Growth of methicillin-resistant Staphylococcus aureus during raw milk soft cheese-production and the inhibitory effect of starter cultures.

The consumption of raw milk or raw milk products might be a potential risk factor for the transmission of methicillin-resistant Staphylococcus aureus (MRSA). Therefore, we studied MRSA growth during raw milk soft cheese-production. Furthermore, we investigated the inhibitory effect of four starter cultures (Lactococcus lactis, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lactobacillus helveticus) on the growth of MRSA in a spot-agar-assay and in raw milk co-culture following a cheesemaking temperature profile. During the initial phases of raw milk cheese-production, MRSA counts increased by 2 log units. In the ripening phase, MRSA counts only dropped slightly and remained high up to the end of the storage. Comparable MRSA counts were found in the rind and core and strain-specific differences in survival were observed. In the spot-agar-assay, all four starter cultures showed strong or intermediate inhibition of MRSA growth. In contrast, in raw milk, only Lactococcus lactis strongly inhibited MRSA, whereas all other starter cultures only had minor inhibitory effects on MRSA growth. Our results indicate that MRSA follow a similar growth pattern as described for other S. aureus during raw milk soft cheese-production and illustrate the potential use of appropriate starter cultures to inhibit MRSA growth during the production of raw milk cheese.

The occurrence of methicillin-resistant non-aureus staphylococci in samples from cows, young stock, and the environment on German dairy farms.

This study aimed to determine the occurrence of methicillin-resistant (MR) non-aureus staphylococci (NAS) on 20 preselected German dairy farms. Farms were selected based on the detection of methicillin-resistant Staphylococcus aureus (MRSA) during previous diagnostic investigations. Bacterial culture of presumptive MR-NAS was based on a 2-step enrichment method that has been recommended for MRSA detection. Quarter milk samples (QMS), bulk tank milk, swab samples from young stock, and environmental samples were collected for bacterial culture. Methicillin-resistant NAS were detected on all study farms. The MR-NAS positive test rate was 3.3% (77/2,347) in QMS, 42.1% (8/19) in bulk tank milk, 29.1% (59/203) in nasal swabs from milk-fed calves, 18.3% (35/191) in postweaning calves, and 7.3% (14/191) in nasal swabs from prefresh heifers. In the environment, MR-NAS were detected in dust samples on 25% (5/20) of the dairy farms as well as in teat liners and suckers from automatic calf feeders. The geometric mean somatic cell count in QMS affected by MR-NAS (183,000 cells/mL) was slightly higher compared with all QMS (114,000 cells/mL). Nine MR-NAS species were identified; Staph. sciuri, Staph. lentus, Staph. fleurettii, Staph. epidermidis, and Staph. haemolyticus were the most common species. In addition, 170 NAS isolates were identified that showed reduced cefoxitin susceptibility (4 mg/L) but did not harbor the mecA or mecC genes. On some farms, similar mobile genetic elements were detected in MR-NAS and MRSA. It was suggested that resistance genes may be transferred between NAS and Staph. aureus on the respective farms.

The occurrence and distribution of livestock-associated methicillin-resistant Staphylococcus aureus ST398 on German dairy farms.

