Genomic characterization of third-generation cephalosporin-resistant Escherichia coli strains isolated from diseased dogs and cats: Report from Japanese Veterinary Antimicrobial Resistance Monitoring.

This study investigates the genomic characteristics of canine and feline cefotaxime (CTX, a third-generation cephalosporin)-resistant Escherichia coli using the JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System, a nationwide monitoring. In this study, whole-genome sequencing (WGS) was performed on 51 canine and 45 feline CTX-resistant E. coli isolates, with certain isolates subjected to pulsed-field gel electrophoresis with S1 nuclease for plasmid-chromosome separation. The most common blaCTX-M genes were blaCTX-M-27 (dogs: 11/51 [21.6 %]; cat: 10/45 [22.2 %]), followed by blaCTX-M-14 (dogs: 10/51 [19.6 %]; cats: 10/45 [22.2 %]), and blaCTX-M-15 (dogs: 9/51 [17.6 %]; cats: 5/45 [11.1 %]). Besides ß-lactamase genes, all isolates harbored mdf(A), a multidrug efflux pump, with resistance genes for aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines. None of the isolates had carbapenemase genes, such as blaOXA-48, blaNDM, and blaIMP, whereas most of the isolates showed double mutations in gyrA and parC, which affected quinolone resistance. For the isolates separately analyzed for plasmid and chromosomal DNA via WGS, the majority of CTX-M genes were present on the plasmids. Some plasmids also harbored the same combination of resistance genes and plasmid replicon type, although they differed from isolates derived from different areas of Japan. The predominant plasmids were blaCTX-M-27,aadA5, aph(6)-Id, aph(3")-Ib, sul1, sul2, tet(A), dfrA17, and mph(A) on IncF. The predominant combination of ST131, O25:H4, and B2 isolates comprised the largest cluster in the minimum spanning tree and the ST131 E. coli harboring blaCTX-M-27 from human in Japan was closely related to these isolates. The results indicated that CTX-resistant canine and feline E. coli harbored multiple plasmids carrying the same combination of resistance genes and emphasizes the need to prevent the spread. DATA AVAILABILITY: All raw short-read sequence data have been deposited in the DNA Data Bank of Japan. (DRR Run No, DRR335726-335821).

Prevalence and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Pigs in Japan.

We investigated the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pig slaughterhouses from 2018 to 2022 in Japan and the isolates were examined for antimicrobial susceptibility and genetic characteristics by whole-genome analysis. Although the positive LA-MRSA rates on farms (29.6%) and samples (9.9%) in 2022 in Japan remained lower than those observed in European countries exhibiting extremely high rates of confirmed human LA-MRSA infections, these rates showed a gradually increasing trend over five years. The ST398/t034 strain was predominant, followed by ST5/t002, and differences were identified between ST398 and ST5 in terms of antimicrobial susceptibility and the resistance genes carried. Notably, LA-MRSA possessed resistance genes toward many antimicrobial classes, with 91.4% of the ST398 strains harboring zinc resistance genes. These findings indicate that the co-selection pressure associated with multidrug and zinc resistance may have contributed markedly to LA-MRSA persistence. SNP analysis revealed that ST398 and ST5 of swine origin were classified into a different cluster of MRSA from humans, showing the same ST in Japan and lacking the immune evasion genes (scn, sak, or chp). Although swine-origin LA-MRSA is currently unlikely to spread to humans and become a problem in current clinical practice, preventing its dissemination requires using antimicrobials prudently, limiting zinc utilization to the minimum required nutrient, and practicing fundamental hygiene measures.

Characterization of Actinobacillus pleuropneumoniae isolated from pigs in Japan using whole genome sequencing.

We conducted whole-genome sequencing to investigate the serotypes, the presence of virulence and antimicrobial resistance genes, and the genetic relationships among isolates of Actinobacillus. pleuropneumoniae derived from diseased pigs. Serotype 2 (71.2%) was the most common, but the prevalence of serotypes 6 (13.6%) and 15 (6.8%) increased. Existing vaccines are considered ineffective on the isolates belonging to serotypes 6 and 15. The phylogenetic tree based on core genome single nucleotide polymorphisms showed that the isolates were clustered by serotype. Of the isolates, 62.5% did not have an antimicrobial resistance gene, including a florfenicol resistance gene, but 32.2% had a tetracycline resistance gene. The antimicrobial resistant phenotype and genotype were almost identical. The plasmid-derived contigs harbored resistance genes of aminoglycosides, tetracyclines, ß-lactams, phenicols, or sulfonamides. It has been suggested that isolates with different genetic properties from vaccine strains are circulating; however, antimicrobial resistance may not be widespread.

