The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.

The results of three standard methods (broth dilution, agar dilution, disk diffusion) and an experimental modification of the microdilution method for determination of resistance to ampicillin, cephalotin, cloxacillin, neomycin, novobiocin, penicillin and streptomycin were compared using 151 Staphylococcus aureus isolates obtained from cases of mastitis. The accuracy of the dilution methods was compared by determination of minimum inhibition concentrations (MIC, MIC50, MIC90 and modal MIC) and by assessment of the agreement within the tolerance of +/-1 dilution step in 2-fold dilution series. The results of the dilution methods were further compared with those of the reference disk diffusion method and the strains were classified as sensitive or resistant using the interpretation criteria for human strains. The comparisons indicated that MIC characteristics and the final classification as sensitive or resistant were method-dependent. Resistance to aminoglycoside antibiotics was observed more often when using broth dilution methods, especially when the broth was supplemented with lactose.

A previously healthy 10-year-old girl with a 2-day history of upper respiratory illness and fever rapidly developed respiratory failure and sepsis with leukopenia, and expired despite attempts at resuscitation. Postmortem examination revealed bilateral necrotizing pneumonia and evidence of disseminated intravascular coagulation. Nasopharyngeal swabs and lung tissue submitted to the Centers for Disease Control and Prevention (CDC) were positive for Enterovirus D68 (EV-D68). Blood and lung cultures were positive for methicillin-resistant Staphylococcus aureus (MRSA). The isolates were submitted to the CDC and were found to be positive for the toxin Panton-Valentine leukocidin. We describe a fatality related to invasive toxin-mediated MRSA associated with EV-D68 coinfection, along with the clinical, laboratory, and autopsy findings, which provided important clues, prompting further investigation at the CDC to arrive at the correct diagnosis.

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