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Aim The aim of this study was to determine whether the practice of co-grazing with cattle and wild life constitutes a risk of transmission of antibiotic resistant bacteria to wild ungulates. Methods and Results Faecal samples were collected from buffalo (n = 35), wildebeest (n = 40), zebra (n = 40) and cattle (N = 20) from Mikumi National Park, Tanzania (MNP), where cattle is prohibited and from Ngorongoro Conservation Area (NCA) where co-grazing is practiced. The number of coliforms and enterococci resistant to selected antibiotics was determined. Wild life generally harboured higher number of resistant Escherichia coli and Enterococci than cattle, but with no general influence in wild life of co-grazing with cattle. Vancomycin-resistant Enterococci were detected in wild life samples, and E. coli resistant to cefotaxime and enrofloxacin were observed among isolates from all wild life, but not from cattle. Culture independent estimates of the number of sulII gene copies obtained by qPCR did not differ between wild life from the two sample sites, while tetW was significantly higher in samples from MPN than from NCA. Conclusions Antibiotic resistant bacteria were not more frequently found in ungulates grazing together with cattle than ungulates without this interaction. Significance and Impact of the Study This study did not indicate that transmission of antibiotic resistant bacteria is a frequent event following co-grazing of wild life and cattle.

This study provides an estimate of antimicrobial resistance in intestinal indicator bacteria from humans (n = 97) and food animals (n = 388) in Tanzania. More than 70% of all fecal samples contained tetracycline (TE), sulfamethoxazole (STX), and ampicillin (AMP)-resistant coliforms, while cefotaxime (CTX)-resistant coliforms were observed in 40% of all samples. The average Log

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCC mec ), the genetic element carrying the β-lactam resistance gene mecA . However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCC mec , we sequenced the genomes of 106 isolates, comprising S. sciuri ( n = 76), Staphylococcus vitulinus ( n = 18), and Staphylococcus fleurettii ( n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCC mec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCC mec : S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCC mec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus .

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for Lawsonia intracellularis -induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested. IMPORTANCE Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.

The aim of this study was to evaluate the population dynamics of CTX-M-producing Enterobacteriaceae in individual pigs on a farm positive for CTX-M-14-producing Escherichia coli . Fecal samples were collected once around the farrowing time from five sows and four times along the production cycle from two of their respective offspring. Multiple colonies per sample were isolated on cefotaxime-supplemented MacConkey agar with or without prior enrichment, resulting in 98 isolates identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry and tested for bla CTX-M . CTX-M-positive isolates ( n = 86) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harboring bla CTX-M were characterized in 22 representative isolates by replicon typing and restriction fragment length polymorphism. Based on the PFGE results, all individuals shed unrelated CTX-M-14-producing E. coli strains during the course of life. Concomitant shedding of CTX-M-2/97-producing Proteus mirabilis or Providencia rettgeri was observed in two sows and two offspring. At least two genetically unrelated CTX-M-producing E. coli strains were isolated from approximately one-fourth of the samples, with remarkable differences between isolates obtained by enrichment and direct plating. A clear decrease in strain diversity was observed after weaning. Dissemination of bla CTX-M-14 within the farm was attributed to horizontal transfer of an IncK plasmid that did not carry additional resistance genes and persisted in the absence of antimicrobial selective pressure. Assessment of strain diversity was shown to be influenced by the production stage from which samples were collected, as well as by the isolation method, providing useful information for the design and interpretation of future epidemiological studies of CTX-M-producing Enterobacteriaceae in pig farms.

Staphylococcus pseudintermedius is a commensal and a common opportunistic pathogen causing mainly infections of the integumentary system in dogs. The recent emergence of multidrug-resistant S. pseudintermedius isolates, in particular methicillin-resistant strains (MRSP) is a threat to small animal health and highlights the need for antimicrobial resistance surveillance to detect trends and potentially perform timeous interventions. We systematically reviewed 202 published articles to investigate temporal changes in antimicrobial resistance in clinical and commensal S. pseudintermedius isolated from dogs in 27 countries between 1980 and 2013. Resistance to the most common antimicrobials tested for in published studies and important for the treatment of staphylococcal infections in dogs were assessed separately for methicillin resistant (MRSP) and methicillin susceptible (MSSP) isolates. Stratified by MSSP and MRSP, no significant increases in antimicrobial resistance were observed over time, except for the penicillinase-labile penicillins (penicillin and ampicillin) among MSSP. However, in recent years, a few studies have reported higher-level of resistance to amikacin, gentamicin and enrofloxacin amongst MSSP. The review highlights inconsistencies between studies as a result of several factors, for example the use of different antimicrobial susceptibility testing methods and interpretation criteria. We recommend that data on susceptibility in important companion animal pathogens are collected and presented in a more harmonized way to allow more precise comparison of susceptibility patterns between studies. One way to accomplish this would be through systematic surveillance either at the country-level or at a larger scale across countries e.g. EU level.