Role of Plasmids in Co-Selection of Antimicrobial Resistances Among Escherichia coli Isolated from Pigs.

Co-selection is thought to occur when resistance genes are located on the same mobile genetic element. However, this mechanism is currently poorly understood. In this study, complete circular plasmids from swine-derived Escherichia coli were sequenced with short and long reads to confirm that resistance genes involved in co-resistance were co-transferred by the same plasmid. Conjugative transfer tests were performed, and multiple resistance genes were transmitted. The genes possessed by the donor, transconjugant, and plasmid of the donor were highly similar. In addition, the sequences of the plasmid of the donor and the plasmid of the transconjugant were almost identical. Resistance genes associated with statistically significant combinations of antimicrobial use and resistance were co-transmitted by the same plasmid. These results suggest that resistance genes may be involved in co-selection by their transfer between bacteria on the same plasmid.

Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice.

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from pigs in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of infection in hospitalized patients and can be prevalent in humans and various animal species. In European countries, MRSA isolates belonging to clonal complex 398 have been detected at high rates in pigs. However, the prevalence of MRSA in pigs and farm environments in Japan remains unclear. MRSA isolates were obtained from pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust. We conducted whole-genome sequencing (WGS) and analyzed the molecular epidemiological relationship between these MRSA isolates using core genome multilocus sequence typing (cgMLST). The prevalence rates of MRSA among pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust were 5.2 %, 3.4 %, 28.8 %, and 0.06 %, respectively. ST 398 isolates that classified as ST398/t034 were isolated from pigs from all sources. The results of cgMLST showed that ST398/t034 isolates originating from domestic pigs clustered into the same cluster as the isolates from imported breeding pigs. However, some clusters only included isolates of domestic pig origin. Most MRSA isolates in this study carried resistance genes for aminoglycosides, ß-lactams, macrolides, tetracyclines, and zinc. None of the MRSA isolates in this study harbored Panton-Valentine leukocidin toxin genes. Molecular epidemiological analysis suggested a relationship between isolates from slaughter pigs and imported breeding pigs and the presence of MRSA isolates of domestic origin. However, more data are needed for elucidation of the origin of these MRSA variants in the pig industry in Japan.

Nationwide Monitoring of Antimicrobial-Resistant Escherichia coli and Enterococcus spp. Isolated From Diseased and Healthy Dogs and Cats in Japan.

The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was established for nationwide monitoring of antimicrobial-resistant bacteria isolated from animals. Here, antimicrobial resistance of Escherichia coli and Enterococcus spp. isolates from diseased and healthy dogs and cats was investigated. Isolates were collected from diseased dogs and cats and from healthy dogs and cats in 2018 to 2020. Minimum inhibitory concentrations were determined for 1873 E. coli and 1383 Enterococcus spp. isolates. E. coli isolates were most commonly resistant to nalidixic acid [diseased dog (DD), 62.1%; diseased cat (DC), 59.9%; healthy dog (HD), 23.5%; healthy cat (HC, 24.0%] and ampicillin (DD, 54.4%; DC, 64.1%; HD, 28.4%; HC, 25.2%), followed by ciprofloxacin (DD, 45.0%; DC, 44.0%; HD, 12.9%; HC, 10.4%). Enterococcus spp. isolates were most resistant to tetracycline (DD, 66.9%; DC, 67.8%; HD, 47.0%; HC, 52.0%), followed by erythromycin (DD, 43.2%; DC, 46.6%; HD, 27.8%; HC, 34.0%) and ciprofloxacin (DD, 27.9%; DC, 43.7%; HD, 9.7%; HC 12.9%). Only a few E. coli isolates were resistant to colistin and none were resistant to meropenem. Also, none of the Enterococcus spp. isolates we have tested were resistant to vancomycin. The significantly higher resistance rates of E. coli and Enterococcus spp. isolates from diseased, as opposed to healthy, dogs and cats against most of the tested antimicrobials indicates that the use of antimicrobials could select resistant E. coli and Enterococcus spp.

