Unexpected common occurrence of transferable extended spectrum cephalosporinase-producing Escherichia coli in Swedish surface waters used for drinking water supply.

The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans, food-producing animals and food world-wide. However, the occurrence and impact of these so-called extended spectrum cephalosporinase (ESC)-producing Enterobacteriaceae in aquatic environments are poorly documented. This study investigated the occurrence, concentrations and characteristics of ESC-producing E. coli (ESC-Ec) in samples of surface water collected at five Swedish water treatment plants that normally have relatively high prevalence and concentration of E. coli in surface water. ESC-Ec was found in 27 of 98 surface water samples analysed. All but two positive samples were collected at two of the water treatment plants studied. The ESC-Ec concentration, 1-3cfu/100mL, represented approximately 4% of the total amount of E. coli in the respective surface water sample. In total, 74% of the isolates were multi-resistant, but no isolate was resistant to carbapenems. Six types of ESBL/pAmpC genes were found in the 27 E. coli isolates obtained from the positive samples, of which four (blaCTX-M-15, blaCMY-2, blaCTX-M-1 and blaCTX-M-14) were found during the whole sampling period, in samples taken at more than one water treatment plant. In addition, the genes were situated on various types of plasmids and most E. coli isolates were not closely related with regard to MLST types. The combinations of ESBL/pAmpC genes, plasmids and E. coli isolates were generally similar to those found previously in healthy and sick individuals in Sweden. In conclusion, the occurrence of ESC-Ec in Swedish surface water shows that resistant bacteria of clinical concern are present in aquatic environments even in a low-prevalence country such as Sweden.

Evidence of household transfer of ESBL-/pAmpC-producing Enterobacteriaceae between humans and dogs - a pilot study.

BACKGROUND: Extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESCRE) are an increasing healthcare problem in both human and veterinary medicine. The spread of ESCRE is complex with multiple reservoirs and different transmission routes. The aim of this study was to investigate if ESCRE carriage in dogs is more prevalent in households with a known human carrier, compared to households where humans are known to be negative for ESCRE. Identical ESCRE strains in humans and dogs of the same household would suggest a possible spread between humans and dogs. METHODS: Twenty-two dog owners with a positive rectal culture for ESCRE each collected a rectal sample from their dog. In addition, a control group of 29 healthy dog owners with a documented negative rectal culture for ESCRE each sampled their household dog. Samples were cultivated for ESCRE using selective methods. In households where both humans and dogs carried ESCRE, isolates were further analysed for antimicrobial susceptibility by disc diffusion or microdilution and for genotype and genetic relatedness using molecular methods. RESULTS: In 2 of 22 households studied, identical ESCRE strains with respect to bacterial species, antibiogram, genotype, and MLVA type were found in humans and dogs. The ESCRE found in the two households were ESBL-producing E. coli with the resistance gene blaCTX-M-27 and AmpC-producing E. coli with blaCMY-2, blaTEM-1. ESCRE were not found in dogs in the control group. CONCLUSIONS: In households where humans are carrying ESCRE, identical strains were to a limited extent found also in household dogs, indicating a transfer between humans and dogs. In contrast, ESCRE were not found in dogs in households without human carriers.

Microevolution during an Anthrax outbreak leading to clonal heterogeneity and penicillin resistance.

Anthrax is a bacterial disease primarily affecting grazing animals but it can also cause severe disease in humans. We have used genomic epidemiology to study microevolution of the bacterium in a confined outbreak in cattle which involved emergence of an antibiotic-resistant phenotype. At the time of death, the animals contained a heterogeneous population of Single Nucleotide Variants (SNVs), some being clonal but most being subclonal. We found that independent isolates from the same carcass had similar levels of SNV differences as isolates from different animals. Furthermore the relative levels of subclonal populations were different in different locations in the same carcass. The heterogeneity appeared to be derived in part from heterogeneity in the infectious dose. The resistance phenotype was linked to clonal mutations in an anti-sigma factor gene and in one case was preceded by an acquisition of a hypermutator phenotype. In another animal, small subclonal populations were observed with counteracting mutations that had turned off the resistance genes. In summary, this study shows the importance of accounting for both acquired and inherited heterogeneity when doing high-resolution infection tracing and when estimating the risks associated with penicillin treatment.

Escherichia coli with extended-spectrum beta-lactamases or transferable AmpC beta-lactamases and Salmonella on meat imported into Sweden.

The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans and animals world-wide. Their occurrence in food-producing animals suggests that meat is a possible link between the two populations. This study investigated the occurrence and characteristics of Salmonella and ESBL- or pAmpC-producing E. coli in 430 samples of beef, pork and broiler meat imported into Sweden, in order to provide data required for assessing the potential public health risk of these bacteria in food. Depending on region of origin, ESBL/pAmpC-producing E. coli were found in 0-8% of beef samples, 2-13% of pork samples and 15-95% of broiler meat samples. The highest prevalence was in South American broiler meat (95%), followed by broiler meat from Europe (excluding Denmark) (61%) and from Denmark (15%). Isolates from meat outside Scandinavia were generally defined as multiresistant. A majority of the ESBL/pAmpC genes were transferable by conjugation. Bla(CTX-M-2) and bla(CTX-M-8) were the dominant genes in E. coli from South American broiler meat, whereas bla(CMY-2) and bla(CTX-M-1) dominated in European meat. The majority of bla(CMY-2) and bla(CTX-M-1) were situated on plasmids of replicon type incK and incI1, respectively. The same combinations of ESBL/pAmpC genes and plasmids have been described previously in clinical human isolates. Salmonella was found in five samples tested, from European pork and broiler meat. No Salmonella isolate was resistant to third-generation cephalosporins. In conclusion, meat imported into Sweden, broiler meat in particular, is a potential source of human exposure to ESBL- and pAmpC-producing E. coli.

