Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses.

Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

Evidence for transmission between humans and the environment of a nosocomial strain of Enterococcus faecium.

An ampicillin- and ciprofloxacin-resistant Enterococcus faecium (ARE) strain, named FMSE1, with a characteristic biochemical phenotype, was in a recent study found to dominate among faecal ARE isolates from patients in several Swedish hospitals. In the present study, the prevalence of this strain among 9676 enterococcal isolates from healthy children, hospital sewage, urban sewage, surface water, slaughtered animals (broilers, pigs and cattle) and pig faeces and manure was investigated. Enterococcal isolates having the same biochemical phenotype as the FMSE1 were most common in samples of hospital sewage (50%), surface water (35%), treated sewage (28%) and untreated sewage (17%), but rare in samples from healthy children (0.8%) and animals (2%). PFGE typing of FMSE1-like isolates from hospital sewage indicated that they were closely related to the nosocomial FMSE1 strain. Thus, this study indicated a possible transmission route for nosocomial E. faecium from patients in hospitals to hospital sewage and urban sewage, and further via treatment plants to surface water and possibly back to humans. This proposed route of circulation of drug-resistant enterococci might be further amplified by antibiotic usage in human medicine. In contrast, such transmission from food animals seems to play a negligible role in Sweden.

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.

Harmonization of antimicrobial susceptibility testing among veterinary diagnostic laboratories in the five Nordic countries.

A total of 100 bacterial strains (25 Escherichia coli, 25 Salmonella enterica, 25 Staphylococcus aureus, and 25 Enterococcus strains) and four reference strains were tested for susceptibility toward 8-12 antimicrobial agents in 12 veterinary diagnostic laboratories in the five Nordic countries using routine methodology. In addition, the 25 Enterococcus strains were identified to species level. A total of 22,598 (97.2%) out of 23,259 test results were in accordance when the data were categorized as susceptible or resistant. When the reported results were categorized according to the National Committee of Clinical Laboratory Standards breakpoints, the percentage of concordant results increased to 98.4% and the performance between laboratories varied between 94.2 and 99.4% concordant results. For E. coli, S. aureus, and Salmonella, all laboratories except one had more than 97% concordant results, whereas for Enterococcus spp., two laboratories had less than 90% concordant results. Susceptibility testing of Salmonella to fluoroquinolones gave rise to almost 0.5% nonconcordant results and susceptibility testing of S. aureus to vancomycin resulted in that 1.8% of the strains were incorrectly reported as vancomycin resistant. Ten laboratories identified the Enterococcus spp. to species level. All five Enterococcus faecium and 10 Enterococcus faecalis selected from the strain collection at the Danish Veterinary Institute were correctly identified by all laboratories, whereas some problems were observed identifying other enterococcal species. This study showed a good performance and agreement in antimicrobial susceptibility testing at the 12 participating laboratories and that surveillance data covering susceptibility test results of E. coli, S. aureus, and Salmonella from animals in the Nordic countries are comparable. But it also showed that some aspects can be improved. In addition, the study showed that the different laboratories are capable of identifying E. faecalis and E. faecium.

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.

High prevalence of vancomycin-resistant enterococci in Swedish sewage.

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.