Correlation of minimum inhibitory concentrations between human and animal antimicrobials against Escherichia coli isolated from livestock.

We analyzed the correlation between minimum inhibitory concentrations (MICs) of antimicrobials used in humans and those used in animals to enable comparison of antimicrobial susceptibility between Escherichia coli isolated from humans and those from animals. We compared the following pairs of MIC data: piperacillin (PIPC) to ampicillin (ABPC), amikacin (AMK) to kanamycin (KM), minocycline (MINO) to oxytetracycline (OTC), and levofloxacin (LVFX) to enrofloxacin (ERFX) using 103 isolates of E. coli from healthy livestock (cattle, pigs, broiler chickens, and layer chickens). Kappa analysis of the agreement for resistance and susceptibility between PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX showed almost perfect (κ = 0.81), slight (κ = 0.12), fair (κ = 0.37), and moderate (κ = 0.46) agreement, respectively. Within the antimicrobial pairs, all isolates resistant to the human antimicrobial were also resistant to the veterinary antimicrobial. However, there was less agreement within the pairs for those isolates that were sensitive to the human antimicrobial. The percentage agreement for susceptibility, defined as the percentage of isolates sensitive to both antimicrobials compared with isolates sensitive to both antimicrobials, as well as those sensitive only to the human antimicrobial, was 89.9%, 87.3%, 64.0%, and 89.9% for PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX, respectively. Our results suggest that the possibility of missing the resistance for antimicrobials used in human medicine by examining MICs for the equivalent antimicrobials used in veterinary medicine is low.

Quantitative Release Assessment of mcr-mediated Colistin-resistant Escherichia Coli from Japanese Pigs.

Colistin is a critically important antibiotic for humans. The Japanese government withdrew colistin growth promoter and shifted therapeutic colistin to a second-choice drug for pigs in 2017. A quantitative release assessment of mcr-mediated colistin-resistant Escherichia coli (E. coli) in Japanese finisher pigs was conducted under the World Organisation for Animal Health (OIE) risk assessment framework. Input data included colistin resistance and mcr-1-5 test results for E. coli isolates in the Japan Veterinary Resistance Monitoring System (JVARM), postal survey results regarding indication disease occurrence and colistin use by swine veterinarians in 2017 and 2018, and colistin resistance and mcr monitoring experiments at four pig farms in 2017-2018. An individual-based model was developed to assess the risk: the proportion of Japanese finisher pigs with mcr-1-5-mediated colistin-resistant E. coli dominant in the gut on an arbitrary day. Before implementing risk management measures, the risk was estimated to be 5.5% (95% CI: 4.2%-10.1%). At 12 months after stopping colistin growth promoter, the proportion of pigs with plasmid-mediated colistin-resistant E. coli declined by 52.5% on the experiment farms (95% CI: 8.7%-80.8%). The probability of therapeutic colistin use at the occurrence of bacterial diarrhea declined from 37.3% (95% CI: 30.3%-42.5%) in 2017 to 31.4% (95% CI: 26.1%-36.9%), and that of edema disease declined from 55.0% (95% CI: 46.0%-63.7%) to 44.4% (95% CI: 36.9%-52.0%). After risk management implementation, the risk was estimated to have declined to 2.3% (95% CI: 1.8%-4.3%; 58.2% reduction). Scenario analyses showed that pen-level colistin treatment effectively reduces the risk from 5.5% to 4.7% (14.5% reduction), an effect similar to stoppage of therapeutic colistin (16.4% reduction to 4.6%).

Comparative Genomic Analysis of Third-Generation-Cephalosporin-Resistant Escherichia coli Harboring the blaCMY-2-Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan.

