Distinct evolutionary origins of common multi-drug resistance phenotypes in Salmonella typhimurium DT104: a convergent process for adaptation under stress.

Antimicrobial resistance makes pathogenic bacteria hard to control, but little is known about the general processes of resistance gain or loss. Here, we compared distinct S. typhimurium DT104 strains resistant to zero, two, five, or more of the tested antimicrobials. We found that common resistance phenotypes could be encoded by distinct genes, on SGI-1 or plasmid. We also demonstrated close clonality among all the tested non-resistant and differently resistant DT104 strains, demonstrating dynamic acquisition or loss (by total deletion or gradual decaying of multi-drug resistance gene clusters) of the genetic traits. These findings reflect convergent processes to make the bacteria resistant to multiple antimicrobials by acquiring the needed traits from stochastically available origins. When the antimicrobial stress is absent, the resistance genes may be dropped off quickly, so the bacteria can save the cost for maintaining unneeded genes. Therefore, this work reiterates the importance of strictly controlled use of antimicrobials.

Salmonella phages and prophages: genomics, taxonomy, and applied aspects.

Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains.

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.

Characterization of blaCMY-2 plasmids in Salmonella and Escherichia coli isolates from food animals in Canada.

One hundred thirty-four bla(CMY-2) plasmids from Salmonella and Escherichia coli strains from animals and food in Canada were characterized. Five plasmid groups were identified based on replicon type and restriction profiles. Three groups contained E. coli plasmids only. IncA/C plasmids included most multiresistant plasmids and all those of bovine origin.

Preliminary investigations of the distribution of Escherichia coli O149 in sows, piglets, and their environment.

Little is known about the sources and kinetics of enterotoxigenic Escherichia coli colonization in pigs during the pre-and post-weaning period. In this study, farrowing pens, sows, and piglets were tested for the presence of E. coli O149 by real-time polymerase chain reaction (PCR) after bacterial culture pre-enrichment on 2 farms, one with a history of post-weaning diarrhea (problem farm - PF) and the other without such a history (non-problem farm - NPF). Unlike those on the PF, the sows from the NPF did not carry E. coli O149 before parturition, although they were colonized to frequencies similar to animals on the PF soon afterwards. Most piglets from the NPF were colonized within a week after birth, whereas only a small proportion of those on the PF were colonized during that period. No difference was observed in the frequency of piglet colonization at the 2 farms either at weaning or during the following week. Post-weaning diarrhea (PWD), which is caused by enterotoxigenic E. coli (ETEC), is a multifactorial disease. The presence of ETEC alone is not always sufficient for the disease to develop. Many other factors are considered to be associated with the occurrence of PWD, including feed type (1,2), feeding regimen (1,3,4), the presence of other infectious agents (3,5), weaning age, and weight (6). Weaning, which is considered to be a major physiological and psychological stress factor, is critical for the disease to occur (7). Although piglets are already colonized with ETEC before weaning (4,8), on many farms, clinical disease occurs only after weaning (1). Both sows (9,10) and the environment (6) could be possible sources of infection for piglets, but results from previous studies have not resolved this issue because of the low sensitivity of ETEC detection methods. This study provides preliminary data based on a sensitive detection method for E. coli O149 in pigs and their environment. The results demonstrate the potential of real-time PCR for future studies on this topic.

Clostridium perfringens in retail chicken.

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% "atypical" genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.

The Escherichia coli O149 rfb gene cluster and its use for the detection of porcine E. coli O149 by real-time PCR.

PCR or hybridization assays are widely used for the identification and detection of various Escherichia coli serogroups and serotypes. This study explored this approach for the detection of E. coli O149 in pigs, a dominant serogroup among those associated with porcine post-weaning diarrhea (PWD) worldwide. For this purpose, we sequenced the O antigen (rfb) gene cluster of a representative enterotoxigenic E. coli (ETEC) O149:H10 strain and identified a 2kbp region specific for the O149 rfb gene cluster. This region lacked similarity with sequences in GenBank, except for the rfb gene cluster of the human pathogen Shigella boydii O1 with which it shares 99% sequence identity. Southern blot hybridization with 91 E. coli strains belonging to 62 serogroups confirmed the specificity of the identified region. Restriction analysis of the rfb gene cluster of 38 geographically and chronologically diverse E. coli O149 strains with the endonucleases HindIII, PstI and XhoI demonstrated that this cluster is highly conserved across the serogroup O149. Analysis of the O antigen of a representative O149:H10 ETEC strain by nuclear magnetic resonance confirmed the identity of the O antigen structure in recent and older O149 ETEC strains. Finally, we developed a real-time PCR assay for the detection of E. coli O149 in pig fecal samples by targeting the part of the rfb sequence specific for this serogroup. This method was suitable for a sensitive qualitative detection of O149 E. coli in pig fecal samples after enrichment, but further investigations are needed to find a suitable method for a direct, reproducible and sensitive quantification of O149 E. coli in pig feces.

