Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae.

Bacterial antimicrobial resistance (AMR) is constantly evolving and horizontal gene transfer through plasmids plays a major role. The identification of plasmid characteristics and their association with different bacterial hosts provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of AMR determinants. Molecular identification of plasmid and strain genotypes elicits a distinction between spread of AMR genes by plasmids and dissemination of these genes by spread of bacterial clones. For this reason several methods are used to type the plasmids, e.g. PCR-based replicon typing (PBRT) or relaxase typing. Currently, there are 28 known plasmid types in Enterobacteriaceae distinguished by PBRT. Frequently reported plasmids [IncF, IncI, IncA/C, IncL (previously designated IncL/M), IncN and IncH] are the ones that bear the greatest variety of resistance genes. The purpose of this review is to provide an overview of all known AMR-related plasmid families in Enterobacteriaceae, the resistance genes they carry and their geographical distribution.

Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut.

Animal health benefits from a stable intestinal homeostasis, for which proper development and functioning of the intestinal microbiota and immune system are essential. It has been established that changes in microbial colonization in early life (the first 2 wk post hatch) impacts the functioning of the adult gut and the associated crosstalk between microbiota and intestinal mucosal cells. The aim of the present study was to study the effect of the administration of antibiotics later in life (d 15 to 20 post hatch) on microbiota and immune parameters. For this purpose, chickens received from 15 d post hatch during 5 d amoxicillin or enrofloxacin through their drinking water. Before and at 6, 16, and 27 d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. At 6 d after the start of the administration of the antibiotics, the composition and diversity of the microbiota were affected significantly (P < 0.05), but this change was small and observed only temporarily since differences disappeared at 16 d after initiating treatment with amoxillin and at 27 d after starting treatment with enrofloxacin. Intestinal morphology and development were not visibly affected since there were no differences between villus/crypt ratios and numbers of PAS+ and PCNA+ cells in the duodenum and jejunum at any time point. At 16 d after the start of antibiotic administration, the number of CD4+ T-cells and CD8+ T-cells in the duodenum was lower compared to the control animals; however, this difference was not significant. At some time points, significant differences (P < 0.05) were observed among the groups to locally expressed IL-8, IL-1ß, IFN-γ, IL-2, and IL-4 mRNA. However, this effect was not long lasting, as differences that were observed at 16 d after starting the treatment had disappeared at 27 d after treatment was started. The results of this study indicate that later in the broiler's life, antibiotics only temporarily affect intestinal microbial and immune parameters.

ESBL/AmpC-producing Enterobacteriaceae in households with children of preschool age: prevalence, risk factors and co-carriage.

OBJECTIVES: ESBL/AmpC-producing Enterobacteriaceae are an emerging public health concern. As households with preschool children may substantially contribute to the community burden of antimicrobial resistance, we determined the prevalence, risk factors and co-carriage of ESBL/AmpC-producing bacteria in preschool children and their parents. METHODS: From April 2013 to January 2015, each month 2000 preschool children were randomly selected from Dutch population registries. The parents were invited to complete an epidemiological questionnaire and to obtain and send a faecal sample from the selected child and from one parent. Samples were tested for ESBL/AmpC-producing bacteria. Logistic regression was used to identify risk factors for ESBL/AmpC carriage in children and parents, and findings were internally validated by bootstrapping. RESULTS: In total, 1016 families were included and ESBL/AmpC prevalence was 4.0% (95% CI 3.2%-5.0%); 3.5% (95% CI 2.5%-4.8%) in children and 4.5% (95% CI 3.4%-6.0%) in parents. Attending a daycare centre (DCC) was the only significant risk factor for children (OR 2.1, 95% CI 1.0-4.3). For parents, the only significant risk factor was having one or more children attending DCCs (OR 2.2, 95% CI 1.2-4.8). For parents of ESBL/AmpC-positive children the OR for ESBL/AmpC carriage was 19.7 (95% CI 9.2-42.4). Co-carriage of specific ESBL/AmpC genotypes in child and parent occurred more often than expected by chance (14.6% versus 1.1%, P < 0.001). CONCLUSIONS: In this study, intestinal carriage with ESBL/AmpCs was detected in ∼4% of households with preschool children. DCC attendance was a risk factor in both children and parents and co-carriage of specific genotypes frequently occurred in child-parent pairs. These findings suggest household transmission or/and family-specific exposure to common sources of ESBL/AmpC-producing bacteria.