The off-label use of third-generation cephalosporins (3GCs) during in ovo vaccination or vaccination of newly hatched chicks has been a common practice worldwide. CMY-2-producing Escherichia coli strains have been disseminated in broiler chicken production. The objective of this study was to determine the epidemiological linkage of blaCMY-2-positive plasmids among broilers both within and outside Japan, because the grandparent stock and parent stock were imported into Japan. We examined the whole-genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002 to 2014. The predominant 3GC resistance gene was blaCMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (n = 21; 24.1%), IncI (n = 12; 13.8%), IncB/O/K/Z (n = 28; 32.2%), and IncC (n = 22; 25.3%). Those plasmids were subjected to gene clustering, network analyses, and plasmid multilocus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single-nucleotide variant (SNV)-based phylogenetic analysis. MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. The sequence type 429 (ST429) cluster harboring blaCMY-2-positive IncB/O/K/Z was closely related to isolates from broilers in Germany harboring blaCMY-2-positive IncB/O/K/Z. pST55-IncI, pST12-IncI1-Iγ, and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to plasmids detected in E. coli isolates from chickens in North America, whereas 26 IncB/O/K/Z types were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria inside and outside Japan.

Evaluation of the relationship between the minimum inhibitory concentration of ceftiofur and third-generation cephalosporins in Escherichia coli isolates from food-producing animals.

To enable future comparison of the antimicrobial susceptibility data between bacteria obtained from animals and humans, it is necessary to compare the relationships between minimum inhibitory concentrations (MICs) of veterinary and human medicine. We evaluated the relationship between the MIC of ceftiofur (CTF) and the MICs of other third-generation cephalosporins (TGCs): cefotaxime (CTX), cefpodoxime (CPDX), and ceftazidime (CAZ), determined by the broth microdilution method using 118 cefazolin-resistant Escherichia coli isolates from food-producing animals. Using the Clinical and Laboratory Standards Institute criteria, very major classification errors were observed only in CAZ (17.8%, 21 of 118); major and minor errors were observed in all TGCs (CTX: 0.8% [1 of 118] and 9.3% [11 of 118]; CPDX: 9.3% [11 of 118] and 6.8% [8 of 118]; CAZ: 2.5% [3 of 118] and 9.3% [11 of 118], respectively). The Spearman correlation coefficients between the MICs of CTF and CTX, CPDX, and CAZ were 0.765, 0.731, and 0.306, respectively. The sensitivity and specificity values were 100.0% and 81.8% for CTX, 99.0% and 27.3% for CPDX, and 76.0% and 86.4% for CAZ compared with CTF. The C-statistic was 0.978 for CTF and CTX, 0.953 for CPDX, and 0.798 for CAZ. For the TGCs evaluated in our study, testing for CTX susceptibility results showed the highest correlation with the results given when testing for CTF susceptibility.

Prevalence of Colistin Resistance Gene mcr-1 and Absence of mcr-2 in Escherichia coli Isolated from Healthy Food-Producing Animals in Japan.

We screened mcr-1 and mcr-2 genes in 9,306 Escherichia coli strains isolated from healthy animals in the Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) system. mcr-1 was detected in 39 strains (5, 20, and 14 strains isolated from cattle, swine, and broilers, respectively), whereas mcr-2 was not detected. mcr-2 was also not detected with the investigation sequence homology search against our curated GenEpid-J database.

Molecular Typing of Fluoroquinolone-Resistant Campylobacter jejuni Isolated from Broilers in Japan Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis.

Fluoroquinolone-resistant Campylobacter jejuni isolates from broilers in Japan were characterized using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) in order to elucidate the genetic relationship between these strains. Forty-three of the isolates were classified into 20 sequence types and were clustered into 21 PFGE types with 70% similarity. The most dominant clonal complex (CC) was CC-21 (41.9%). Diverse PFGE patterns were observed within the same CC, but the combined analysis of PFGE type and CC revealed that the strains with the same combination were isolated from the same district or neighboring districts. On the other hand, strains with the same combination pattern were also isolated from geographically distant districts. Our results elucidate two possible reasons for the prevalence of fluoroquinolone-resistant C. jejuni among broiler farms: (1) the resistant C. jejuni is clonally disseminated within the limited area, and (2) susceptible C. jejuni acquired fluoroquinolone resistance during the use of fluoroquinolone on the farms.

Multivariable Analysis of the Association Between Antimicrobial Use and Antimicrobial Resistance in Escherichia coli Isolated from Apparently Healthy Pigs in Japan.