The objective of this study was to investigate the occurrence and distribution of methicillin-resistant Staphylococcus aureus (MRSA) on 20 German dairy farms. Farms were selected based on previous MRSA reports from phenotypic susceptibility testing of mastitis pathogens. Samples were collected from predefined groups of cows, young stock, farm personnel, and the environment. A high MRSA-positive test rate was detected in swab samples from milk-fed calves (22.7%; 46/203). In postweaning calves, the MRSA-positive test rate was 9.1% (17/187). From prefresh heifers, both nasal swabs and udder cleft swabs were collected if possible. Including both sample types, the MRSA-positive test rate in prefresh heifers was 13.0% (26/200). The positive test rate was 8.9% (17/191) in nasal swabs and 6.5% (11/170) in udder cleft swabs. In quarter milk samples (QMS), the MRSA-positive test rate was 2.9% (67/2347), and on cow level, 7.9% (47/597) of the dairy cows were affected. Among all cows included in this study, the geometric mean of somatic cell counts was higher in QMS that carried MRSA (345,000 cells/mL) in comparison to all QMS (114,000 cells/mL). No differences in parity or the affected mammary quarter position on the udder were observed among the 47 infected cows. Methicillin-resistant S. aureus was also detected in boot swab samples (dust), teat liners, and in suckers from automatic calf feeders. All isolates belonged to livestock-associated sequence type 398 and most common staphylococcal protein A (spa)-types were t011 and t034. Most isolates harbored the staphylococcal cassette chromosome mec (SCCmec)-type V, with the exception of some isolates with SCCmec-type IVa on 1 farm. Similar MRSA genotypes in samples from humans and dairy cows underline the possible zoonotic and reverse-zoonotic transmission of livestock-associated MRSA strains from dairy farms. Similar MRSA genotypes in pig and cattle barns were detected on only 1 of 5 farms that kept both cattle and pigs. Similar MRSA spa-types were detected in samples from different sources (dairy cows, young stock, environment, and humans), suggesting a possible contagious transmission on some of the farms. Sporadically, up to 3 different MRSA spa-types were detected in QMS from the respective farms. On MRSA-affected farms, improper milking hygiene procedures and elevated bulk-tank milk somatic cell counts (>250,000 cells/mL) were observed. The occurrence of livestock-associated MRSA ST398 in different samples from dairy farms, and especially in young calves, should be considered for future MRSA-monitoring programs and biosecurity guidelines.

Characterization of VIM-1-Producing E. coli Isolated From a German Fattening Pig Farm by an Improved Isolation Procedure.

A few reports indicate that livestock might represent a new reservoir for carbapenemase-producing Enterobacteriaceae (CPE). In 2015, VIM-1-producing Escherichia coli were detected at slaughter in colon contents of animals from a German fattening pig farm within the national monitoring on ESBL-producing E. coli. In this study, pooled faces samples from pigs, as well as samples from the barn surrounding environment of this fattening farm were taken, to evaluate the dissemination of CPEs. Several modifications of the culture-dependent detection procedure were investigated for their potential to improve the sensitivity of the CPE isolation method. The current reference procedure was adapted by adding a real-time PCR pre-screening and additional enrichment steps. It was possible to isolate 32 VIM-1-producing E. coli from four fecal samples of three different barns using two serial enrichment steps in combination with real-time PCR and selective agar plates. By genetic typing, we confirmed the presence of two E. coli clonal lineages circulating on this particular farm: one was harboring the bla VIM- 1 on an IncHI2 plasmid while the second lineage carried the gene on the chromosome. Despite its different locations, the bla VIM- 1 gene was harbored on a class 1 integron in both clonal lineages. Whole-genome sequencing revealed that the VIM-1-carrying plasmids exhibited only slight variability in its compositions and sizes. We assume that the prevalence of CPEs in animal production in Germany and other European countries might be underestimated and there is a concern of further spread of VIM-1-producing bacteria in German livestock and food.

Detection and quantification of methicillin-resistant Staphylococcus aureus in fresh broiler meat at retail in Germany.

The aim of this study was to detect, to quantify and to characterize MRSA in broiler meat samples with skin. Furthermore, we compared an isolation method using a second selective enrichment step (method A) with a simpler method omitting this step (method B). For quantification we used a direct plating method on selective agar plates and a "Most probable number" (MPN) technique for estimation of low numbers of MRSA. Presumptive MRSA colonies were confirmed by MALDI-TOF and by PCR. After confirmation the isolated MRSA were characterized by spa-typing and, if necessary, by multi-locus sequence typing. Method B detected more MRSA-positive samples (16.7%, n = 215) than method A (12.1%). However, method B also produced more false positive results (28.4%).The highest estimated number of MRSA in fresh broiler meat with skin was 1100 MPN/g, but in most positive samples (80.1%) the estimated numbers of MRSA were lower than 10 MPN/g. Thus, the numbers of MRSA in the samples were too low to detect using the spread plate technique. Ten different spa-types were identified. Six of these with 69% of the isolates were assigned to the clonal complex CC398 (t034; t011; t2576; t571; t5452; t1457). Spa-types t1430, t13177 and t899 can be assigned to CC9. Spa-type t304 was identified as MLST-type ST6. In conclusion, we provide quantitative data on low level contamination of fresh broiler meat with MRSA. Most isolated MRSA were from livestock associated spa-types. Omitting the second enrichment step was associated with an increase in sensitivity but lower specificity of the cultural method.