A genome map of Salmonella enterica serovar Agona: numerous insertions and deletions reflecting the evolutionary history of a human pathogen.

Salmonella enterica serovar Agona is an important zoonotic pathogen, causing serious human illness worldwide, but knowledge about its genetics and evolution, especially regarding the genomic events that might have contributed to the formation of S. Agona as an important pathogen, is lacking. As a first step toward understanding this pathogen and characterizing its genomic differences with other salmonellae, we constructed a physical map of S. Agona in strain SARB1 using I-CeuI, XbaI, AvrII and Tn10 insertions with pulsed-field gel electrophoresis techniques. On the 4815-kb genomic map, we located 82 genes, revealed one inversion of about 1000 kb and resolved seven deletions and seven insertions ranging from 10 to 67 kb relative to the genome of Salmonella typhimurium LT2. These genomic features clearly distinguish S. Agona from other previously analyzed salmonellae and provide clues to the molecular basis for its genomic divergence. Additionally, these kinds of physical maps, combined with emerging high-speed sequencing technologies, such as the Solexa or SOLiD techniques, which require a pre-existing high-resolution physical map such as the S. Agona map reported here, will play important roles in genomic comparative studies of bacteria involving large numbers of strains.

Occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern Ontario: A preliminary study.

The prevalence and patterns of antimicrobial susceptibility of fecal Escherichia coli, Salmonella spp., extended beta-lactamase producing E. coli (ESBL-E. coli), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus pseudintermedius (MRSP) were determined for healthy dogs (n = 188) and cats (n = 39) from veterinary hospitals in southern Ontario that had not had recent exposure to antimicrobials. The prevalence of antimicrobial resistance in E. coli was as follows: streptomycin (dogs - 17%, cats - 2%), ampicillin (dogs - 13%, cats - 4%), cephalothin (dogs - 13%, cats - < 1%), and tetracycline (dogs - 11%, cats - 2%). Eleven percent of dogs and 15% of cats had isolates that were resistant to at least 2 antimicrobials. Cephamycinase (CMY)-2 producing E. coli was cultured from 2 dogs. No Salmonella spp., ESBL-E. coli, MRSA, or MRSP isolates were recovered. The observed prevalence of resistance in commensal E. coli from this population was lower than that previously reported in companion animals, but a small percentage of dogs may be a reservoir for CMY-2 E. coli.

A longitudinal study of the Salmonella status on Ontario swine farms within the time period 2001-2006.

In order to describe the farm-level Salmonella status, 113 Ontario swine farms were tested for Salmonella one to five times within the time period 2001-2006. During 422 visits, 6844 fecal samples were collected and cultured for Salmonella. Salmonella was recovered from 437 (6.38%) of the fecal samples, and 69 (61%) of the farms had at least one positive sample over the entire period of the study. Salmonella was not recovered on 11 farms of the 54 farms visited five times, nor from 7 of the 17 farms visited four times. On seven farms Salmonella was not recovered over the first four visits but were cultured on the fifth visit. The isolates belonged to 30 different serovars, and serogroup B and C1 were the most common serogroups. Salmonella Typhimurium (including var. Copenhagen) was the most common serovar recovered from 35.5% of the farms with DT104 as the most frequent phage type. Only 24% of the total random variance in prevalence of Salmonella was due to repeated measurement, while the variation in prevalence of Salmonella Typhimurium (including var. Copenhagen) and DT104 due to repeated measurement was 37% and 52% of total random variance, respectively. Although the observed trends may be partly attributed to the different culturing methods, different types of samples, and sampling strategies used in each year, it may also denote the dynamics of Salmonella as a bacterial population on swine farms. These findings indicate that monitoring over time may be useful to detect changes in Salmonella on swine farms.