Intranasal fentanyl for analgesia in the paediatric emergency department.

Intranasal opiate analgesia is the first choice in children presenting to paediatric emergency departments with severe pain. A prospective observational study was performed which showed that intranasal fentanyl is a safe effective alternative to diamorphine.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study.

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions.

Vancomycin-resistant enterococci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 microg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.

Comparison of enterococcal populations in animals, humans, and the environment--a European study.

The objectives of the present study were to generate knowledge of enterococcal populations in the food chain, by studying the population structure (in measures of abundance and diversity) among enterococci in different geographical regions and in different parts of the food chain, as well as the similarities between different enterococcal populations. Altogether, 2868 samples were collected from humans (healthy and hospitalised individuals and clinical isolates), animals (slaughterhouse carcasses and farm animals), and the environment (pig farms, sewage, and surface water) in four European countries-Sweden, Denmark, UK, and Spain. The samples were characterised with regard to presence and numbers of enterococci, and eight (for faecal samples) or 24 (for environmental samples) isolates per sample were phenotyped and preliminarily identified with the PhP-RF system. In total, more than 20,000 isolates were typed. A majority of the samples (77%) showed the presence of presumed enterococci. The diversities of enterococci in environmental samples were generally high, and also faecal samples normally showed presence of more than one enterococcal strain. The most common species found were Enterococcus faecium (33%), E. faecalis (29%), and E. hirae (24%), but different enterococcal populations differed in their species distribution. Clinical isolates, hospitalised patients, and hospital sewage in Sweden showed a clear dominance of E. faecalis (80%, 57%, and 54%, respectively) whereas healthy individuals and urban sewage contained less E. faecalis (39% and 40%, respectively). The species distribution among isolates from slaughterhouses varied between animal species and also between countries, but E. faecalis seemed to be mainly associated with broiler, and E. hirae with cattle and pigs. The results from the study have indicated a simplified method to study the diversity of bacterial populations. Instead of collecting many samples and analysing one or a few isolates per sample, it is possible to collect fewer samples and analyse several isolates per sample. Both approaches yielded similar information on the diversity of the populations. Another useful information was that since samples from hospital sewage, urban sewage, and manure contained enterococcal populations that reflected those in faecal samples of hospitalised patients, healthy humans, and animals, respectively, such samples may be used as pooled faecal samples and may replace cumbersome samplings from many individuals.

A tissue cage model in calves for studies on pharmacokinetic/pharmacodynamic interactions of antimicrobials.

An in vivo model for studies of pharmacokinetic/pharmacodynamic (PK/PD) interactions of antimicrobials was developed. Tissue cages with a constant surface area but with different volumes were implanted in calves and infected with Mannheimia haemolytica. Penicillin was injected directly into the cages. With this procedure, different concentration-time profiles could be simulated so that the effect of a range of PK/PD indices on the infection could be monitored. The area under the curve to minimum inhibitory concentration (MIC) and time above MIC were equally predictive for effect, but Cmax to MIC was not. If drug dosages in relation to the MIC of strains used for infection are optimised, the model offers an interesting alternative to explore relevant factors for drug dosage optimisation.

Pharmacokinetic/pharmacodynamic relationship of danofloxacin against Mannheimia haemolytica in a tissue-cage model in calves.

OBJECTIVE: To evaluate an experimental model of the pharmacokinetic/pharmacodynamic (PK/PD) relationship of danofloxacin against Mannheimia haemolytica infection, using subcutaneously implanted tissue cages in calves. METHODS: Tissue cages implanted subcutaneously in calves were infected with M. haemolytica and different concentration-time profiles of danofloxacin were simulated. Drug concentrations and bacterial counts were monitored over time and various PK/PD parameters calculated. RESULTS: By using different types of cage and various doses, a range of PK/PD indices were simulated. The PK/PD index that best predicted the antimicrobial effect was the AUC/MIC ratio. The magnitude of this index needed for near-maximum effect (80%), assessed using the area under the bacterial kill curve to 48 h, was 244 h. CONCLUSIONS: The model described enabled different concentration-time profiles to be simulated, and PK/PD interactions to be studied in the presence of the host's defences. The validity of this model needs to be confirmed by clinical studies, but the results suggest that it may be a useful intermediary step between in vitro and clinical studies.

Antimicrobial resistance among enterococci from pigs in three European countries.

Enterococci from pigs in Denmark, Spain, and Sweden were examined for susceptibility to antimicrobial agents and copper and the presence of selected resistance genes. The greatest levels of resistance were found among isolates from Spain and Denmark compared to those from Sweden, which corresponds to the amounts of antimicrobial agents used in food animal production in those countries. Similar genes were found to encode resistance in the different countries, but the tet(L) and tet(S) genes were more frequently found among isolates from Spain. A recently identified transferable copper resistance gene was found in all copper-resistant isolates from the different countries.