Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.     

Detecting the Enterotoxigenicity of Staphylococcus aureus Strains

An optimal sensitivity plate method for examining large number of staphylococcal strains for production of the known enterotoxins (A-E) is presented. Small volumes of relatively concentrated enterotoxin are produced by the semi-solid agar, cellophane-over-agar, or sac culture techniques. Detection of the enterotoxin in the supernatant fluid is accomplished with the optimal sensitivity plate method. In this method small plastic petri dishes (50 mm) were used for a modified Ouchterlony of high sensitivity.     

Biochemical Characteristics and Enterotoxigenicity of Staphylococcus aureus Strains Isolated from Poultry

About half (49%) of strains of Staphylococcus aureus isolated from poultry were non-typable with the international human set of phages, and 55% were biotype B according to the biochemical identification scheme of Hájek & Maršálek (1971, 1973). A furthest neighbour clustering strategy and principal coordinate analysis based on 17 biochemical tests made clear distinctions between biotype B strains and a group of biotype A and intermediate strains. Overall 62% of strains were enterotoxigenic, the majority producing enterotoxin A. Significantly fewer intermediate strains than biotype A or B strains were enterotoxigenic. Starch gel zymograms of intracellular esterases showed a general correlation with the biotyping and phage typing results.     

Isolation and Characterization of Methicillin-Resistant Staphylococcus aureus Strains from Louisiana Retail Meats

We investigated the prevalence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in 120 retail meat samples from 30 grocery stores in Baton Rouge, LA. S. aureus strains were recovered from 45.6% of pork samples and 20% of beef samples, whereas MRSA strains were isolated from six meat samples (five pork samples and one beef sample). The MRSA isolates were of two strain types (clones), one harboring Panton-Valentine leucocidin and belonging to pulsed-field gel electrophoresis type USA300 and the other one belonging to USA100.     

Antimicrobial Resistance of Staphylococcus aureus Strains Acquired by Pig Farmers from Pigs

Carriage of animal-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) is common among pig farmers. This study was conducted (i) to investigate whether pig farmers are colonized with pig-specific S. aureus genotypes other than CC398 and (ii) to survey antimicrobial resistance of S. aureus isolates from pigs and pig farmers. Forty-eight S. aureus isolates from pig farmers and veterinarians and 130 isolates from pigs collected in Western Switzerland were genotyped by spa typing and amplified fragment length polymorphism (AFLP). Antimicrobial resistance profiles were determined for representative sample of the isolates. The data obtained earlier on healthy S. aureus carriers without exposure to agriculture were used for comparison. The genotype composition of S. aureus isolates from pig farmers and veterinarians was similar to isolates from pigs with predominant AFLP clusters CC398, CC9, and CC49. The resistance to tetracycline and macrolides (clarithromycin) was common among the isolates from farmers and veterinarians (52 and 21%, respectively) and similar to resistance levels in isolates from pigs (39 and 23%, respectively). This was in contrast to isolates from persons without contact with agriculture, where no (0/128) isolates were resistant to tetracycline and 3% of the isolates were resistant to clarithromycin. MRSA CC398 was isolated from pigs (n = 11) and pig farmers (n = 5). These data imply that zoonotic transmission of multidrug-resistant S. aureus from pigs to farmers is frequent, and well-known MRSA transmission merely represents the tip of the iceberg for this phenomenon. We speculate that the relatively low frequency of MRSA isolation is related to lower antimicrobial use in Switzerland compared to, for example, the Netherlands.     

Comparison of Several Selective Media for Isolation and Differentiation of Coagulase-Positive Strains of Staphylococcus aureus

Six coagulase-positive strains of Staphylococcus aureus which had been cultivated in Brain Heart Infusion broth, milk, and brine were plated on seven isolation media. A significant difference in the growth patterns of the individual strains was found as well as a significant effect resulting from the previous cultivation history before plating. Brine and, to a lesser extent, milk were found to reduce maximal cell concentrations attained, but strains grown in brine and milk showed greater ability to withstand the selective action of the isolation media. Fibrinogen applied to the surface of five of the media allowed the formation of characteristic halos by coagulase-positive strains of S. aureus. Only half of the strains studied produced a zone of precipitation on SM110-Egg Yolk agar. The isolation medium containing cycloheximide and a high level of polymxin B was most inhibitory to the organisms.     

