A total of 669 individual cow milk samples originating from asymptomatic cows from 16 dairy farms were examined for the presence of microorganisms with the potential to cause mastitis. Coagulase-negative staphylococci clearly predominated (53.5% positive samples) followed by streptococci and enterococci (both occurring in 16.1% samples). Among streptococci, so-called mastitis streptococci (S. uberis, S. dysgalactiae and S. agalactiae) prevailed (11.7% positive samples). Enterobacteriaceae were found in 10.0% samples, most of which (6.6% samples) were positive for Escherichia coli. Yeasts (mainly Candida spp.) were found in 8.2% samples. One of the major mastitis pathogens, Staphylococcus aureus subsp. aureus, was isolated from 9.0% of samples. S. aureus isolates were further characterised in terms of their capability to form biofilm, antimicrobial susceptibility and clonality (PFGE). All S. aureus isolates were capable of biofilm formation and were generally susceptible to the majority of tested antibiotics. The exception was ampicillin, resistance to which was observed in 27.7% isolates. Therefore, the relatively frequent occurrence of S. aureus could be attributed to persistent intramammary infections due to biofilm formation rather than low efficacy of particular antibiotics. PFGE analysis revealed clonal spread of certain S. aureus isolates within and between farms indicating that certain lineages of S. aureus mastitis strains are particularly successful.

Schlegelová J., Babák V., Holasová M., Konstantinová L., Necidová L., Šišák F., Vlková H., Roubal P., Jaglic Z. (2010): Microbial contamination after sanitation of food contact surfaces in dairy and meat processing plants. Czech J. Food Sci., 28: 450-461.The occurrence of listeria monocytogenes, Salmonella spp., Bacillus cereus, Staphylococcus spp., enterococcus spp., and escherichia coli in raw food materials, food products, and on food contact surfaces after sanitation was investigated during the period of 2005-2006 in three dairy cattle farms (120 samples), one dairy (124 samples), and two meat processing plants (160 samples). A total of 1409 isolates were identified. The epidemiological characterisation and determination of the virulence factors and antimicrobial resistance were performed on selected isolates. The level of bacterial contamination generally decreased during the production process (the contamination of food products was lower than that of raw material). However, the contamination of food contact surfaces was relatively high even after sanitation. Moreover, specific microbiological profiles were found on the inside equipment surfaces in dairy facilities, where genetically closely related multi-resistant strains persisting in biofilm communities may occur as demonstrated for staphylococci. Although the occurrence of potentially significant pathogens was not high, the microorganisms such as l. monocytogenes, Salmonella spp., and shiga-toxin positive e. coli principally contaminated the meat processing plants. B. cereus isolates, among which 76% were positive for diarrhogenic enterotoxin, typically occurred on the inside equipment surfaces and in the heat-treated products.

Geographically related Staphylococcus epidermidis isolates from human patients (n=30), dairy farms (farmers and individual raw milk from cattle, n=36) and a dairy plant (n=55) were examined for epidemiological relatedness by pulsed-field gel electrophoresis and, using in vitro methods, for the ability to produce biofilm and antimicrobial resistance. Methicillin-resistant isolates (MRSE) were also identified and characterized. Isolates from farmers and dairy cattle were found to be genetically related, while isolates from human patients were highly diverse. Some dairy plant isolates (18.2%) were closely related to those from dairy farms. Biofilm production and resistance to antimicrobial agents were most typical for isolates from human patients, of which 76.7% were MRSE. Methicillin resistance was also widespread in farm-related isolates (61.1%). This study indicates the possible transmission of S. epidermidis between cattle and farmers. Dairy products were not proven to be an important source of either human infections or methicillin-resistant strains.

