Britta Kraushaar

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 methicillin-resistant Staphylococcus aureus in a fresh meat pork production chain.

The objective of this study was to investigate the prevalence of methicillin-resistant Staphylococcus aureus on different stages of a fresh pork production chain to reveal potential carryover from live animals to meat. Samples were collected at different stages of the production process in a large German abattoir with an integrated processing unit for fresh pork. Samples included nasal swabs from pigs at stunning, environmental samples from the slaughter line, surface samples from carcasses, environmental and meat samples from the processing unit, and samples from final products. Samples were analyzed with an established two-step selective enrichment method, and isolates were characterized with respect to their S. aureus protein A gene (spa) and staphylococcal cassette chromosome mec (SCCmec; which harbors the mecA gene) types. Contamination rate was highest (64.7%) in nasal swabs and lower (6.0%) on carcasses, meat at processing (4.2%), and final products (2.8%). Environmental samples were positive along the slaughter line (12%) but not in the processing unit. spa types t011 and t034 and SCCmec type V predominated the isolates. Heterogeneity of spa types was highest in nasal swabs. Results show that methicillin-resistant S. aureus can be identified at all stages of the production chain. Further studies are needed to identify potential control points to reduce the carryover from farm animals to the final products.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

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.

Heavy metal and disinfectant resistance genes among livestock-associated methicillin-resistant Staphylococcus aureus isolates

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged in animal production worldwide. Most LA-MRSA in Europe belong to the clonal complex (CC) 398. The reason for the LA-MRSA emergence is not fully understood. Besides antimicrobial agents used for therapy, other substances with antimicrobial activity applied in animal feed, including metal-containing compounds might contribute to their selection. Some of these genes have been found in various novel SCCmec cassettes. The aim of this study was to assess the occurrence of metal-resistance genes among a LA-S. aureus collection [n=554, including 542 MRSA and 12 methicillin-susceptible S. aureus (MSSA)] isolated from livestock and food thereof. Most LA-MRSA isolates (76%) carried at least one metal-resistance gene. Among the LA-MRSA CC398 isolates (n=456), 4.8%, 0.2%, 24.3% and 71.5% were positive for arsA (arsenic compounds), cadD (cadmium), copB (copper) and czrC (zinc/cadmium) resistance genes, respectively. In contrast, among the LA-MRSA non-CC398 isolates (n=86), 1.2%, 18.6% and 16.3% were positive for the cadD, copB and czrC genes, respectively, and none were positive for arsA. Of the LA-MRSA CC398 isolates, 72% carried one metal-resistance gene, and the remaining harboured two or more in different combinations. Differences between LA-MRSA CC398 and non-CC398 were statistically significant for arsA and czrC. The czrC gene was almost exclusively found (98%) in the presence of SCCmec V in both CC398 and non-CC398 LA-MRSA isolates from different sources. Regarding the LA-MSSA isolates (n=12), some (n=4) were also positive for metal-resistance genes. This study shows that genes potentially conferring metal-resistance are frequently present in LA-MRSA.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

ABSTRACT Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

First description of PVL-positive methicillin-resistant Staphylococcus aureus (MRSA) in wild boar meat

Staphylococcus aureus is an important food-borne pathogen due to the ability of enterotoxigenic strains to produce staphylococcal enterotoxins (SEs) in food. Methicillin-resistant S. aureus (MRSA) is also an important pathogen for humans, causing severe and hard to treat diseases in hospitals and in the community due to its multiresistance against antimicrobials. In particular, strains harbouring genes encoding for the Panton-Valentine leukocidin (PVL) toxin are of concern from a public health perspective as they are usually capable of causing severe skin and soft tissue infections (sSSTIs) and occasionally necrotizing pneumonia which is associated with high mortality. This is the first report on the detection of MRSA with genes encoding for PVL in wild boar meat. Among the 28 MRSA isolated from wild boar meat in the course of a national monitoring programme in Germany, seven harboured PVL-encoding genes. Six of the isolates were identical according to the results of spa-, MLST-, microarray- and PFGE-typing. They could be assigned to the epidemic MRSA clone USA300. Epidemiological investigations revealed that people handling the food were the most likely common source of contamination with these MRSA. These findings call again for suitable hygienic measures at all processing steps of the food production chain. The results of the study underline that monitoring along the food chain is essential to closely characterise the total burden of MRSA for public health.

Antimicrobial resistances and virulence markers in Methicillin-resistant Staphylococcus aureus from broiler and turkey: A molecular view from farm to fork.

Little is known about the characteristics of MRSA occurring along the broiler and turkey production chains. The aim of this study was to characterise and compare MRSA of turkey and broiler origin sampled on different production levels using a DNA microarray and antimicrobial susceptibility testing. Main differences could be observed in the prevalence of the resistance genes erm(C), aacA-aphD and tet(K) and the number of non-wild type strains with minimum inhibitory concentration values (MICs) above the epidemiological cut-off values (ECOFFs) for gentamicin and kanamycin. Overall, MRSA with non-wild type phenotype for the macrolide-lincosamide-streptogramin group, tetracycline, and trimethoprim were found in more than 70% of poultry isolates. Non-wild type isolates carrying the qacC gene conferring resistance to quaternary ammonium compound disinfectants were found at all production stages in similar frequencies. Regarding the presence of enterotoxin genes in poultry-derived MRSA the enterotoxin gene cluster (egc) was only found in Non-CC (clonal complex) 398 strains. Three CC398 strains harboured the genes sed (from turkey at slaughter and broiler meat) and sea-N315 (from a chicken carcass). One Non-CC398 strain isolated from turkey meat was found positive for the seb gene and also enterotoxin production. Similarity analysis based on selected resistance and virulence markers revealed a high clonality among Non-CC398 isolates. Isolates of the same clonal complex clustered together according to their common virulence and resistance profiles. Strains of CC9 were grouped in two subclusters due to different resistance genes. Our results underline, that there are other poultry associated clones of MRSA (mainly CC9 and CC5) besides the predominant CC398 which are similar in both poultry species.

Comparison of spa types, SCCmec types and antimicrobial resistance profiles of MRSA isolated from turkeys at farm, slaughter and from retail meat indicates transmission along the production chain.

The prevalence of MRSA in the turkey meat production chain in Germany was estimated within the national monitoring for zoonotic agents in 2010. In total 22/112 (19.6%) dust samples from turkey farms, 235/359 (65.5%) swabs from turkey carcasses after slaughter and 147/460 (32.0%) turkey meat samples at retail were tested positive for MRSA. The specific distributions of spa types, SCCmec types and antimicrobial resistance profiles of MRSA isolated from these three different origins were compared using chi square statistics and the proportional similarity index (Czekanowski index). No significant differences between spa types, SCCmec types and antimicrobial resistance profiles of MRSA from different steps of the German turkey meat production chain were observed using Chi-Square test statistics. The Czekanowski index which can obtain values between 0 (no similarity) and 1 (perfect agreement) was consistently high (0.79–0.86) for the distribution of spa types and SCCmec types between the different processing stages indicating high degrees of similarity. The comparison of antimicrobial resistance profiles between the different process steps revealed the lowest Czekanowski index values (0.42–0.56). However, the Czekanowski index values were substantially higher than the index when isolates from the turkey meat production chain were compared to isolates from wild boar meat (0.13–0.19), an example of a separated population of MRSA used as control group. This result indicates that the proposed statistical method is valid to detect existing differences in the distribution of the tested characteristics of MRSA. The degree of similarity in the distribution of spa types, SCCmec types and antimicrobial resistance profiles between MRSA isolates from different process stages of turkey meat production may reflect MRSA transmission along the chain.