Lactate Dehydrogenase Activity in Certain Strains of Staphylococcus aureus

Lactate dehydrogenase (LDH) was studied in phage-propagating strains 29, 3A, 6, 81, and 42D of Staphylococcus aureus selected from the five groups in the International-Blair series. Cells were cultivated in Brain Heart Infusion (Difco) under nearly anaerobic conditions and were harvested near the end of the log phase. LDH activity was maximal at the end of the exponential growth period and was measured spectrophotometrically by reduction of p-nitro-blue tetrazolium, with phenazine methosulfate as a coupling agent. Crude enzyme extracts were prepared both by an acetone extraction technique and by sonic treatment. LDH activity for these enzyme preparations was determined by the colorimetric method mentioned and also by measuring the rate of nicotinamide adenine dinucleotide reduction at 340 mmu. The order of activity observed, by use of both assay methods, was 29 > 81 > 6 > 3A > 42D. LDH forms (possibly isoenzymes) for each of 15 strains, which represent the five phage-propagating groups of the International-Blair series, were separated by acrylamide gel electrophoresis. Five forms were distinguished and arbitrarily numbered on the basis of their rate of migration, no. 5 being the slowest component. No one strain had more than four, nor fewer than two, LDH forms. Form 3 appeared in 13 of the 15 strains and was followed in frequency by no. 2, 1, 4, and 5.     

Derivation of High Enterotoxin B-Producing Mutants of Staphylococcus aureus from the Parent Strains

Certain pH-sensitive (membrane) mutants of Staphylococcus aureus, strains 14458 and 778, produce significantly more type-B enterotoxin (SEB) than the parent type. Some carbohydrate mutants (car) from these parent strains also are superior to the parent in SEB formation. By isolating car mutants from high-SEB-producing membrane mutants, it is possible to derive a double mutant producing from six to 50 times as much SEB as the parent type. Inversion of the sequence by isolating pH-sensitive mutants from car mutants does not yield clones with strikingly higher SEB production than the parent strain. The successful isolation sequence (pH-sensitive mutant first and car mutants derived from it) is relatively simple and virtually assures detection of a truly high-SEB-producing clone. The total number of clones whose direct assay for SEB formation is necessary for detection of a high-producing mutant is on the order of 50 to 60.     

The Incidence of Enterotoxin Production in Strains of Staphylococcus aureus Isolated from Foods

S ummary . The production of staphylococcal enterotoxins A, B, C, D and E among 200 strains of Staphylococcus aureus was surveyed using a double diffusion immunoprecipitation technique. Enterotoxin A was found to occur most frequently and enterotoxin B least frequently. The distribution of enterotoxigenicity in strains isolated from meat products differed from that of strains isolated from dairy products. The correlations of porcine plasma coagulation and of bacteriophage pattern with enterotoxigenicity were determined.     

An Infrared Spectrophotometric Study of Some Strains of Staphylococcus aureus

A method of obtaining expanded differential spectra of staphylococcal strains for comparative studies is described. Some differences found through the comparison of a large number of strains isolated from humans, turkeys, and other hosts are shown. No spectral characteristics that would associate a particular strain with its host were discerned. Differences in the strength of a band at 11.6 to 11.7 μ allowed division of the strains examined into two groups.     

Production of the Bsa Lantibiotic by Community-Acquired Staphylococcus aureus Strains