The objective of this study was to investigate the association between antimicrobial agent use and antimicrobial resistance in Escherichia coli isolated from healthy pigs using data from 2004 to 2007 in the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM). Fecal E. coli isolates from 250 pigs (one isolate each from a pig per farm) were examined for antimicrobial resistance. Information on the use of antimicrobials within preceding 6 months and types of farms recorded in JVARM was collected and statistically analyzed against the resistance patterns. In the univariate analysis, associations between both therapeutic and feed additive use of antimicrobials, and resistance to dihydrostreptomycin, gentamicin, kanamycin, ampicillin, cefazolin, ceftiofur, oxytetracycline, chloramphenicol, trimethoprim, nalidixic acid, enrofloxacin, colistin, and bicozamycin, and husbandry factors were investigated. In multivariable analysis, generalized estimating equations were used to control geographical intraclass correlation. Confounding for structurally unrelated associations was tested using generalized linear models. The results suggested direct and cross selections in the associations between use of aminoglycosides in reproduction farms and resistance to kanamycin, use of tetracyclines in larger farms and resistance to oxytetracycline, use of beta-lactams and resistance to ampicillin, use of phenicols and resistance to chloramphenicol, and use of fluoroquinolones and resistance to nalidixic acid and enrofloxacin. Coselection was suggested in the use of tetracyclines and chloramphenicol resistance. The associations between use of beta-lactams and dihydrostreptomycin resistance, use of macrolides and ampicillin and oxytetracycline resistance, and use of colistin and kanamycin resistance were significant, but were confounded by the simultaneous use of homologous antimicrobials.

Control of the development and prevalence of antimicrobial resistance in bacteria of food animal origin in Japan: a new approach for risk management of antimicrobial veterinary medicinal products in Japan.

Antimicrobial agents are essential for controlling bacterial disease in food-producing animals and contribute to the stable production of safe animal products. The use of antimicrobial agents in these animals affects the emergence and prevalence of antimicrobial resistance in bacteria isolated from animals and animal products. As disease-causing bacteria are often transferred from food-producing animals to humans, the food chain is considered a route of transmission for the resistant bacteria and/or resistance genes. The Food Safety Commission of Japan (FSC) has been assessing the risk posed to human health by the transmission of antimicrobial-resistant bacteria from livestock products via the food chain. In addition to the FSC's risk assessments, the Japanese Ministry of Agriculture, Forestry and Fisheries has developed risk-management guidelines to determine feasible risk-management options for the use of antimicrobial veterinary medicinal products during farming practices. This report includes information on risk assessment and novel approaches for risk management of antimicrobial veterinary medicinal products for mitigating the risk of development and prevalence of antimicrobial resistance in bacteria originating from food-producing animals in Japan.

Detection of aac(6')-Ib-cr in avian pathogenic Escherichia coli isolates in Japan.

We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in avian pathogenic Escherichia coli (APEC) strains in Japan. A total of 117 APEC strains collected between 2004 and 2007 were examined for PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA and oqxAB) by polymerase chain reaction. None of the APEC strains carried qnrA, qnrB, qnrC, qnrD, qnrS, qepA or oqxAB, but one of the isolates was identified as an AAC (6')-Ib-cr producer. Phylogenetic grouping, multi-locus sequence typing and serotyping showed that this isolate belonged to phylogenetic group A, sequence type 167 and untypable serogroup. To our knowledge, this is the first report of the aac (6')-Ib-cr gene in bacteria from food-producing animals in Japan.

Diversity of plasmid replicons encoding the bla(CMY-2) gene in broad-spectrum cephalosporin-resistant Escherichia coli from livestock animals in Japan.