Denmark is a country with high prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 in pigs. Even though pig farming is regarded as the main source of human infection or colonization with MRSA CC398, 10-15% of the human cases appear not to be linked to pigs. Following the recent reports of MRSA CC398 in horses in other European countries and the lack of knowledge on S. aureus carriage in this animal species, we carried out a study to investigate whether horses constitute a reservoir of MRSA CC398 in Denmark, and to gain knowledge on the frequency and genetic diversity of S. aureus in horses, including bothmethicillin-resistant and -susceptible S. aureus (MSSA). Nasal swabs were collected from 401 horses originating from 74 farms, either at their farms or prior to admission to veterinary clinics. Following culture on selectivemedia, species identification by MALDI-TOF MS and MRSA confirmation by standard PCR-based methods, S. aureus and MRSA were detected in 54 (13%) and 17 (4%) horses originating from 30 (40%) and 7 (9%) farms, respectively. Based on spa typing, MSSA differed genetically from MRSA isolates. The spa type prevalent among MSSA isolates was t127 (CC1), which was detected in 12 horses from 11 farms and represents the most common S. aureus clone isolated from human bacteremia cases in Denmark. Among the 17 MRSA carriers, 10 horses from three farms carried CC398 t011 harboring the immune evasion cluster (IEC), four horses from two farms carried IEC-negative CC398 t034, and three horses from two farms carried a mecC-positive MRSA lineage previously associated with wildlife and domestic ruminants (CC130 t528). Based on whole-genome phylogenetic analysis of the 14 MRSA CC398, t011 isolates belonged to the recently identified horse-adapted clone in Europe and were closely related to human t011 isolates fromthree Danish equine veterinarians, whereas t034 isolates belonged to pig-adapted clones. Our study confirms that horses carry an equine-specific clone of MRSA CC398 that can be transmitted to veterinary personnel, and reveals that these animals are exposed to MRSA and MSSA clones that are likely to originate from livestock and humans, respectively.

Current knowledge on extended-spectrum beta-lactamases (ESBLs) in animals is based largely on cross-sectional studies and qualitative data. The aim of this longitudinal study was to elucidate carriage proportions and fecal counts of ESBL-producing Escherichia coli in pigs during the production cycle. At each of three ESBL-positive single-sited farrow-to-finisher pig farms (farms A, B, and C) included in the study, individual fecal samples were taken from 17 to 20 sows 1 week before farrowing and from 2 piglets of each sow's litter four times from birth to slaughter (as piglets, weaners, and finishers). Cefotaxime (CTX)-resistant coliforms in feces were counted on MacConkey agar containing 2 μg/ml CTX and characterized for the presence of ESBL-encoding genes by PCR and sequencing. CTX-M-positive pigs were detected in all age groups at farms A ( bla CTX-M-9 group, compatible with bla CTX-M-14/17 ) and B ( bla CTX-M-1 group, compatible with bla CTX-M-1/61 ), whereas only three weaners were positive at farm C ( bla CTX-M-1 group, compatible with bla CTX-M-1/61 ). A significant decrease in carriage was detected during the production cycle, with on average 50% carriage immediately after birth, 58% just before weaning, 29% during weaning, and 12% during finishing. The observed reduction in numbers of CTX-M-positive pigs was accompanied by a significant reduction in mean fecal counts of CTX-resistant coliforms from ∼10 7 CFU/g in piglets to ∼10 3 CFU/g in finishers ( P < 0.001). These findings provide novel information about the epidemiology of ESBLs at the farm level and have important implications for assessments of risks of meat contamination during slaughter.

Denmark is a country with high prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 in pigs. Even though pig farming is regarded as the main source of human infection or colonization with MRSA CC398, 10–15% of the human cases appear not to be linked to pigs. Following the recent reports of MRSA CC398 in horses in other European countries and the lack of knowledge on S. aureus carriage in this animal species, we carried out a study to investigate whether horses constitute a reservoir of MRSA CC398 in Denmark, and to gain knowledge on the frequency and genetic diversity of S. aureus in horses, including both methicillin-resistant and -susceptible S. aureus (MSSA). Nasal swabs were collected from 401 horses originating from 74 farms, either at their farms or prior to admission to veterinary clinics. Following culture on selective media, species identification by MALDI-TOF MS and MRSA confirmation by standard PCR-based methods, S. aureus and MRSA were detected in 54 (13%) and 17 (4%) horses originating from 30 (40%) and 7 (9%) farms, respectively. Based on spa typing, MSSA differed genetically from MRSA isolates. The spa type prevalent among MSSA isolates was t127 (CC1), which was detected in 12 horses from 11 farms and represents the most common S. aureus clone isolated from human bacteremia cases in Denmark. Among the 17 MRSA carriers, 10 horses from three farms carried CC398 t011 harboring the immune evasion cluster (IEC), four horses from two farms carried IEC-negative CC398 t034, and three horses from two farms carried a mecC-positive MRSA lineage previously associated with wildlife and domestic ruminants (CC130 t528). Based on whole-genome phylogenetic analysis of the 14 MRSA CC398, t011 isolates belonged to the recently identified horse-adapted clone in Europe and were closely related to human t011 isolates from three Danish equine veterinarians, whereas t034 isolates belonged to pig-adapted clones. Our study confirms that horses carry an equine-specific clone of MRSA CC398 that can be transmitted to veterinary personnel, and reveals that these animals are exposed to MRSA and MSSA clones that are likely to originate from livestock and humans, respectively.