Subinhibitory concentrations of tetracycline affect virulence gene expression in a multi-resistant Salmonella enterica subsp. enterica serovar Typhimurium DT104.

Treatment of salmonellosis with antibiotics is controversial and may prolong carriage and shedding. Therefore, this study sought to investigate if exposure to antimicrobials influences the expression of factors involved in virulence and host colonization. The effect of subinhibitory tetracycline treatment (16 microg/ml, 30 min) on a multi-drug resistant Salmonella Typhimurium DT104 strain was investigated using a targeted microarray. Real-time reverse transcriptase PCR was used to confirm and further assess transcription of 10 selected genes. An in vitro cell invasion assay was performed to assess the invasiveness of the tetracycline-treated isolate. Out of 323 genes, 11 were significantly up-regulated and four were down-regulated in the microarray assays. The hilD and hilA genes, both regulators of Salmonella Pathogenicity Island 1, were up-regulated. Other up-regulated genes included the fliC, fliD, motA and motB genes, involved in motility, the fur gene, an important regulator of iron acquisition systems and of acid tolerance. The drug-exposed replicates showed a 2.5-fold increase in intracellular bacteria over the non-exposed control in cell cultures. These findings suggest a drug-induced expression profile consistent with the early stages of Salmonella infection and invasion concomitant with an increased ability to invade epithelial cells in vitro.

Molecular epidemiology and antimicrobial resistance of Salmonella typhimurium DTI04 on Ontario swine farms.

This study was conducted to examine antimicrobial resistances, plasmid profiles, and pulsed-field gel electrophoresis patterns of 80 Salmonella Typhimurium (including var. Copenhagen) DT104 strains (including DT104a and DT104b) recovered from pig and environmental fecal samples on 17 swine farms in Ontario. No resistance was observed to amoxicillin/clavulanic acid, apramycin, carbadox, cephalothin, ceftriaxone, ceftiofur, cefoxitin, ciprofloxacin, nalidixic acid, trimethoprim, and tobramycin. However, the isolates exhibited resistance against 4 to 10 antimicrobials with the most frequent resistance being to sulfonamides (Su), ampicillin (A), streptomycin (S), spectinomycin (Sp), chloramphenicol (C), tetracycline (T), and florfenicol (F). Thirteen distinct resistance patterns were determined but 88% of isolates shared the typical resistance pattern "ACSpSSuT." Twelve different plasmid profiles were observed; the 62 MDa virulence-associated plasmid was detected in 95% of the isolates. The 2.1 MDa plasmid was the second most frequent one, which was harbored by 65% isolates. The isolates were classified into 23 distinct genotypes by PFGE-SpeI + BlnI when difference in at least one fragment was defined as a distinct genotype. In total, 39 distinct "types" were observed when defining a "type" based on the combination of antimicrobial resistance, plasmid pattern, and PFGE-SpeI + BlnI for each isolate. The highest diversity was 0.96 (95% CI: 0.92, 0.96) for the "type" described above followed by 0.92 (95% CI: 0.88, 0.93) for PFGE-SpeI + BlnI. The diversity of DT104 isolates indicates there might be multiple sources for this microorganism on swine farms. This knowledge might be used to track these sources, as well as to study the extent of human salmonellosis attributed to pork compared to food products derived from other food-producing animals.

Prevalence and patterns of antimicrobial resistance of fecal Escherichia coil among pigs on 47 farrow-to-finish farms with different in-feed medication policies in Ontario and British Columbia.

The main objectives of this study were to determine the prevalence and patterns of antimicrobial resistance in pigs on farms that medicated swine ration and those that did not. A total of 940 isolates of Escherichia coli from 188 pooled fecal samples obtained from weaner and finisher pigs on 47 farrow-to-finish swine farms (34 farms used in-feed medication and 13 did not) were tested for susceptibility to 21 antimicrobials using a breakpoint concentration method. The prevalence of resistance varied widely (0.0% to 81.3%) among the antimicrobials tested. Ninety percent of all the isolates tested were resistant to one or more antimicrobials. The most common multi-drug resistance patterns were to 2 to 6 antimicrobials. Resistance was significantly more frequent (P < 0.01) on farms that used in-feed medication compared to those that did not, and significantly more frequent (P < 0.01) in weaner pigs compared to finisher pigs. These findings indicate that resistance to a broad range of antimicrobials was prevalent among fecal E. coli isolates of pigs on study farms, and that this constitutes a potential reservoir for resistance genes that could spread to pathogens. The findings also provide further evidence that use of medication in swine rations provides selective pressure for antimicrobial resistance in E. coli in pigs.