Broad-spectrum cephalosporin (BSC) resistance has increased in Escherichia coli isolates from broiler chickens in Japan since 2004. The purpose of this study was to understand the epidemiology of BSC-resistant E. coli in livestock animals. Among 3274 E. coli isolates from 1767 feces of apparently healthy animals on 1767 farms between 2004 and 2009, 118 ceftiofur (CTF)-resistant isolates (CTF MIC ≥4 µg/mL) were identified on 74 farms. After elimination of apparently clonal isolates from a single animal, 75 selected CTF-resistant isolates (62 isolates from 61 broiler chickens, 10 isolates from 10 layer chickens, two isolates from two cows, and one isolate from a pig) were characterized. The bla(CMY-2) gene was most frequently detected in 50 isolates, followed by bla(CTX-M) (CTX-M-2: six isolates; CTX-M-14: four isolates; CTX-M-25: two isolates; CTX-M-1: one isolate) and bla(SHV) (SHV-12: seven isolates; SHV-2, SHV-2a, SHV-5: one isolate each). In particular, 42 of 62 broiler chicken isolates harbored bla(CMY-2). Pulsed-field gel electrophoresis analyses using XbaI revealed divergent profiles among the BSC-resistant isolates. The incompatibility groups of bla(CMY-2) plasmids from 34 of the 42 broiler chicken isolates belonged to IncIγ (10 isolates), IncA/C (nine isolates), IncB/O (seven isolates) and IncI1 (six isolates), or were nontypeable (two isolates). Co-transmission of resistance to non-ß-lactam antibiotics was observed in transconjugants with IncA/C plasmids, but not with IncI1, IncIγ, and IncB/O plasmids except for one isolate with IncB/O. Our findings suggest that the bla(CMY-2) gene is a key player in BSC-resistant E. coli isolates and that coselection is unlikely to be associated with the abundance of bla(CMY-2) plasmids, except for IncA/C plasmids.

Relationships between mutant prevention concentrations and mutation frequencies against enrofloxacin for avian pathogenic Escherichia coli isolates.

Clinical efficacies of mutant prevention concentration (MPC) and mutant selection window (MSW) hypotheses have been evaluated for human clinical isolates. We tested the MSW hypothesis by evaluating the relationships between MPCs and mutation frequencies against enrofloxacin for avian pathogenic Escherichia coli (APEC) isolates. Mutation frequencies of strains with MPC:MIC ratios of 8 to 16 were significantly higher than those of strains with an MPC:MIC ratio of 4. Mutation frequencies and MPCs of serogroup O2 strains were lower than those of the other strains; these results may correlate with the absence of fluoroquinolone-resistant O2 strains. Our results support the MSW hypothesis that the range of the MSW is involved in selection of resistant mutants.

Evaluation of transferability of R-plasmid in bacteriocin-producing donors to bacteriocin-resistant recipients.

Bacteriocin-producing Escherichia coli (donors) rapidly kill conventional recipient E. coli DH5α in conjugation experiments. To evaluate plasmid transferability of bacteriocin-producing donors, we established 2 different bacteriocin-resistant mutants derived from E. coli DH5α and used them as recipients. When the bacteriocin-resistant mutants were used in conjugation experiments, the transconjugant recovery from 20 bacteriocin-producing donors increased from 5% (1/20) to 65% (13/20), and the transfer frequencies increased. These results showed that bacteriocins inhibited the transfer of the R-plasmid from bacteriocin-producing donors. Thus, application of bacteriocin-resistant recipients might aid the evaluation of the potential transferability of plasmids from bacteriocin-producing donors.

Prevalence and mechanism of antimicrobial resistance in Staphylococcus aureus isolates from diseased cattle, swine and chickens in Japan.

Antimicrobial administration is essential for the control and treatment of diseases in animals, but the emergence and prevalence of antimicrobial-resistant Staphylococcus aureus is a significant concern during animal production. Here we investigated the antimicrobial susceptibility of S. aureus from diseased food-producing animals and molecularly characterized the methicillin-resistant and fluoroquinolone-resistant isolates. A total of 290 S. aureus isolates obtained from cattle (n=246), swine (n=16), and chickens (n=28) between 2003 and 2009 were examined for antimicrobial susceptibility against 9 antimicrobials using an agar dilution method. Resistance to penicillin (PC) was most frequently found (24.8%), followed by oxytetracycline (OTC, 10.0%), dihydrostreptomycin (4.1%), erythromycin (EM, 3.1%), enrofloxacin (ERFX, 2.1%), and kanamycin (1.7%). The PC resistance rate was significantly higher in swine than in cattle (P<0.01) and chickens (P<0.01). The resistance rates to OTC, EM and ERFX were significantly higher in swine and chickens than in cattle (P<0.05). Methicillin-resistant S. aureus (MRSA) was recovered from milk derived from a cow with mastitis in 2003; sequence type 8, SCCmec type IV and spa type t024. In the six ERFX-resistant strains isolated after 2003, amino acid substitutions in ParC with/without GyrA were detected. As the prevalence of MRSA and FQ-resistant S. aureus in the animals should be noticed, continuous monitoring is necessary to control resistance to clinically important antimicrobials in S. aureus from food-producing animals.