Lameness is the most commonly reported health problem in horses, and lameness investigations which include local anaesthetic injections are routinely performed by equine practitioners. Through this process, bacteria can enter the tissues perforated by the needle and may cause local infections at the injection site. The objective of this in vitro study was to investigate if local anaesthetics at concentrations available in commercially available solutions could inhibit growth and/or kill bacteria that could be inoculated into the synovial space or soft tissues during injection. This study evaluated the antimicrobial activity of the local anaesthetics bupivacaine, lidocaine and mepivacaine against 40 equine clinical bacterial isolates of the Actinobacillus, Corynebacterium, Enterobacter, Escherichia, Pseudomonas, Rhodococcus, Staphylococcus and Streptococcus genera. Minimum inhibitory and minimum bactericidal concentrations (MICs and MBCs) were determined by the broth microdilution method. Clinically applied concentrations of bupivacaine, lidocaine, and mepivacaine inhibited visual growth of 93%, 93%, and 80% of isolates tested, respectively. For the majority (80%) of the inhibited isolates, the concentrations were also bactericidal. The tested local anaesthetics possessed antimicrobial activity against equine pathogens at concentrations that are routinely applied in clinical cases. However, this antimicrobial activity should not discourage antiseptic preparation prior to local anaesthetic injections.

Multidrug-resistant bacteria pose a serious threat to public health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers were found to display activity against Gram-negative multidrug-resistant bacteria. In the present work, the influence of hydrophobicity, fluorination, and distribution of cationic/hydrophobic residues on antimicrobial, hemolytic, and cytotoxic properties of α-peptide/β-peptoid hybrids were investigated. An array of 22 peptidomimetics was tested. Analogues with enhanced hydrophobicity were found to exhibit increased activity against Gram-positive bacteria. Incorporation of fluorinated residues into the peptidomimetics conferred increased potency against Gram-positive bacteria, while hemolytic properties and activity against Gram-negative bacteria depended on the degree and type of fluorination. Generally, shorter oligomers were less potent than the corresponding longer analogues. However, some short analogues exhibited equal or higher antimicrobial activity. The alternating hydrophobic/cationic design proved superior to other distribution patterns of cationic side chains and hydrophobic moieties.

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the β-lactam resistance gene mecA. However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus.