Oral administration of antimicrobials increase antimicrobial resistance in E. coli from chicken--a systematic review.

Antimicrobials play an important role in animal and human health care. It was the aim of this systematic review to assess the effects of oral administration of antimicrobials on the development of antimicrobial resistance (AMR) in Escherichia coli (E. coli) from chickens. Moreover, the effects of the administration of more than one antimicrobial and of different dosages were studied. Literature was searched in November 2012 from the electronic databases ISI Web of Science, PubMed, Scopus and a national literature database (DIMDI) as well as the database ProQuest LLC. The search was updated in March 2014. Original studies describing a treatment (A) and a control group of either non-treatment (C) or initial value (0) and determining AMR in E. coli at different sample points (SP) were included. The literature search resulted in 35 full text articles on the topic, seven (20%) of which contained sufficient information on the administered antimicrobial and the impact of treatment on AMR. Most papers described the use of more than one antimicrobial, several dosages, controls (non-treatment or pre-treatment) and measured AMR at different SPs leading to a total of 227 SPs on the impact of the use of antimicrobials on AMR in chickens. 74% of the SPs (168/227) described a higher AMR-rate in E. coli from treated animals than from controls. After the administration of a single antimicrobial, AMR increased at 72% of the SPs. Administration of more than one antimicrobial increased AMR at 82% of the SPs. Higher dosages were associated with similar or higher AMR rates. The limited number of studies for each antimicrobial agent and the high variability in the resistance effect call for more well designed studies on the impact of oral administration on AMR development and spread.

Validation of bovine oestrous-specific synthetic molecules with trained scent dogs; similarities between natural and synthetic oestrous smell.

Oestrous detection is crucial for successful dairy cow reproduction. Bulls identify cows in oestrus by oestrous-specific odours especially in urine and vaginal fluid. These have been used to train dogs to detect cows in heat. To improve and simplify the dog training, a spray containing synthetic oestrous molecules was developed. The objective of this study was to test the spray on similarities to the natural substance thus to assess its suitability as a training substance for heat detection dogs. Ten privately owned dogs of various breeds were trained. Dogs should be trained either to differentiate natural vaginal fluid from cows in oestrus and dioestrus (n = 5), or spray with or without synthetic oestrous molecules (n = 5). Dogs trained on natural fluid and on spray could detect the oestrous odour they had been trained on with an overall accuracy of 69.0% and 82.4%, respectively (p = 0.019). To validate the synthetic molecules, dogs trained with synthetic molecules had to detect oestrous odour in natural fluid without further training (accuracy 37.6%). Dogs trained on natural fluid detected the synthetic molecules with an accuracy of 50.0% (50% vs 37.4%, p < 0.05). Dogs can recognize natural vaginal fluid from cows in oestrus after they have been trained with synthetic oestrous molecules, but accuracy needs to be improved.

Methicillin-resistant Staphylococcus aureus in cattle food chains - prevalence, diversity, and antimicrobial resistance in Germany.

Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. It was the objective of this study to estimate the prevalence of MRSA in different cattle food chains (milk, beef, and veal) in Germany, to analyze the MRSA diversity along each food chain and to compare the characteristics of the different subtypes. Samples were collected between 2009 and 2012 from dairy herds (bulk tank milk), veal herds (dust from the stables), veal calves, and beef cattle at slaughter (nasal swabs) and carcasses of veal calves (surface cuts) and beef as well as veal at retail. Sampling was proportionally distributed over the country according to the cattle population (on-farm sampling), slaughterhouse capacity (abattoir samples), and the human population (meat at retail). Methicillin-resistant S. aureus were isolated using harmonized methods from all sample types and populations investigated. The highest proportion of positive samples was found in nasal swabs from veal calves at slaughter in 2012 (144/320; 45.0%) and the lowest rate in bulk tank milk in 2009 (14/388; 4.1%). Most isolates, irrespective of the origin, were from spa types t011 and t034. Both have been assigned to the clonal complex (CC) 398. Few isolates (15/632; 2.4%) were from spa types not associated with the CC398. Spa-type patterns were similar along individual food chains but differed between food chains. Antimicrobial resistance patterns differed between isolates from the different food chains and spa types. Isolates from the veal chain displayed the highest resistance rates. We conclude that there is substantial diversity in the MRSA prevalence across different cattle production sectors.

