Ørjan Olsvik

Continuing high prevalence of VanA‐type vancomycin‐resistant enterococci on Norwegian poultry farms three years after avoparcin was banned

Avoparcin was used as a feed additive in Norwegian broiler and turkey production from 1986 until 1995. It was banned due to the selection of VanA‐type vancomycin‐resistant enterococci (VRE) in animal husbandry and to reduce the potential for human exposure to VRE. The aim of the present study was to investigate the prevalence of VRE carriage in Norwegian poultry farmers and their poultry three years after avoparcin was banned. Corresponding faecal samples from poultry and humans on farms where avoparcin had previously been used (exposed farms, n = 73) and farms where avoparcin had never been used (unexposed farms, n = 74) were analysed for the presence of VRE. For each farm, one sample from the poultry house and one sample from the farmer were obtained. VRE were isolated from 72 of 73 (99%) and eight of 74 (11%) poultry samples from exposed and unexposed farms, respectively. VRE were isolated from 13 of 73 (18%) and one of 74 (1%) farmer samples from exposed and unexposed farms, respectively. All VRE isolates were highly resistant to vancomycin and possessed the vanA gene, as shown by PCR. The high prevalence of VRE is in accordance with previous Norwegian studies, and shows a remarkable stability of the VanA resistance determinant in an apparently non‐selective environment.

Immunomagnetic separation of Salmonella from foods

Salmonella could be separated from different inoculated foods using antibody-coated immunomagnetic beads. When applied on suitable foods, the immunomagnetic separation technique showed a sensitivity of 10-20 Salmonella cells/g of the original sample. The technology appeared less useful for some food items.

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.

Pathogenic Escherichia coli found in food

The bacteria constituting the species Escherichia coli are commonly found in the intestinal flora of man and animals, and were until late 1950s recognized as non-pathogenic normal cohabitants. However, certain strains might induce disease, and E. coli should therefore be regarded as a potential pathogenic organism. The pathogenic strains can cause distinct disease syndrome as different diarrheal diseases, wound infections, meningitis, septicemia, artherosclerosis, hemolytic uremic syndrome and immunological diseases such as reactive and rheumatoid arthritis. Several different groups of diarrhea-inducing strains are known. The enterotoxigenic E. coli (ETEC) strains produce one or more of toxins from the heat-labile and the heat-stable enterotoxin families. These strains possess specific adhesion fimbria for intestinal attachment and colonization. Some enteropathogenic E. coli strains (EPEC) produce one or more of the cytotoxins, but adhere also to intestinal cells interfering with the electrolyte transport system. The group of strains possessing invasive properties are designated enteroinvasive E. coli (EIEC). Recently, the enterohemorrhagic E. coli (EHEC) strains have been identified and shown to produce one or more of the cytotoxins (vero-cytotoxins, shiga-like toxins). Food originating from warm-blooded animals may be contaminated with E. coli, but contamination from human sources are more common for food involved in outbreak of disease. In general, strains causing disease in animals do possess other colonization factors than those found on human pathogenic strains. EIEC strains are, like Shigella, only known to induce disease in man. However, both healthy and sick cattle are suspected to be a major reservoir for EHEC strains, and several outbreaks have been associated with consumption of meat or meat products. Cheeses have been the source of outbreaks of both ETEC and EIEC in Europe and the USA, while water seems to be a major source for the different diarrheic E. coli strains affecting children and tourists in the 3rd world. Strains causing non-enteric disease are less known as being transmitted to humans with food as a vector, but the importance of some of these diseases, should implicate further research on what role food plays in spreading these organisms. The recipient of the potential pathogenic E. coli through food, the humans, are also of different risk of contracting diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

Stability, persistence, and evolution of plasmid-encoded VanA glycopeptide resistance in enterococci in the absence of antibiotic selection in vitro and in gnotobiotic mice.

Long-term persistence of VanA glycopeptide-resistant enterococci (GRE) has been observed in the absence of antibiotic selection. In the present study, we examined fitness parameters of a glycopeptide-susceptible Enterococcus faecium parent strain and its plasmid-mediated, VanA-resistant derivative before and after 1,000 generations in serial transfer broth cultures with or without antibiotic selection. With the exception of the vanA-containing plasmid, the strains were otherwise isogenic. The stability of the plasmid-encoded vanA resistance determinant was also investigated in vitro and in gnotobiotic mice. Competition experiments revealed that GRE with newly acquired VanA resistance had a 4% reduction in fitness relative to their susceptible parental counterpart. The relative difference in competitive fitness between resistant and susceptible strains was not significantly changed after 1,000 generations. Environmental adaptation was observed in all strains and exceeded the biological cost of resistance. Thus, the evolved VanA-resistant E. faecium populations out-numbered their unevolved ancestral susceptible E. faecium strain in mixed cultures, but remained less competitive than the evolved parent. The glycopeptide resistance determinant was similarly stably maintained during long-term colonization in gnotobiotic mice without antibiotic selection. In vivo vanA plasmid transfer was observed. The results suggest that environmental adaptation, in vivo gene transfer, and plasmid maintenance system(s) favor long-term VanA GRE persistence without antibiotic selection and compensate for the biological costs of possessing the resistance genes.

Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.