Associations of antimicrobial uses with antimicrobial resistance of fecal Escherichia coli from pigs on 47 farrow-to-finish farms in Ontario and British Columbia.

This study assessed the associations between antimicrobial use and other management practices in pigs and antimicrobial resistance in generic Escherichia coli recovered from feces of weaner and finisher pigs on 39 purposefully selected farrow-to-finish farms in Ontario and 8 in British Columbia. Antimicrobials (n = 13), most frequently penicillins and tetracycline, wer dministered to different age groups of pigs on study farms through various routes of administration. Logistic regression was used to model risk factors to antimicrobial resistance in fecal E. coli of pigs for the following antimicrobials: ampicillin, apramycin, carbadox, cephalothin, chloramphenicol, kanamycin, neomycin, nitrofurantoin, spectinomycin, streptomycin, sulfamethoxazole tetracycline, and cotrimoxazole (trimethoprim and sulfamethoxazole). Use of antimicrobials in weaner pigs compared with use in finisher pigs was associated with resistance in most models. There was phenotypic evidence of different mechanisms of resistance selection, including direct selection [use of carbadox was associated with carbadox resistance (OR = 6.48)]; cross-resistance [use of spectinomycin was associated with streptomycin resistance (OR = 2.29)]; and possible co-selection [ceftiofu use was associated with tetracycline resistance (OR = 6.12)]. These results provide further evidence that use of antimicrobials in pigs selects for resistance among fecal E. coli within and between classes of antimicrobials.

Effect of plasmid pTENT2 on severity of porcine post-weaning diarrhoea induced by an O149 enterotoxigenic Escherichia coli.

A particularly virulent O149:H10 enterotoxigenic Escherichia coli clone harbours a newly characterized plasmid pTENT2 carrying the tetracycline-resistance tetA and the virulence genes estA, paa, and sepA that were not present in less virulent clones. The objectives of this study were to assess whether the additional genes on pTENT2 played a role in the increased severity of post-weaning diarrhoea and if they provided any potential advantage for the emergence of the highly virulent clone. Groups of pigs were dosed orally with isogenic pTENT2-positive and pTENT2-negative ETEC strains, and the clinical and pathological changes were compared between the groups. Two additional groups were given the pTENT2-positive strains and maintained on feed with or without chlortetracycline to assess the effect of subtherapeutic levels of tetracycline on the short-term persistence of the ETEC O149:H10 clone. The severity of diarrhoea within the first few hours post-inoculation was significantly increased (p=0.0408) in animals receiving pTENT2-positive strains as compared to animals receiving pTENT2-negative strains. There were no consistent or significant histopathological differences between any of the groups and no significant difference in the persistence of ETEC between groups.

Chloramphenicol and kanamycin resistance among porcine Escherichia coli in Ontario.

OBJECTIVES: The purpose of this study was to compare the distribution of chloramphenicol and kanamycin resistance genes across three populations of porcine Escherichia coli. METHODS: PCR was used to assess the distribution of the major chloramphenicol and kanamycin resistance genes catA1, cmlA and floR, and aphA1, aphA2 and aadB in enterotoxigenic E. coli (ETEC), non-ETEC isolates from cases of diarrhoea and commensal E. coli from healthy pigs. Associations between these genes and resistance genes for other antimicrobials or virulence genes were assessed. RESULTS: The chloramphenicol and kanamycin resistance genes were distributed differently among the three E. coli populations. While aphA1, aphA2 and aadB were evenly distributed among resistant ETEC, non-ETEC and commensals, the catA1 gene was significantly more frequent in ETEC than in non-ETEC and commensals. Transformation experiments confirmed statistical associations by demonstrating that elt, estB, astA, aadA and sul1 were located with catA1 on a large ETEC plasmid. Plasmids carrying cmlA also carried sul3 and aadA. Other plasmids carrying floR and aadB also carried tet(A), sul2, strA/strB, bla(CMY-2) and occasionally aac(3)IV. CONCLUSIONS: The clustering of genes observed is a likely cause for chloramphenicol resistance persistence. Similar to tetracycline, chloramphenicol resistance genes are physically linked to virulence genes. This is not the case for kanamycin resistance determinants, which were linked to other resistance genes only.