Oral antimicrobials increase antimicrobial resistance in porcine E. coli--a systematic review.

Administration of antimicrobials to livestock increases the risk of antimicrobial resistance (AMR) in commensal bacteria. Antimicrobials in pig production are usually administered per pen via feed which implies treatment of sick alongside with healthy animals. The objective of this systematic literature review was to investigate the effect of orally administered antimicrobials on AMR in Escherichia coli of swine. Studies published in peer reviewed journals were retrieved from the international online databases ISI Web of Knowledge, PubMed, Scopus and the national electronic literature data base of Deutsches Institut für Medizinische Dokumentation und Information. The studies were assessed using the eligibility criteria English or German language, access to full paper version, defined treatment and control group (initial value or non-treatment) as well as administration and resistance testing of the same antimicrobial class. In the qualitative synthesis, only studies were included presenting the summary measures odds ratio or prevalence of resistance, the category of the applied antimicrobial and the dosage. An effect of the antimicrobial on AMR in E. coli was evaluated as an "increase", "no effect" or "decrease" if the odds or alternatively the prevalence ratio were >1.0, 1.0 or <1.0, respectively. Eleven studies, describing 36 different trials, fulfilled the eligibility criteria and were finally assessed. An increase of AMR in E. coli was found in 10 out of 11 trials comparing AMR after with AMR prior to oral treatment and in 22 of the 25 trials comparing orally treated with untreated groups. Effects expressed as odds or prevalence ratios were highest for the use of aminoglycosides, quinolones and tetracycline. There was no clear association between the reported dosage and AMR towards tetracycline. Information on antimicrobial substance and dosage was missing in 4 and 5 of the 11 finally selected studies. The 36 identified trials were inhomogenous in usage and provision of information on sample size. Oral administration of antimicrobials increases the risk of AMR in E. coli from swine. There is however a lack of studies on the impact of dosage and longitudinal effects of treatment. The published studies have a number of issues concerning their scientific quality. More high quality research is needed to better address and quantifiy the effect of orally administered antimicrobials on AMR in swine.

Occurrence of livestock-associated methicillin-resistant Staphylococcus aureus in Turkey and broiler barns and contamination of air and soil surfaces in their vicinity.

The emission of microorganisms, especially resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), from poultry farms is of public interest, and its occurrence and relevance are controversially discussed. So far, there are limited data on this issue. In this study, we investigated the occurrence of livestock-associated (LA)-MRSA inside and outside previously tested MRSA-positive poultry barns in Germany. In total, five turkey and two broiler fattening farms were investigated four and three times, respectively. In a longitudinal study during one fattening period, samples were collected from animals, the animals' environment inside the barn, including the air, and the barns' surroundings, such as ambient air and boot swabs of ground surfaces at different distances from the barn. Moreover, a cross-sectional study was carried out once inside the barns on five turkey and four broiler farms during the last third of the fatting period. In the cross-sectional study, LA-MRSA was detected in the air of most barns (7 of 9, 77.8%), as well as in many samples originating from animals, with detections levels of 50 to 54% in broiler and 62 to 77% in turkey farms. In the longitudinal study, LA-MRSA was found in the ambient air outside two turkey barns and on the ground surface on the downwind side of many (44.4%) turkey and broiler farms. The same spa types of isolates were observed inside and outside the barns. Transmission of MRSA within poultry farms, as well as emission via the airborne route, seems to be possible.

Short communication: Ability of dogs to detect cows in estrus from sniffing saliva samples.

Efficient estrus detection in high-producing dairy cows is a permanent challenge for successful reproductive performance. In former studies, dogs have been trained to identify estrus-specific odor in vaginal fluid, milk, urine, and blood samples under laboratory conditions with an accuracy of more than 80%. For on-farm utilization of estrus-detection dogs it would be beneficial in terms of hygiene and safety if dogs could identify cows from the feed alley. The objective of this proof of concept study was to test if dogs can be trained to detect estrus-specific scent in saliva of cows. Saliva samples were collected from cows in estrus and diestrus. Thirteen dogs of various breeds and both sexes were trained in this study. Five dogs had no experience in scent detection, whereas 8 dogs had been formerly trained for detection of narcotics or cancer. In the training and test situation, dogs had to detect 1 positive out of 4 samples. Dog training was based on positive reinforcement and dogs were rewarded with a clicker and food for indicating saliva samples of cows in estrus. A false indication was ignored and documented in the test situation. Dogs with and without prior training were trained for 1 and 5 d, respectively. For determining the accuracy of detection, the position of the positive sample was unknown to the dog handler, to avoid hidden cues to the dog. The overall percentage of correct positive indications was 57.6% (175/304), with a range from 40 (1 dog) to 75% (3 dogs). To our knowledge, this is the first indication that dogs are able to detect estrus-specific scent in saliva of cows.