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.Staphylococcus aureus can be a human commensal bacterium, colonizing the skin and mucosal surfaces such as the nares, pharynx, and vagina in approximately 25 to 40% of the population. However, it is also a human pathogen that can cause epidemics of invasive disease. Genome sequencing of S. aureus strains has highlighted that the species is highly clonal, with approximately 78% of the genes being conserved and representing the core genome. The remaining 22% of the genes, which are variable and include those present on genomic islands, pathogenicity islands, prophages, integrated plasmids, and transposons, can in turn be regarded as an accessory genome (for a review, see reference 19) that provides a means via which S. aureus can evolve to adapt to particular niches and environmental pressures. The environmental pressure that has most strongly influenced S. aureus evolution in the past century has been the development and application of different antibiotics. These advancements have dictated that the strains that have flourished in hospitals, most notably hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strains, tend to be multidrug resistant but suffer from a concomitant reduction in fitness relative to isolates from the community, due to being encumbered with staphylococcal cassette chromosome mec (SCCmec) types I to III and additional antibiotic resistance genes (48, 55). The negative consequences of this reduction in fitness are, however, mitigated by the reduction in competition from the human commensal microbiota by antibiotic exposure.Since the late 1990s, MRSA infections have been detected among the general population and among healthy individuals (typically children and young adults) who lack traditional risk factors (26). It was apparent that the S. aureus strains responsible for these community-acquired MRSA (CA-MRSA) infections were genetically distinct from their HA counterparts, possessing the more simple type IV (and to a lesser extent, type V and VII) allelic versions of SCCmec (13, 55) and fewer antibiotic resistance genes (20). While this fact indicated that these strains might represent less of a health care challenge than the HA strains, it quickly became apparent that the enhanced competitiveness of these strains, resulting in rapid growth (CA-MRSA strains grow much faster than HA-MRSA strains) (4) and increased virulence (67) of CA-MRSA, meant that any delay in switching from the β-lactam antibiotics normally used to treat infections of unknown etiology could have very serious medical implications, including death. Indeed, paradoxically, CA-MRSA strains have since spread to hospitals and have been responsible for a number of infections.In contrast to HA-MRSA strains, which by virtue of their multidrug-resistant nature, coupled with exposure to antibiotics, have a selective advantage over other microorganisms in the hospital environment, CA-MRSA strains, like commensal S. aureus strains, often face stiff competition from the natural flora of healthy individuals. It has been speculated that the production of an antimicrobial compound may provide CA-MRSA isolates with a competitive advantage in such environments (4, 14). The theory was first suggested when sequencing of strain FPR3757 (part of the virulent USA300 clonal group) revealed the presence of bsa (bacteriocin of S. aureus) genes, which resembled those associated with production of the epidermin subgroup of lantibiotics (2, 60). Lantibiotics are ribosomally produced, posttranslationally modified peptide antibiotics that are generally active against bacterial species which are closely related to the producing organism, and these antimicrobials are thought to have a role in niche competition in many natural environments (41). Lantibiotics have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications (10, 72). Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, there have also been a few examples of lantibiotic production by pathogens (11, 46, 69). In this instance, despite the identification of the bsa genes, the production of a lantibiotic by CA-MRSA isolates has remained speculative. Indeed, to date, there has been only one confirmed example of a lantibiotic, i.e., staphylococcin C55 (46), produced by S. aureus and no definitive evidence that CA- (or HA)-MRSA strains produce such compounds. There is, however, some evidence to suggest that staphylococcin Au-26, which is produced by a vaginal isolate of S. aureus and has an inhibitory spectrum encompassing lactobacilli isolated from the endocervix and representative strains of Staphylococcus hominis, Staphylococcus warneri, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus mutans, Lactococcus spp., and oral Neisseria spp., may also be a lantibiotic (63). Here, 17 years after its initial characterization, we have carried out a closer inspection of staphylococcin Au-26 and the associated producer and have established that the staphylococcin Au-26 and Bsa genetic loci are almost identical. Prompted by this finding, we employed a combination of mutagenesis and mass spectrometry (MS) to reveal that these genes are functional in a number of other staphylococci, including a large percentage of CA-MRSA isolates. We suggest that, as a consequence of eliminating competing human microbiota, this lantibiotic contributes strongly to the fitness of these community-associated isolates.     

Lack of Staphylococcal Enterotoxin Production among Strains of Staphylococcus aureus from Bovine Mastitis in Denmark

Staphylococcal enterotoxins (SE’s) are a group of small exoproteins produced by some strains of Staphylococcus aureus. The SE’s, designated A to E according to their antigenic specificities, are important causes of food poisoning worldwide. Milk and dairy products are frequently associated with S. aureus enter-otoxin food poisoning, and it is supposed that infected milk from mastitic animals constitute the main source of enterotoxigenic S. aureus of animal origin (Bryon 1983, Gilmour & Harvey 1990, Bergdoll 1989). Indeed, S. aureus is the most common cause of bovine mastitis worldwide, and if mastitis strains produce SE this makes up an enormous reservoir of potential enterotoxin producers. The production of SE by S. aureus isolated from bovine mastitis have been investigated in several countries (Matsunaga et al. 1993, Kenny et al. 1993, Olson et al 1970, Orden et al. 1992, Olsvik et al. 1981, Adekeye 1980, Garcia et al. 1980, Abbar 1986, Harvey & Gilmour 1985). Since no studies have been performed on the prevalence of enterotoxigenic strains of S. aureus isolated from bovine mastitis in Denmark, a well characterized collection of S. aureus (Aarestrup et al. 1995) was investigated with respect to this property.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.