The association between measurements of antimicrobial use and resistance in the faeces microbiota of finisher batches.

The objectives were to present three approaches for calculating antimicrobial (AM) use in pigs that take into account the rearing period and rearing site, and to study the association between these measurements and phenotypical resistance and abundance of resistance genes in faeces samples from 10 finisher batches. The AM use was calculated relative to the rearing period of the batches as (i) 'Finisher Unit Exposure' at unit level, (ii) 'Lifetime Exposure' at batch level and (iii) 'Herd Exposure' at herd level. A significant effect on the occurrence of tetracycline resistance measured by cultivation was identified for Lifetime Exposure for the AM class: tetracycline. Furthermore, for Lifetime Exposure for the AM classes: macrolide, broad-spectrum penicillin, sulfonamide and tetracycline use as well as Herd Unit Exposure for the AM classes: aminoglycoside, lincosamide and tetracycline use, a significant effect was observed on the occurrence of genes coding for the AM resistance classes: aminoglycoside, lincosamide, macrolide, ß-lactam, sulfonamide and tetracycline. No effect was observed for Finisher Unit Exposure. Overall, the study shows that Lifetime Exposure is an efficient measurement of AM use in finisher batches, and has a significant effect on the occurrence of resistance, measured either by cultivation or metagenomics.

High frequencies of antibiotic resistance genes in infants' meconium and early fecal samples.

The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to ß-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies' mothers, as well as a mother-infant series including meconium, fecal samples collected through the infant's 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds' samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant's GIT as a noteworthy reservoir of this gene.

The use of third and fourth generation cephalosporins affects the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli in Danish pig herds.

Extended-spectrum cephalosporinase resistance is currently the fastest emerging antimicrobial resistance problem worldwide; however, evidence documenting the effect of potential risk factors is limited. The main objective of this study was to investigate the effect of using third and fourth generation cephalosporins on the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli (ESC-Ec) in Danish pig herds. Conventional, integrated, medium to large herds were selected based on information from the Danish Central Husbandry Register and two groups were formed based on the use of third and fourth generation cephalosporins within a specified period, namely, 20 herds with no cephalosporin use (non-exposed) and 19 herds with frequent use (exposed). Data on prescribed antimicrobials were obtained from the National database (VetStat). Management data were obtained through a questionnaire. At the herd level, three pooled faecal samples were collected from sows with their piglets (farrowing pens), weaners, and finishers. ESC-Ec were then identified using selective enrichment. Because several of the herds only had a low number of weaners and/or finishers, analysis was only performed on samples from the farrowing pens. Logistic regression showed a significant effect of using cephalosporins-III/IV on the occurrence of ESC-Ec in the farrowing pens, even when adjusted for use of other antimicrobials 1 year prior to sampling. No confounding effect was identified in relation to management data. The relative risk ESC-Ec in exposed compared to non-exposed was 4.7 (95% confidence interval 2.0-11.5), confirming that regular use of cephalosporins-III/IV was a significant risk factor for the occurrence of ESC-Ec.

Comparison of air samples, nasal swabs, ear-skin swabs and environmental dust samples for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds.

To identify a cost-effective and practical method for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds, the relative sensitivity of four sample types: nasal swabs, ear-skin (skin behind the ears) swabs, environmental dust swabs and air was compared. Moreover, dependency of sensitivity on within-herd prevalence was estimated. spa-typing was applied in order to study strain diversity. The sensitivity of one air sample was equal to the sensitivity of ten pools of five nasal swabs and relatively independent of within-herd prevalence [predicted to be nearly perfect (99%) for within-herd prevalence ⩾25%]. The results indicate that taking swabs of skin behind the ears (ten pools of five) was even more sensitive than taking nasal swabs (ten pools of five) at the herd level and detected significantly more positive samples. spa types t011, t034 and t4208 were observed. In conclusion, MRSA detection by air sampling is easy to perform, reduces costs and analytical time compared to existing methods, and is recommended for initial testing of herds. Ear-skin swab sampling may be more sensitive for MRSA detection than air sampling or nasal swab sampling.