Background: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged globally in companion animals in the last decade. In Europe, the multidrug-resistant sequence type (ST) 71 is widespread, but recently other clones have appeared. The objective of this study was to examine genotypic diversity and antimicrobial resistance of clinical MRSP isolates obtained from dogs, including dogs sampled on multiple occasions, in Denmark over a six-year period. For that purpose a total of 46 clinical MRSP isolates obtained from 36 dogs between 2009 and 2014 were subjected to antimicrobial susceptibility testing, multilocus-sequence typing (MLST) and SCCmec typing.Results: Twenty-three sequence types were identified with ST71, mostly associated with SCCmec II-III, as the most common occurring in 13 dogs. Among the remaining 33 isolates, 19 belonged to clonal complex (CC) 258 comprising ST258-SCCmec IV and its single-and double-locus variants. These were susceptible to 4-7 of the 22 antibiotics tested, whereas CC71 isolates were susceptible to only 2-5 antibiotics. Clone-specific differences were especially pronounced for fluoroquinolones and aminoglycosides with most CC71 isolates being resistant and almost all CC258 isolates being susceptible. Sixteen of the 19 CC258 isolates had oxacillin MICs of 0.5 g/L, whereas MICs for CC71 isolates were consistently above 4 g/L. Four of five dogs representing multiple isolates had distinct STs on different sampling events.Conclusions: The overall genotypic diversity of MRSP is high in Denmark indicating multiple acquisitions of SCCmec into distinct clones, and mutational evolution, which appears to be particularly rapid for certain ancestral clones such as ST258. ST71-SCCmec II-III is the most common MRSP lineage and is typically multidrug-resistant. CC258-SCCmec IV isolates, which emerged in Denmark since 2012, display susceptibility to a wider range of antimicrobials. The isolation of distinct STs in individual dogs over time suggests repeated exposure or short-term genetic evolution of MRSP clones within patients. Background: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged globally in companion animals in the last decade. In Europe, the multidrug-resistant sequence type (ST) 71 is widespread, but recently other clones have appeared. The objective of this study was to examine genotypic diversity and antimicrobial resistance of clinical MRSP isolates obtained from dogs, including dogs sampled on multiple occasions, in Denmark over a six-year period. For that purpose a total of 46 clinical MRSP isolates obtained from 36 dogs between 2009 and 2014 were subjected to antimicrobial susceptibility testing, multilocus-sequence typing (MLST) and SCCmec typing.Results: Twenty-three sequence types were identified with ST71, mostly associated with SCCmec II-III, as the most common occurring in 13 dogs. Among the remaining 33 isolates, 19 belonged to clonal complex (CC) 258 comprising ST258-SCCmec IV and its single-and double-locus variants. These were susceptible to 4-7 of the 22 antibiotics tested, whereas CC71 isolates were susceptible to only 2-5 antibiotics. Clone-specific differences were especially pronounced for fluoroquinolones and aminoglycosides with most CC71 isolates being resistant and almost all CC258 isolates being susceptible. Sixteen of the 19 CC258 isolates had oxacillin MICs of 0.5 g/L, whereas MICs for CC71 isolates were consistently above 4 g/L. Four of five dogs representing multiple isolates had distinct STs on different sampling events.Conclusions: The overall genotypic diversity of MRSP is high in Denmark indicating multiple acquisitions of SCCmec into distinct clones, and mutational evolution, which appears to be particularly rapid for certain ancestral clones such as ST258. ST71-SCCmec II-III is the most common MRSP lineage and is typically multidrug-resistant. CC258-SCCmec IV isolates, which emerged in Denmark since 2012, display susceptibility to a wider range of antimicrobials. The isolation of distinct STs in individual dogs over time suggests repeated exposure or short-term genetic evolution of MRSP clones within patients.

The objective of this longitudinal study was to investigate the occurrence and genetic background of faecal Escherichia coli resistant to cefotaxime (CTX) in horses receiving broad-spectrum antimicrobial prophylaxis after admission to a veterinary teaching hospital. The ten horses enrolled in the study were treated with cefquinome either alone (n=4) or in combination with metronidazole (n=3) or other antimicrobial agents (n=3). CTX-resistant coliforms in faeces collected before, during and after treatment were quantified on selective MacConkey agar supplemented with CTX, and a colony isolated randomly from each positive sample was characterized by pulsed-field gel electrophoresis, and by PCR detection and sequencing of bla(TEM), bla(SHV), bla(CTX-M) and bla(CMY). All horses were negative for CTX-resistant coliforms at admission but became positive within the first three days of treatment. The average faecal densities of CTX-resistant coliforms increased significantly following antimicrobial prophylaxis (P

Cephalexin is a first generation cephalosporin commonly used in dogs for treatment of pyoderma. The objective of this study was to evaluate the in vivo effects of cephalexin on selection of Escherichia coli resistant to extended-spectrum cephalosporins. A cohort study was conducted on 13 dogs presenting clinical signs of pyoderma and treated with cephalexin and 22 healthy dogs that had not been treated with antibiotics during the previous six months. Selective plating of faeces on MacConkey agar plates containing cefotaxime (CTX) yielded growth of CTX-resistant E. coli for eight of the 13 treated dogs (62%), whereas no growth was observed for any of the control dogs (Fisher exact test, P

SUMMARYWithin-household transmission ofEscherichia colimay contribute to the pathogenesis of urinary tract infection but understanding of transmission is limited by the lack of longitudinal data on individual shedding patterns. In this study, faecalE. coliwas isolated over 6 months from 18 humans and 13 dogs in eight households. Typing 322E. coliisolates by amplified fragment length polymorphism showed high overall diversity as indicated by the average diversity index (0·66). However, individual shedding patterns varied considerably: two persons carried a single residentE. coliclone throughout the study whereas distinct clones were isolated from other individuals on each sampling time. Nineteen clones were shared within six of the eight households and seven of these clones were shared between humans and dogs. The frequent sharing of clones belonging to phylotypes B2 (n=7) or D (n=4) supports the hypothesis that urovirulentE. coliare transmitted between household members, including dogs, or may be acquired by a common source such as food.