Emerging antimicrobial resistance in commensal Escherichia coli with public health relevance.

In 2009, 1462 Escherichia coli isolates were collected in a systematic resistance monitoring approach from primary production, slaughterhouses and at retail and evaluated on the basis of epidemiological cut-off values. Besides resistance to antimicrobial classes that have been extensively used for a long time (e.g. sulphonamides and tetracyclines), resistance to (fluoro)quinolones and third-generation cephalosporins was observed. While in the poultry production chain the majority (60%) of isolates from laying hens was susceptible to all antimicrobials tested, most isolates from broilers, chicken meat and turkey meat showed resistance to at least one (85-93%) but frequently even to several antimicrobial classes (73-84%). In the cattle and pig production chain, the share of isolates showing resistance to at least one antimicrobial was lowest (16%) in dairy cows, whereas resistance to at least one antimicrobial ranged between 43% and 73% in veal calves, veal and pork. Resistance rates to ciprofloxacin and nalidixic acid in isolates from broilers were 41.1% and 43.1%, respectively. Likewise, high resistance rates to (fluoro)quinolones were observed in isolates from chicken meat and turkey meat. In contrast, ciprofloxacin resistance was less frequent in E. coli isolates from the cattle and pig production chain with highest rate in veal calves (13.3%). Highest resistance rates to cephalosporins were observed in broilers and chicken meat, with 5.9% and 6.2% of the isolates showing resistance. In dairy cattle and veal, no isolates with cephalosporin resistance were detected, whereas 3.3% of the isolates from veal calves showed resistance to ceftazidime. Resistance to (fluoro)quinolones and cephalosporins in E. coli isolates is of special concern because they are critically important antimicrobials in human antimicrobial therapy. The emergence of this resistance warrants increased monitoring. Together with continuous monitoring of antimicrobial usage, management strategies should be regularly assessed and adapted.

Prevalence, antimicrobial resistance, and molecular characterization of methicillin-resistant Staphylococcus aureus from bulk tank milk of dairy herds.

It was the objective of the study to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in bulk tank milk from German dairy herds and to characterize isolates from bulk tank milk with respect to their Staph. aureus protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) type, their phenotypic antimicrobial resistance and resistance- resp. virulence-associated genes using broth microdilution and a microarray for Staph. aureus. Bulk tank milk samples (25 mL) were tested for MRSA using a 2-step selective enrichment protocol. Presumptive MRSA were confirmed by PCR. Thirty-six isolates collected from bulk tank milk of dairy herds in 2009 and 2010 were included in the characterization. All isolates displayed spa-types assigned to the clonal complex CC398. Based on the epidemiological cut-off values for the interpretation of minimum inhibitory concentrations isolates were resistant to tetracycline (100%), clindamycin (58%), erythromycin (52%), quinupristin/dalfopristin (36%), and kanamycin (27%). Isolates did not carry genes associated with typical virulence factors for Staph. aureus such as the Panton-Valentine leukocidin. However, they did carry hemolysin genes. Livestock-associated MRSA of CC398 does occur in German dairy herds and the strains have similar properties as described for strains from pigs.

Prevalence of types of methicillin-resistant Staphylococcus aureus in turkey flocks and personnel attending the animals.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have been isolated from a number of livestock species and persons involved in animal production. We investigated the prevalence of LA-MRSA in fattening turkeys and people living on farms that house fattening turkeys. Eighteen (90%) of 20 investigated flocks were positive for MRSA, and on 12 of the farms 22 (37·3%) of 59 persons sampled were positive for MRSA. People with frequent access to the stables were more likely to be positive for MRSA. In most flocks MRSA that could be assigned to clonal complex (CC) 398 were detected. In five flocks MRSA of spa-type t002 that is not related to CC398 were identified. Moreover, other methicillin-resistant Staphylococcus spp. were detected on 11 farms and in eight people working on the farms.

