salmonella is monitored by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) and The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). In this study, we determined the susceptibility to cephalosporins by broth microdilution among 5,041 non-Typhi Salmonella enterica isolated from food animals, retail meats, and humans. In the United States, 109 (4.6%) of isolates collected from humans, 77 (15.7%) from retail meat, and 140 (10.6%) from food animals displayed decreased susceptibility to cephalosporins (DSC). Among the Canadian retail meat and food animal isolates, 52 (13.0%) and 42 (9.4%) displayed DSC. All isolates displaying DSC were screened for ß-lactamase genes (bla(TEM), bla(SHV), bla(CMY), bla(CTX-M), and bla(OXA-1)) by polymerase chain reaction. At least one ß-lactamase gene was detected in 74/109 (67.9%) isolates collected from humans, and the bla(CMY) genes were most prevalent (69/109; 63.3%). Similarly, the bla(CMY) genes predominated among the ß-lactamase-producing isolates collected from retail meats and food animals. Three isolates from humans harbored a bla(CTX-M-15) gene. No animal or retail meat isolates harbored a bla(CTX-M) or bla(OXA-1) gene. A bla(TEM) gene was found in 5 human, 9 retail meat, and 17 animal isolates. Although serotype distributions varied among human, retail meat, and animal sources, overlap in bla(CMY)-positive serotypes across sample sources supports meat and food-animal sources as reservoirs for human infection.