The use of voided urine specimens for bacteriological culture in dogs is discouraged because contamination from external genitalia could lead to misinterpretation of laboratory results. Quantitative culturing and defining significant bacteriuria could increase the usefulness of voided specimens. However, limited evidence exists for the cut-offs currently recommended. The aim of this study was to evaluate the accuracy of current veterinary cut-off values for significant bacteriuria in voided canine urine. A secondary aim was to investigate if accuracy improved when applying qualitative criteria used in humans. Paired urine specimens were collected by both cystocentesis and voiding, and quantitative bacteriological cultures were performed within the same day. Cystocentesis was used as the reference standard with a cut-off for significant bacteriuria of ≥1000 colony forming units (CFU)/mL. Voided specimens were compared to cystocentesis using: (1) the veterinary cut-off of ≥100,000 CFU/mL; and (2) various cut-offs depending on qualitative criteria (sex, clinical signs and complicating factors), adapted from human guidelines. Ninety-four dogs with suspected urinary tract infection (UTI) were included for analysis. The veterinary cut-off yielded an accuracy of 94% with a sensitivity and specificity of 94% (95% confidence intervals [CI] 0.81, 0.99) and 94% (95% CI 0.86, 0.98), respectively. Applying the human guidelines did not improve overall accuracy (89%), and yielded a sensitivity and specificity of 97% (95% CI 0.86, 1.00) and 86% (95% CI 0.77, 0.92), respectively. The veterinary cut-off value of ≥100,000 CFU/mL for voided urine is appropriate for determining significant bacteriuria in the majority of dogs with suspected UTI if specimens are refrigerated and cultured on the day of collection.

Background: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are increasingly reported in dogs. The objective of this study was to provide data on the prevalence of ESBL-producing E. coli in dog faecal deposits in public gardens. Results: A total of 209 faecal deposits collected in nine public gardens in Copenhagen, Denmark were screened by selective enrichment followed by plating on MacConkey agar supplemented with cefotaxime. Presumptive ESBLproducing E. coli were confirmed by MALDI-TOF MS and polymerase chain reaction (PCR) for detection of common cefotaxime resistance determinants (bla TEM , bla SHV , bla CTX-M and bla CMY-2 ). ESBL-producers were further characterized by multilocus sequence typing (MLST) and antimicrobial susceptibility testing using broth microdilution. Plasmids harbouring ESBL genes were characterized by S1 nuclease pulsed field gel electrophoresis, PCR-based replicon typing, and pMLST. Cefotaxime-resistant E. coli were detected in four (1.9 %) samples. Three samples harboured CTX-M-1-producing isolates, and one sample contained two CTX-M-15-producing isolates displaying distinct colony morphology. All isolates belonged to distinct sequence types (STs), including one E. coli lineage previously associated to a humanspecific pathotype (ST59). bla CTX-M-1 was carried on IncI1 plasmids classified as ST3 or ST58 by pMLST, whereas bla CTXM-15 was located on IncF/Y and non-typeable plasmids in the two strains isolated from the same sample. Conclusions: The study shows that dog faeces are a vector for dissemination of CTX-M-producing E. coli within urban areas. The risk derived from human exposure to dog faeces in public gardens depends on the prevalence of these bacteria in the local dog population as well as on the owners’ practice to remove and dispose their dog’s faeces.

Preterm infants are susceptible to infection and necrotizing enterocolitis (NEC) and are often treated with antibiotics. Simultaneous administration of enteral and parenteral antibiotics during the first days after preterm birth prevents formula-induced NEC lesions in pigs, but it is unknown which administration route is most effective. We hypothesized that only enteral antibiotics suppress gut bacterial colonization and NEC progression in formula-fed preterm pigs. Caesarean-delivered preterm pigs (90–92% of gestation) were fed increasing amounts of infant formula from birth to day 5 and given saline (CON) or antibiotics (ampicillin, gentamicin, and metronidazole) via the enteral (ENT) or parenteral (PAR) route ( n = 16–17). NEC lesions, intestinal morphology, function, microbiology, and inflammatory mediators were evaluated. NEC lesions were completely prevented in ENT pigs, whereas there were high incidences of mild NEC lesions (59–63%) in CON and PAR pigs ( P < 0.001). ENT pigs had elevated intestinal weight, villus height/crypt depth ratio, and goblet cell density and reduced gut permeability, mucosal adherence of bacteria, IL-8 levels, colonic lactic acid levels, and density of Gram-positive bacteria, relative to CON pigs ( P < 0.05). Values in PAR pigs were intermediate with few affected parameters (reduced lactic acid levels and density and adherence of Gram-positive bacteria, relative to CON pigs, P < 0.05). There was no evidence of increased antimicrobial resistance following the treatments. We conclude that enteral, but not parenteral, administration of antibiotics reduces gut bacterial colonization, inflammation, and NEC lesions in newborn, formula-fed preterm pigs. Delayed colonization may support intestinal structure, function, and immunity in the immediate postnatal period of formula-fed preterm neonates.