For monitoring the occurrence of MRSA (methicillin resistant Staphylococcus aureus) and MR-CNS (methicillin resistant coagulase-negative staphylococci), cow’s, goat’s, and sheep’s milks (bulk milks and individual samples) were investigated. Human nasal and throat swabs of the farm staff and nasal swabs of animals were also investigated as well. In total 1729 samples were examined and 634 strains were isolated by means of the cultivation method and used in this study. Generic identification of the staphylococci isolates was done performed by biochemical tests and all S. aureus and CNS isolates were checked by the PCR method for the presence of mecA gene which is responsible for methicillin resistance. The presence of the staphylococcal cassette chromosome mec (SCCmec), Panton-Valentine leukocidin (pvl) and genes encoding toxic shock syndrome toxin (tst) was detected in all strains confirmed as MRSA. The species were also examined for antimicrobial susceptibility by using disk diffusion method with antibiotic disks. S. aureus was the most frequently identified species from the samples tested (n = 557; 32.2%), followed by S. haemolyticus (n = 32; 1.9%), S. chromogenes (n = 24; 1.4%), S. epidermidis (n = 20; 1.2%), and S. caprae (n = 1; 0.16%). Among the resistant staphylococci (n = 49), S. aureus (n = 25; 51%) was found the most frequently, followed by S. epidermidis (n = 17; 34.7%), S. chromogenes (n = 6; 12.2%), and S. haemolyticus (n = 1; 2%). The resistant Staphyloccocus sp. occurred mainly in cow’s milk (MRSA, S. epidermidis, S. chromogenes, S. haemolyticus) and in animal’s swabs (S. epidermidis). One MRSA was also found in goat’s milk and one was isolated from human swab. No resistant strains were found in sheep’s milk. The negative results of the analysed genes presence (pvl, tst) were identical with all MRSA tested. The staphylococcal cassette chromosome mec (SCCmec) was classified as type IV or V.

The aim of this study was to determine both the occurrence and the genetic basis of resistance to erythromycin among 1 235 <I>Staphylococcus</I> spp. isolates obtained between 2000 and 2006 from (a) raw milk and meat (1 704 samples), (b) foodstuffs produced from these (451 samples), and (c) contact surfaces at processing plants and dairy farms (363 samples) in the Czech Republic. Isolates were screened by broth microdilution method for resistance to erythromycin and further 11 antimicrobial agents. In addition, isolates were screened by agar dilution (erythromycin range 1–128 mg/l) and D-zone test for inducible resistance to macrolides, lincosamides and streptogramin B (iMLS_B). Forty isolates were found to be either resistant, or intermediate, to erythromycin (3.2% of isolates); of these, more than 50% were identified as <I>S. epidermidis</I>. A total of 15 (1.2%) resistant isolates of staphylococci originated from foodstuffs. Resistance mediated by methylation – i.e. iMLS_B-resistance (10 isolates with the <I>erm</I>(A) or <I>erm</I> (C) gene) and constitutive MLS_B-resistance (one isolate with the <I>erm</I> (B) and <I>erm</I> (C) genes) – exhibited a significantly high level of resistance to erythromycin with minimum inhibitory concentrations (MIC) of 64 – >128 mg/l (MIC_mode = >128 mg/l). In contrast, the efflux mechanism encoded by the <I>msr</I>(A) gene (13 isolates; MIC_range = 4–128, MIC_mode = 128 mg/l), the inactivation mechanisms of resistance encoded by the <I>mph</I>(C) gene (three isolates; MIC_range = 8–32 mg/l), and/or their combination (13 isolates; MIC_range = 4–128, MIC_mode = 64 mg/l) led to lower MIC values. The efflux gene <iomsr</I>(A) dominated among the erythromycin-resistant isolates (65% of resistant isolates). This first report on the resistance of <I>Staphylococcus</I> spp. to erythromycin in the Czech Republic illustrates that, while occurrence was low, isolates from food were nevertheless carriers of <I>erm</I> (A), <I>erm</I> (B), <I>erm</I> (C),<I> msr</I>(A) and <I>mph</I>(C) genes for resistance to erythromycin and, therefore, represent a potential thread to humans.