Reduction of veterinary antimicrobial use in the Netherlands. The Dutch success model.

Use of antimicrobials in animals poses a potential risk for public health as it contributes to the selection and spread of antimicrobial resistance. Although knowledge of the negative consequences of extensive antimicrobial use in humans and animals accumulated over the decades, total therapeutic antimicrobial use in farm animals in the Netherlands doubled between 1990 and 2007. A series of facts and events formed a window of opportunity to reduce antimicrobial use in farm animals. The recent discovery of significant reservoirs of antimicrobial-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase-producing bacteria (ESBL) in farm animals, with potential public health implications, combined with an increasing lack of confidence of the public in intensive livestock industries, and discrepancy between the very low antimicrobial use in humans and high use in animals, resulted in intensive collaboration between the government, veterinary professional organizations and important stakeholders within the livestock sector. A combination of compulsory and voluntary actions with clear reduction goals resulted in a 56% reduction in antimicrobial use in farm animals in the Netherlands between 2007 and 2012 and aims at accomplishing a 70% reduction target in 2015. This article describes and analyses the processes and actions behind this transition from an abundant antimicrobial use in farm animals towards a more prudent application of antimicrobials in farm animals in the Netherlands.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: prevalence, risk factors and molecular characteristics.

OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.

Antimicrobial resistance in commensal Escherichia coli in veal calves is associated with antimicrobial drug use.

The aim of this study was to determine the association between farm management factors, including antimicrobial drug usage, and resistance in commensal Escherichia coli isolates from the faeces of white veal calves. Ninety E. coli isolates from one pooled sample per farm (n = 48) were tested for their phenotypical resistance against amoxicillin, tetracycline, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole (TMP/SMX). Logistic regression analysis revealed the following risk factors (P < 0·05); farmer wearing the same work clothes for several days [ciprofloxacin, odds ratio (OR) 2·6; tetracycline, OR 2·4], administration of trimethoprim-sulfonamide combinations (TMP/SMX, OR 3·0; amoxicillin, OR 3·1; tetracycline, OR 2·6), ⩾0·3 animal daily dosage per production cycle (ADD/pc), quinolones (ciprofloxacin, OR 2·8), ⩾1·3 ADD/pc, penicillins (ciprofloxacin, OR 3·3; tetracycline, OR 3·4), 20-40 ADD/pc, tetracyclines (tetracycline, OR 3·2) and >40 ADD/pc, tetracyclines (tetracycline, OR 13·1; amoxicillin, OR 6·5). In this study antimicrobial resistance in commensal E. coli was mainly associated with antimicrobial drug use.

Complete sequence of pSAM7, an IncX4 plasmid carrying a novel blaCTX-M-14b transposition unit isolated from Escherichia coli and Enterobacter cloacae from cattle.

The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.

Decreased ciprofloxacin susceptibility in Salmonella Typhi and Paratyphi infections in ill-returned travellers: the impact on clinical outcome and future treatment options.

The emergence of decreased ciprofloxacin susceptibility (DCS) in Salmonella enterica serovar Typhi and serovar Paratyphi A, B or C limits treatment options. We studied the impact of DCS isolates on the fate of travellers returning with enteric fever and possible alternative treatment options. We evaluated the clinical features, susceptibility data and efficacy of empirical treatment in patients with positive blood cultures of a DCS isolate compared to patients infected with a ciprofloxacin-susceptible (CS) isolate in the period from January 2002 to August 2008. In addition, the pharmacokinetic and pharmacodynamic parameters of ciprofloxacin, levofloxacin and gatifloxacin were determined to assess if increasing the dose would result in adequate unbound fraction of the drug 24-h area under the concentration-time curve/minimum inhibitory concentration (ƒAUC(0-24)/MIC) ratio. Patients with DCS more often returned from the Indian subcontinent and had a longer fever clearance time and length of hospital stay compared to patients in whom the initial empirical therapy was adequate. The mean ƒAUC(0-24)/MIC was 41.3 ± 18.8 in the patients with DCS and 585.4 ± 219 in patients with a CS isolate. For DCS isolates, the mean ƒAUC0-24/MIC for levofloxacin was 60.5 ± 28.7 and for gatifloxacin, it was 97.9 ± 28.0. Increasing the dose to an adequate ƒAUC(0-24)/MIC ratio will lead to conceivably toxic drug levels in 50% of the patients treated with ciprofloxacin. Emerging DCS isolates has led to the failure of empirical treatment in ill-returned travellers. We demonstrated that, in some cases, an adequate ƒAUC(0-24)/MIC ratio could be achieved by increasing the dose of ciprofloxacin or by the use of alternative fluoroquinolones.

Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales.

OBJECTIVES: To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. METHODS: UK CTX-M-14-producing E. coli (n=70) from cattle (n=33), turkeys (n=9), sheep (n=2) and humans (n=26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of the bla(CTX-M-14) gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence. RESULTS: The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 isolates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxigenic E. coli ('VTEC'); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of the bla(CTX-M-14) gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n=9), turkeys (n=8) and humans (n=4). All pCT-like plasmids were conjugative, and most were IncK (n=21) and had the same local genetic environment flanking the bla(CTX-M-14) gene (n=23). RFLP analysis demonstrated ≥ 75% similarity among most plasmids (n=22). CONCLUSIONS: pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla(CTX-M-14) genes to different E. coli isolates from humans, cattle and turkeys.

Occurrence and characteristics of extended-spectrum-ß-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses.

OBJECTIVES: To investigate the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. METHODS: Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤ 25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. RESULTS: Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were bla(CTX-M-1, -2, -9, -14, -15,) bla(TEM-52), bla(CMY-2) and bla(CMY-)(39). bla(CTX-M-1) was predominant (n = 17). bla(CTX-M-9) occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying bla(CTX-M-1) belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. CONCLUSIONS: This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains.

Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine milk samples.

The aim of this study was to examine whether antimicrobial resistance profiles of coagulase-negative Staphylococcus (CNS) species isolated from milk of dairy cows differed between bacterial species, and to compare results obtained by phenotypic and genotypic profiling of resistance to penicillin, oxacillin and macrolide-lincosamide (ML) antibiotics. Of 170 CNS isolates, 83 (48.8%) were phenotypically susceptible to all antimicrobial agents tested in minimum inhibitory concentration (MIC) assays, 40.6% expressed resistance to a single compound or a single class of compounds, and 10.6% to multiple drug classes. Nine percent, 68%, 19%, 4% and 1% of isolates were negative for all resistance genes tested by PCR or positive for one, two, three or four resistance genes, respectively. Phenotypic resistance and detection of resistance genes other than blaZ were relatively rare in Staphylococcus chromogenes, which was the most common CNS species (36% of 170 genotypically identified isolates). In Staphylococcus epidermidis, which was the second most common CNS species (14% of isolates), phenotypic penicillin resistance was significantly more common than in other CNS species. Almost half of the S. epidermidis isolates carried multiple resistance genes and 30% carried the methicillin resistance gene mecA. Survival analysis using MIC values showed significant associations between phenotypic and genotypic resistance profiles. We conclude that CNS species from bovine milk differ significantly in phenotypic and genotypic antimicrobial resistance profiles, which has implications for treatment and management decisions.

MRSA CC398 in the pig production chain.

In 2005, a distinct clone of methicillin resistant Staphylococcus aureus (MRSA CC398) was found in pigs and people in contact with pigs. The structure of the pig production chain in high technology pig husbandry enables pathogens to spread during animal trading, with an increasing prevalence in herds further down the chain. The objective of this study was to quantify the effect of the MRSA status of the supplying herd on the MRSA status of the receiving herd in order to gain more insight into the role of animal trading as a transmission route for MRSA CC398. Nasal samples (60-80 pigs per herd) were collected from 38 herds; in 20 herds, environmental samples were collected as well. Ten MRSA-positive herds (based on the results of nasal swabs of 10 individual pigs per herd) from a prior study were included in the data analysis. Herds were classified as MRSA positive if at least one sample tested positive. The 48 herds were part of 14 complete (40 herds) and 4 incomplete (8 herds) pig production chains. Fifty-six percent of the herds were classified as MRSA positive. MRSA-positive herds were observed at the start (breeding herds), middle (farrowing herds) and the end (finishing herds) of the pig production chain. All of the herds in 8 chains tested MRSA positive;, all of the herds in 5 chains tested MRSA negative and in the remaining 5 chains, MRSA-positive and MRSA-negative herds were detected. Seven spa types were found, which were all previously confirmed to belong to CC398. All of the isolates were susceptible to mupirocin, linezolid, rifampicin, fusidic acid and cotrimoxazole. Resistance against tetracycline, erythromycin and clindamycin was found in 100, 74 and 76% of the isolates, respectively. Seventy-nine percent of herds with a MRSA-positive supplier of pigs were MRSA positive, whereas 23% of herds with a MRSA-negative supplier were MRSA positive (OR=10.8; 95% CI: 1.5-110.1; P=0.011). The presence of entirely MRSA-positive and MRSA-negative chains and the strong association between the MRSA status of herds and their suppliers illustrates a large risk associated with purchasing pigs from MRSA-positive herds; a top-down strategy for future control programs is, therefore, a basic requirement. However, 23% of herds with a MRSA-negative supplier were MRSA positive and furthermore, 46% of the herds at the top of the pig production chain without a supplier tested MRSA positive. This underlined the need for the identification of additional risk factors for MRSA.

Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain?

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Inaccuracy of routine susceptibility tests for detection of erythromycin resistance of Campylobacter jejuni and Campylobacter coli.

In The Netherlands, both an increase in and regional differences in erythromycin resistance of Campylobacter jejuni and Campylobacter coli have been reported. To determine the accuracy of routine tests for erythromycin resistance, 48 erythromycin-resistant isolates from various laboratories that participate in the Dutch surveillance of Campylobacter infections were reinvestigated. Initial susceptibility testing for erythromycin had been performed by disk diffusion in six and MIC-based methods in two laboratories. Reinvestigation was carried out using broth microdilution as a reference standard, as well as E-test and genetic resistance testing. Of 36 C. jejuni isolates reported by the initial laboratories as erythromycin-resistant, four (11%) and five (14%) were confirmed as erythromycin-resistant using broth microdilution according to CLSI and EUCAST resistance criteria, respectively. Erythromycin resistance was found in eight of 12 (67%) C. coli isolates according to both criteria. Results of E-tests were in accordance with these results in all isolates. Resistance-associated mutations in the 23S rRNA gene (A2059G and A2058T) were found in all isolates showing high-level resistance, whereas none were found in susceptible isolates. Routine determination of the erythromycin resistance of C. jejuni and C. coli shows unacceptable interlaboratory variation. In the absence of standardized protocols and interpretive criteria for disk diffusion, and while we await the development of easily applicable and reliable methods for molecular resistance testing, the use of broth microdilution remains the best method.

Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: evidence for lack of penicillin-resistance in Agr-type II strains.

The increasing evidence for a role of biofilm formation in bovine mastitis caused by Staphylococcus aureus led to further investigations on biofilm formation by S. aureus isolates from mastitis in two growth media (TSBg and bovine milk serum). The ability of 99 S. aureus strains that were recently isolated or obtained from a culture collection (historical strains) to form biofilm, in both growth media as well as the correlation of biofilm formation with the presence of the ica-, bap-, and IS257 genes are described. These genes have been correlated with biofilm formation by human S. aureus isolates. All strains were also genotyped with respect to their Agr-type and -subtype, and for the presence of the antibiotic resistance genes blaZ and smr by PCR. The prevalence of the Agr-types and the investigated genes and their correlation with biofilm formation were statistically evaluated. The Agr-type of a strain had a marked effect on the biofilm formation, by that strain, however in contrast to human isolates no significant effect of ica- and IS257 genes on biofilm formation was observed. The bap gene was not found in any of the investigated strains. The presence of biofilm related genes showed a high correlation with the Agr-type of the strains. The data give evidence for a very strong correlation of Agr-type I strains and penicillin-resistance in the bovine S. aureus mastitis strains; none of the Agr-type II strains was found to harbor penicillin-resistance genes. These data indicate that the most prevalent Agr-types in S. aureus bovine mastitis, Agr-type I and II, can be regarded as different subspecies, with different abilities for the formation of biofilm in bovine milk serum. The very high correlation between Agr-type II and penicillin-susceptibility strongly suggests that these strains are not able to accommodate blaZ genes.

[Multiresistant Brachyspira hyodysenteriae in a Dutch sow herd]. / Multiresistentie bij Brachyspira hyodysenteriae-isolaten op een varkensvermeerderingsbedrijf in Nederland.