The complex relationship between drug concentrations and bacterial growth rates require not only the minimum inhibitory concentration but also other parameters to capture the dynamic nature of the relationship. To analyse this relationship between tetracycline concentration and growth of Escherichia coli representative of those found in the Danish pig population, we compared the growth of 50 randomly selected strains. The observed net growth rates were used to describe the in vitro pharmacodynamic relationship between drug concentration and net growth rate based on E max model with three parameters: maximum net growth rate (α max ); concentration for a half-maximal response (E max ); and the Hill coefficient (γ). The net growth rate in the absence of antibiotic did not differ between susceptible and resistant isolates (P = 0.97). The net growth rate decreased with increasing tetracycline concentrations, and this decline was greater in susceptible strains than resistant strains. The lag phase, defined as the time needed for the strain to reach an OD600 value of 0.01, increased exponentially with increasing tetracycline concentration. The pharmacodynamic parameters confirmed that the $$ \alpha_{max} $$ between susceptible and resistant strains in the absence of a drug was not different. EC 50 increased linearly with MIC on a log–log scale, and γ was different between susceptible and resistant strains. The in vitro model parameters described the inhibition effect of tetracycline on E. coli when strains were exposed to a wide range of tetracycline concentrations. These parameters, along with in vivo pharmacokinetic data, may be useful in mathematical models to predict in vivo competitive growth of many different strains and for development of optimal dosing regimens for preventing selection of resistance.

PCR mapping of staphylococcal cassette chromosome mec type IVa and adjacent mobile elements in 94 methicillin-resistant Staphylococcus aureus (MRSA) strains identified two primary structures (A and B) that could be further classified into two (A1 and A2) and five (B1 to B5) variants, primarily based on structural differences in the orfX -J3 region. While spa type t008 (USA300) invariably contained the A variants, other spa types belonging to clonal complex 8 and unrelated lineages generally contained B variants. These findings have important implications for the typing and identification of MRSA strains containing B variants.

Minimum bactericidal concentrations (MBCs) of a commercial ear antiseptic containing chlorhexidine 0.15% and Tris-EDTA (Otodine) were determined by broth microdilution for 150 isolates representing the most common pathogens associated with canine otitis. The microorganisms were classified into three groups according to their levels of susceptibility. The most susceptible group included Staphylococcus pseudintermedius, Malassezia pachydermatis, Streptococcus canis and Corynebacterium auriscanis, which were generally killed by 1 : 64 dilution of the antiseptic product (MBC = 23/0.8 microg/mL of chlorhexidine/Tris-EDTA). The most resistant organism was Proteus mirabilis, which survived up to 1 : 8 dilution of the product (MBC = 375/12 microg/mL). Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus displayed intermediate MBCs ranging between 188/6 and 47/1.5 microg/mL. Interestingly, S. pseudintermedius was more susceptible than S. aureus, and no significant difference was observed between meticillin-resistant and meticillin-susceptible isolates within each species, indicating that antiseptic use is unlikely to co-select for meticillin resistance. Although the concentrations required for killing (MBCs) varied considerably with microorganism type, the combination of chlorhexidine 0.15% and Tris-EDTA was active against all the pathogens most commonly involved in canine otitis.

Ampicillin resistance is a marker for hospital-associated Enterococcus faecium . Feces from 208 dogs were selectively screened for the occurrence of ampicillin-resistant E. faecium (AREF). AREF was detected in 42 (23%) of 183 dogs screened in a cross-sectional study in the United Kingdom and in 19 (76%) of 25 dogs studied longitudinally in Denmark. AREF carriage was intermittent in all dogs studied longitudinally. Multilocus sequence typing of 63 canine AREF isolates revealed the presence of 13 distinct sequence types. Approximately 76% of the isolates belonged to hospital-adapted clonal complex 17 (CC17), including those of sequence types ST-78 and ST-192, which are widespread in European and Asian hospitals. Longitudinal screening of 18 healthy humans living in contact with 13 of the dogs under study resulted in the identification of a single, intermittent CC17 carrier. This person carried one of the sequence types (ST-78) recovered from his dog. Based on PCR and Southern hybridization analyses, the putative virulence gene cluster from orf903 to orf907 was widespread in canine AREF isolates (present in 97%), whereas orf2351 (present in 26% of isolates) and orf2430 (present in 31%) were strongly associated with CC17-related sequence types ( P < 0.05). Surprisingly, esp and hyl were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates generally differed from those previously described for clinical human isolates. The results indicate that dogs are frequent carriers of CC17-related lineages and may play a role in the spread of this nosocomial pathogen. The distinctive virulence and antimicrobial resistance profiles observed among canine AREF isolates raise interesting questions about the origin and evolution of the strains causing human infections.