Effect of subinhibitory concentrations of four commonly used biocides on the conjugative transfer of Tn916 in Bacillus subtilis.

OBJECTIVES: Large amounts of biocides are used to reduce and control bacterial growth in the healthcare sector, food production and agriculture. This work explores the effect of subinhibitory concentrations of four commonly used biocides (ethanol, hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite) on the conjugative transposition of the mobile genetic element Tn916. METHODS: Conjugation assays were carried out between Bacillus subtilis strains. The donor containing Tn916 was pre-exposed to subinhibitory concentrations of each biocide for a defined length of time, which was determined by an analysis of the transcriptional response of the promoter upstream of tet(M) using ß-glucuronidase reporter assays. RESULTS: Ethanol significantly (P = 0.01) increased the transfer of Tn916 by 5-fold, whereas hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite did not significantly affect the transfer frequency. CONCLUSIONS: These results suggest that exposure to subinhibitory concentrations of ethanol may induce the transfer of Tn916-like elements and any resistance genes they contain.

Identification of Tet 39, a novel class of tetracycline resistance determinant in Acinetobacter spp. of environmental and clinical origin.

A novel tetracycline resistance determinant named Tet 39 was found in unrelated Acinetobacter strains isolated from freshwater trout farms (n=4) and sewage (n=6) in Denmark, and from a clinical specimen in the Netherlands (n=1). The determinant was located on transferable plasmids and consisted of tetA(39), most likely conferring resistance by active efflux, and a putative repressor gene tetR(39).

Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark.

Enterococcus faecalis and E. faecium isolated from humans in the community (98 and 65 isolates), broilers (126 and 122), and pigs (102 and 88) during 1998 were tested for susceptibility to 12 different antimicrobial agents and for the presence of selected genes encoding resistance using PCR. Furthermore, the presence of vancomycin resistant enterococci was examined in 38 human stool samples using selective enrichment. Widespread resistance to chloramphenicol, macrolides, kanamycin, streptomycin, and tetracycline was found among isolates from all three sources. All E. faecium isolates from humans and pigs were susceptible to avilamycin, whereas 35% of isolates from broilers were resistant. All E. faecium isolates from humans were susceptible to vancomycin, whereas 10% and 17% of isolates from broilers and pigs, respectively, were resistant. A vancomycin resistant E. faecium isolate was found in one of the 38 human fecal samples examined using selective enrichment. All vancomycin resistant isolates contained the vanA gene, all chloramphenicol resistant isolates the cat(pIP501) gene, and all five gentamicin resistant isolates the aac6-aph2 gene. Sixty-one (85%) of 72 erythromycin resistant E. faecalis examined and 57 (90%) of 63 erythromycin resistant E. faecium isolates examined contained ermB. Forty (91%) of the kanamycin resistant E. faecalis and 18 (72%) of the kanamycin resistant E. faecium isolates contained aphA3. The tet(M) gene was found in 95% of the tetracycline resistant E. faecalis and E. faecium isolates of human and animal origin, examined. tet(K) was not observed, whereas tet(L) was detected in 17% of tetracycline resistant E. faecalis isolates and in 16% of the E. faecium isolates. tet(O) was not detected in any of the isolates from pigs, but was observed in 38% of E. faecalis isolates from broilers, in two E. faecalis isolates from humans and in three E. faecium isolates from broilers. tet(S) was not detected among isolates from animals, but was observed in 31% of E. faecalis and one E. faecium isolate from humans. This study showed a frequent occurrence of antimicrobial resistance and the presence of selected resistance genes in E. faecalis and E. faecium isolated from humans, broilers and pigs. Differences in the occurrence of resistance and tetracycline resistance genes were observed among isolates from the different sources. However, similar resistance patterns and resistance genes were detected frequently indicating that transmission of resistant enterococci or resistance genes takes place between humans, broilers, and pigs.