This case study describes the isolation ofa multiresistant strain ofBrachyspira hyodysenteriae in April 2007 in a Dutch sow herd with recurrent diarrhoea. Examination of faecal samples taken from 7-month-old breeding gilts with diarrhoea revealed the presence of resistance against tiamulin, lincomycin, tylosin, doxycycline, and tylvalosin (the active substance in Aivlosin) in four of five samples. Tiamulin resistance has not been reported in The Netherlands before. The repeated use of tiamulin on the affected farm was assumed to be the main cause of the development of resistance to the drug. The farmer was advised to adopt a medication strategy and to implement management practices that would prevent an ongoing cycle of infection on the farm. It is important that the Dutch swine industry appreciates that tiamulin-resistant strains of B. hyodysenteriae may be found on other farms as well. The appropriate and prudent use of antibiotics is essential in order to prevent the development of resistance against the last option left to cure B. hyodysenteriae infections: valnemulin.

In vitro antibiotic susceptibility of Dutch Mycoplasma synoviae field isolates originating from joint lesions and the respiratory tract of commercial poultry.

The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.

Contaminants and microorganisms in Dutch organic food products: a comparison with conventional products.

Organic products were analysed for the presence of contaminants, microorganisms and antibiotic resistance and compared with those from conventional products. No differences were observed in the Fusarium toxins deoxynivalenol and zearalenone in organic and conventional wheat, during both a dry period and a very wet period which promoted the production of these toxins. Nitrate levels in head lettuce produced organically in the open field were much lower than those in conventional products. In iceberg lettuce and head lettuce from the greenhouse, no differences were detected. Organically produced carrots contained higher nitrate levels than conventional products. Both organic and conventional products contained no residues of non-polar pesticides above the legal limits, although some were detected in conventional lettuce. Organic products contained no elevated levels of heavy metals. Salmonella was detected in 30% of pig faeces samples obtained from 30 organic farms, similar to the incidence at conventional farms. At farms that switched to organic production more then 6 years ago no Salmonella was detected, with the exception of one stable with young pigs recently purchased from another farm. No Salmonella was detected in faeces at the nine farms with organic broilers, and at one out of ten farms with laying hens. This is comparable with conventional farms where the incidence for Salmonella lies around 10%. Campylobacter was detected in faeces at all organic broiler farms, being much higher than at conventional farms. One of the most remarkable results was the fact that faeces from organic pigs and broilers showed a much lower incidence of antibiotic resistant bacteria, except for Campylobacter in broilers. It is concluded that the organic products investigated scored as equally well as conventional products with regard to food safety and at the same time show some promising features with respect to antibiotic resistance.

[Sensitivity to various antibiotics of coagulase-negative staphylococci isolated from samples of milk from Dutch dairy cattle]. / Gevoeligheid voor diverse antibiotica van coagulase negatieve staphylococcen, geïsoleerd uit melkmonsters van Nederlands rundvee.

During recent years the prevalence of coagulase-negative staphylococci in milk samples from Dutch dairy cows has increased. In 1999 16.2% of the bacteria isolated from milk collected from cows with subclinical mastitis were coagulase-negative staphylococci. In 2004 this proportion was 42.2%. The proportion of coagulase-negative staphylococci of the bacteria isolated from milk samples from cows with clinical mastitis was 7.3% in 1999 and 14.1% in 2004. In this study, the susceptibility of 108 coagulase-negative staphylococci to oxacillin, cefquinome, streptomycin, neomycin, penicillin, and the combination of nafcillin, penicillin, and streptomycin was tested. The isolates were cultured from milk collected from cows with mastitis and typed using the Api-Staph system. Eight species were identified. Staphylococcus chromogenes was the predominant species (41.7%), followed by Staphylococcus xylosus (15.7%) and Staphylococcus simulans (10.2%). With the agar dilution method all strains proved to be sensitive to cefquinome and 90% to oxacillin. Three isolates (2.8%) were mecA-positive. Despite the agar dilution results, these three isolates should be considered resistant to all beta-lactam antibiotics (penicillins, penicillins combined with a beta-lactamase inhibitor and all generations of cephalosporins). In the agar diffusion test, all isolates proved to be sensitive to the combination of nafcillin-penicillin-streptomycin, 99% were sensitive to neomycin and 1% intermediate sensitive, and 95% were sensitive to streptomycin, 4% resistant, and 1% intermediate sensitive. The coagulase-negative staphylococci were highly resistant to penicillin (37.4%), although the level of resistance varied between species, from 0% for Staphylococcus simulans to 100% for Staphylococcus saprophyticus. Because coagulase-negative staphylococci are resistant to several antibiotics, sensitivity testing is important for targeted treatment of mastitis.