National surveillance programs on antimicrobial usage and antimicrobial resistance in animals have been established in various countries but few of them include bacteria from pets. The objectives of this study were to assess the prevalence of antimicrobial resistance in healthy dogs and to search for resistance phenotypes of clinical relevance. Escherichia coli and Enterococcus spp. were isolated from faecal swabs obtained from 127 dogs. Disk diffusion was used to measure antimicrobial susceptibility in 117 Escherichia coli, 10 Enterococcus faecium and 51 Enterococcus faecalis of canine origin. Resistance was relatively low compared with food animal species in Denmark. All Escherichia coli isolates were susceptible to broad-spectrum aminopenicillins, third generation cephalosporins and fluoroquinolones. Despite the low prevalence of resistance, statistical analysis of questionnaire data revealed a significant association (p=0.02) between recent antimicrobial treatment and resistance in Escherichia coli. Interestingly, two dogs were found to shed Enterococcus faecium resistant to ampicillin. Multilocus sequence typing of these isolates indicated that the two isolates belonged to sequence types associated with human nosocomial infections, and one (ST-192) was genetically related to human epidemic clonal complex 17. The detection of ampicillin-resistant Enterococcus faecium warrants further studies on the prevalence of these bacteria in dogs and on the possible implications to both animal and human health. The results suggest that distinct methods for detection and assessment of antimicrobial resistance in animals should be considered depending on the target animal species and the purposes of the study.

Human ( n = 33) and canine ( n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains, indicating that dogs may not be the main source of human infection.

Background There is a lack of studies comparing topical antiseptics to systemic antibiotics in the treatment of canine superficial pyoderma. Hypothesis/Objectives To compare the efficacy of topical chlorhexidine with systemic amoxicillin–clavulanic acid for the treatment of canine superficial pyoderma. Animals A randomized controlled trial was conducted in dogs with superficial pyoderma. Group T (n = 31) was treated topically with 4% chlorhexidine digluconate shampoo (twice weekly) and solution (once daily) for 4 weeks. Group S (n = 20) was treated orally with amoxicillin–clavulanic acid (25 mg/kg) twice daily for 4 weeks. Methods Bacterial culture and susceptibility testing were performed on clinical specimens collected before treatment. Severity of lesions and number of intracellular bacteria were evaluated using four-point scales to calculate a total pyoderma score for each dog. Pruritus was assessed by owners using a visual analog scale (range 0–10). Scores were analysed for statistical differences between groups T and S. Results Staphylococcus pseudintermedius was isolated from 48 dogs, including eight meticillin-resistant strains (MRSP). Although the number of dogs was small, no significant differences in pyoderma and pruritus scores were observed between groups throughout the study except for day 1, when group S had a significantly higher total score than group T (P = 0.03). Treatment with chlorhexidine products resulted in resolution of clinical signs in all dogs including those infected with MRSP. Conclusion and clinical importance Topical therapy with chlorhexidine digluconate products may be as effective as systemic therapy with amoxicillin–clavulanic acid. This finding supports the current recommendations to use topical antiseptics alone for the management of superficial pyoderma.

BACKGROUND Antimicrobial resistance may be overestimated in bacterial isolates from clinical samples because veterinarians often submit samples in cases of treatment failure or recurrent cases, which are more frequently associated with resistant strains. HYPOTHESIS/OBJECTIVES To assess to what extent the prevalence of antimicrobial resistance in Staphylococcus pseudintermedius isolated from first-time superficial pyoderma differs from canine skin isolates from clinical samples with unknown clinical background. ANIMALS Two study groups were enrolled in Denmark between March 2012 and October 2013: 57 dogs with first-time superficial pyoderma and no prior antimicrobial treatment (Group A); and 289 different dogs for which skin specimens were submitted for culture during the study (Group B). METHODS One S. pseudintermedius isolate from each dog was confirmed by MALDI-TOF mass spectrometry and tested for antimicrobial susceptibility by broth microdilution. Resistance levels in the two groups were compared by Fisher's exact test. RESULTS Clindamycin resistance was less frequent in Group A (14%) than in Group B (27%) (P = 0.02). Similar trends were observed for amoxicillin-clavulanic acid (1.8 versus 4.8%), chloramphenicol (8.8 versus 14.5%), enrofloxacin (1.8 versus 3.5%), oxacillin (1.8 versus 4.8%) and trimethoprim/sulfamethoxazole (3.5 versus 5.9%). Oxacillin resistance was significantly associated with resistance to six of seven non-β-lactams. CONCLUSIONS AND CLINICAL IMPORTANCE The prevalence of lincosamide resistance is markedly influenced by patients' clinical and antimicrobial treatment history. To limit selection of multidrug-resistant bacteria, lincosamides are appropriate empirical choices for treatment of first-time superficial pyoderma even though resistance is not infrequent. Culture and susceptibility testing are, however, recommended for all pyoderma patients.

Campylobacter jejuni was recovered from four dogs (11%) and four cats (33%) living with Danish human patients infected with C. jejuni . Pulsed-field gel electrophoresis (PFGE) analysis revealed the occurrence of the same quinolone-resistant strain in a girl and her dog. C. jejuni isolates with closely related (>95% similarity) PFGE profiles occurred in humans and pets from different Danish counties.