Quo vadis? - Monitoring Campylobacter in Germany.

Campylobacter is a poorly recognized foodborne pathogen, leading the statistics of bacterially caused human diarrhoea in Europe during the last years. In this review, we present qualitative and quantitative German data obtained in the framework of specific monitoring programs and from routine surveillance. These also comprise recent data on antimicrobial resistances of food isolates. Due to the considerable reduction of in vitro growth capabilities of stressed bacteria, there is a clear discrepancy between the detection limit of Campylobacter by cultivation and its infection potential. Moreover, antimicrobial resistances of Campylobacter isolates established during fattening of livestock are alarming, since they constitute an additional threat to human health. The European Food Safety Authority (EFSA) discusses the establishment of a quantitative limit for Campylobacter contamination of broiler carcasses in order to achieve an appropriate level of protection for consumers. Currently, a considerable amount of German broiler carcasses would not comply with this future criterion. We recommend Campylobacter reduction strategies to be focussed on the prevention of fecal contamination during slaughter. Decontamination is only a sparse option, since the reduction efficiency is low and its success depends on the initial contamination concentration.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Methicillin-resistant Staphylococcus aureus (MRSA) in three dairy herds in southwest Germany.

The objective of this study was to analyse the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in three dairy herds in the southwest of Germany that had experienced individual cases of clinical and subclinical mastitis associated with MRSA. The herds were identified by the detection of MRSA during routine resistance testing of mastitis pathogens. All quarters of all cows in the herds that were positive on California Mastitis Test were sampled for bacteriological analysis on two occasions. Bulk tank milk samples were also tested. Furthermore, nasal swabs were collected from people working on the farms and from cattle. Environmental samples were collected from associated pig holdings. Isolates were characterized using spa-typing and testing for antimicrobial resistance. Our results revealed a substantial spread of MRSA in the three dairy herds. In the first of the two investigations carried out on all cows in the three herds, milk samples of 5.1-16.7% of dairy cows were found positive for MRSA. The respective proportions in the second herd level investigation were 1.4-10.0%. Quarters harbouring MRSA had higher somatic cell counts than quarters that were negative on culture. Methicillin-resistant Staphylococcus aureus were also detected in nasal swabs of staff (7/9), cows (7/15) and calves (4/7), bulk tank milk samples (3/3) and environmental samples from pig premises (4/5) on the farm. Herds B and C had no contact to herd A. However, in all three herds MRSA of spa-type t011 were detected in milk samples. Results show that MRSA of spa-type t011 is a problem in dairy farms that needs urgent attention.

Livestock associated methicillin-resistant Staphylococcus aureus (LaMRSA) isolated from lesions of pigs at necropsy in northwest Germany between 2004 and 2007.

An increasing number of reported detections of methicillin-resistant Staphylococcus aureus (MRSA) in food animals since 2007 has led to the assumption that there is an emerging zoonotic problem with livestock associated (la)MRSA potentially aggravating the MRSA problem in humans. It was the objective of the study to investigate, whether MRSA was present in clinical specimens of pigs collected at post-mortem since 2004 and to further characterize these isolates. We studied 138 isolates of S. aureus collected between 2004 and 2007 from various pathological lesions of pigs at necropsy. Potential MRSA were identified by growth on selective chromogenic media. Isolates were confirmed as MRSA using multiplex PCR. Confirmed isolates were spa- and SCCmec-typed and were tested for antimicrobial resistance. Overall, 60 (43%) S. aureus isolates were identified as MRSA. The majority (57/60) of the MRSA isolates found in the altered porcine tissues were spa-types associated with MRSA ST398. Three MRSA were ST97 isolates, a type that has not been described as an MRSA in pigs before. Other clonal complexes (ST9, ST30) dominated among the methicillin-sensitive S. aureus. MRSA were found in similar frequency in all 4 years. We assume that MRSA in pigs may have occurred earlier than 2004 and might be not really 'emerging', but rather have been overlooked until recently. The potentially causative role of the MRSA in the lesions warrants further investigation.

High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types.

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.