SEROSURVEY FOR ORTHOPOXVIRUSES IN RODENTS AND SHREWS FROM NORWAY

Two hundred and twenty one blood samples representing eight different rodent species and the common shrew (Sorex araneus), collected in Norway between 1993 and 1995, were examined for anti-orthopoxvirus antibodies by a competition enzyme linked imunnosorbent assay (ELISA) and, when possible, an indirect immunofluorescence assay. The serological results indicated that the bank vole (Clethrionomys glareolns), woodmouse (Apodemus sylvaticus) and Norway lemming (Lemmus lemmus) may be reservoir species for orthopoxviruses in Norway, with antibody prevalences of 17 (12/69), 30 (24/81) and 56% (19/34), respectively. Orthopoxvirus infection in lemmings has not been reported previously. On some other small rodent species such as field voles (Microtus agrestis), common rats (Rattus norvegicus), and common shrews, seropositive individuals were detected. However, the total number of tested animals was low, and the role of these species in the epidemiology of orthopoxvirus infections remains unclear. Attempts to isolate orthopoxviruses from these small mammals failed, although orthopoxvirus specific DNA sequences were detected previously in the same animals by the polymerase chain reaction (PCR). The serological results were compared with and discussed in the context of the occurrence of orthopoxvirus-specific DNA sequences, and it is concluded that orthopoxviruses are widely distributed among wildlife in Norway.

Educational intervention for parents and healthcare providers leads to reduced antibiotic use in acute otitis media.

We used a controlled before-and-after design with the aims of reducing both the total consumption of antibiotics and the use of broad-spectrum antibiotics against acute otitis media (AOM), and to study to what extent prescriptions for antibiotics against AOM were dispensed. Information on evidence-based treatment of uncomplicated AOM was provided to doctors and nurses, and written guidelines were implemented. Pamphlets and oral information concerning symptomatic treatment and the limited effect of antibiotic use in AOM were given to parents. Eligible patients were 819 children aged 1-15 y. The proportion of patients receiving a prescription for antibiotics was reduced from 90% at baseline to 74% during the study period. The proportion of prescriptions for penicillin V increased from 72% at baseline to 85% during the study period. There were no significant changes at the control site. The proportion of dispensed prescriptions was 70% both at baseline and during the study period. Educational efforts reduced the total consumption of antibiotics and the use of broad-spectrum antibiotics for AOM in children aged 1-15 y at an emergency call service. Data on antibiotic use in AOM based only on prescribing overestimates the use of antibiotics.

Rapid PCR Detection of the Methicillin Resistance Gene, mecA, on the Hands of Medical and Non-medical Personnel and Healthy Children and on Surfaces in a Neonatal Intensive Care Unit

The hands of medical personnel are the chief vectors for transmission of antibiotic-resistant bacteria and probably serve as an important reservoir for antibiotic resistance genes in hospitals. In this survey we examined different reservoirs of the methicillin resistance gene, mecA, using a simplified PCR method. Samples (n = 151) were taken from the hands of medical and non-medical personnel and healthy children and from surfaces in a neonatal intensive care unit (NICU). We also performed sampling from 4 different body sites in 5 of the medical personnel. Fifteen out of 16 nurses (94%) from the ICU carried the mecA gene on their hands, whereas only 35% of the paediatric nurses were mecA-positive. Of all medical personnel, 44% carried the mecA gene on their hands. There was a significant difference (p < 0.015) between medical and non-medical personnel in terms of the carriage rate of mecA. Four samples from surfaces in a NICU--2 ventilators, 1 bench and 1 telephone--were positive for mecA. Our results are comparable with those from previous studies on reservoirs of methicillin-resistant coagulase-negative staphylococci using conventional culture techniques.The hands of medical personnel are the chief vectors for transmission of antibiotic-resistant bacteria and probably serve as an important reservoir for antibiotic resistance genes in hospitals. In this survey we examined different reservoirs of the methicillin resistance gene, mecA, using a simplified PCR method. Samples (n = 151) were taken from the hands of medical and non-medical personnel and healthy children and from surfaces in a neonatal intensive care unit (NICU). We also performed sampling from 4 different body sites in 5 of the medical personnel. Fifteen out of 16 nurses (94%) from the ICU carried the mecA gene on their hands, whereas only 35% of the paediatric nurses were mecA-positive. Of all medical personnel, 44% carried the mecA gene on their hands. There was a significant difference (p < 0.015) between medical and non-medical personnel in terms of the carriage rate of mecA. Four samples from surfaces in a NICU - 2 ventilators, 1 bench and 1 telephone - were positive for mecA. Our results are comparable with those from previous studies on reservoirs of methicillin-resistant coagulase-negative staphylococci using conventional culture techniques.

Low Faecal Carrier Rate of Vancomycin Resistant Enterococci in Norwegian Hospital Patients

The faecal carrier rate of vancomycin resistant enterococci (VRE) was surveyed among 616 patients in selected departments of 7 Norwegian hospitals. One Enterococcus gallinarum isolate harbouring a vanB2 element was recovered from a child with malignant disease treated with vancomycin and ceftazidime. No vancomycin resistant Enterococcus faecalis or Enterococcus faecium were detected and no VRE isolates of the VanA type were identified. The low level of VRE carriage corresponds to the limited use of glycopeptide antibiotics for human therapeutic purposes in Norway. It indicates a low risk of acquiring VRE infections in Norwegian hospitals.