Background Staphylococcus pseudintermedius is an opportunistic pathogen that causes mainly skin infections in dogs. Although vertical transmission of S. pseudintermedius strains from dam to offspring has been reported, the persistence of the dam's strains in offspring over long periods of time is virtually unknown. Hypothesis/objectives The aim of this study was to investigate to what extent S. pseudintermedius clones resident on the dam may be transmitted to and persist in offspring. Animals A total of 18 dogs and 50 of their offspring, which had been separated from their mother for between 1 month and 9 years, were enrolled in the study. Methods Mouth and perineal swabs taken from all dogs were subjected to selective enrichment and plating on blood agar. Presumptive S. pseudintermedius colonies were confirmed by a species-specific PCR and typed by pulsed-field gel electrophoresis. Results Staphylococcus pseudintermedius was isolated from both the dam and at least one offspring in 12 of the 18 dog families. Pulsed-field gel electrophoresis confirmed carriage of indistinguishable or closely related strains in the dam and offspring within four families, despite the fact that they had lived separately for between 2 and 48 months before sampling. Conclusions and clinical importance Our results indicate that S. pseudintermedius clones that are transmitted from the dam to the puppies around birth may persist in the offspring for long periods of time. The study contributes to the current understanding of the mechanisms of spread of S. pseudintermedius in dogs and highlights the need for research to elucidate the role of bacterial and host genetic factors in colonization.

The current knowledge of in vitro adherence of Staphylococcus pseudintermedius to canine corneocytes is limited to comparative analyses between strains, staphylococcal species or corneocytes collected from different breeds, body sites and hosts. However, the role played by colonization status of corneocyte donors remains unknown. The aim of this study was to evaluate the adherence properties of commensal S. pseudintermedius strains to corneocytes collected from dogs with different colonization status. For this purpose, corneocytes were collected from five dogs that were classified as persistently colonized (D1 and D2), intermittently colonized (D3 and D4) or non-colonized (D5) on the basis of the results of a previous longitudinal study. Adherence to corneocytes originating from each of the five dogs was assessed by an in vitro adhesion assay using four genetically unrelated strains isolated from the colonized dogs (S1 to S4). Irrespective of their host of origin, all strains adhered significantly better to corneocytes from D1 and D2 than to corneocytes from D3, D4 and D5 (P

Thirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avian E. coli belonging to sequence type complex 10 and of IncN and IncI1 plasmids carrying bla CTX-M or bla SHV .

BACKGROUND: Urinary tract infection (UTI) is a common reason for antimicrobial prescription in dogs and cats. The objective of this study was to optimize and evaluate a culture-based point-of-care test for detection, identification and antimicrobial susceptibility testing of bacterial uro-pathogens in veterinary practice.METHODS: Seventy-two urine samples from dogs and cats with suspected UTI presenting to seven veterinary facilities were used by clinical staff and an investigator to estimate sensitivity and specificity of Flexicult Vet A compared to laboratory reference standards for culture and susceptibility testing. Subsequently, the test was modified by inclusion of an oxacillin-containing compartment for detection of methicillin-resistant staphylococci. The performance of the modified product (Flexicult Vet B) for susceptibility testing was evaluated in vitro using a collection of 110 clinical isolates.RESULTS: Bacteriuria was reported by the laboratory in 25 (35 %) samples from the field study. The sensitivity and specificity of Flexicult Vet A for detection of bacteriuria were 83 and 100 %, respectively. Bacterial species were correctly identified in 53 and 100 % of the positive samples by clinical staff and the investigator, respectively. The susceptibility results were interpreted correctly by clinical staff for 70 % of the 94 drug-strain combinations. Higher percentages of correct interpretation were observed when the results were interpreted by the investigator in both the field (76 %) and the in vitro study (94 %). The most frequent errors were false resistance to β-lactams (ampicillin, amoxicillin-clavulanate and cephalotin) in Escherichia coli for Flexicult Vet A, and false amoxicillin-clavulanate resistance in E. coli and false ampicillin susceptibility in Staphylococcus pseudintermedius for Flexicult Vet B. The latter error can be prevented by categorizing staphylococcal strains growing in the oxacillin compartment as resistant to all β-lactams.CONCLUSIONS: Despite the shortcomings regarding species identification by clinical staff and β-lactam susceptibility testing of E. coli, Flexicult Vet B (commercial name Flexicult(®) Vet) is a time- and cost-effective point-of-care test to guide antimicrobial choice and facilitate implementation of antimicrobial use guidelines for treatment of UTIs in small animals, provided that clinical staff is adequately trained to interpret the results and that clinics meet minimum standards to operate in-house culture.