Antimicrobial resistance in Staphylococcus aureus

Recognized since 1883 as a common cause of infection, Staphylococcus aureus' preantimicrobial-era bacteremia mortality rate was 82%. The mortality of that era threatens to return as evidence of growing vancomycin resistance undermines the utility of vancomycin therapy. Successful treatment of S. aureus infections requires knowledge of its antimicrobial resistance capacity.     

Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.     

Antimicrobial substances produced by Staphylococcus aureus strains isolated from cattle in Brazil

Among 46 isolates of Staphylococcus aureus obtained from cattle in the State of Paraíba, Brazil, four were shown to produce antimicrobial substances (AMS). The two best AMS producers carried single plasmids of about 8·0 kbp and 50 kbp, respectively, which were designated pRJ34 and pRJ35. Curing experiments and molecular analysis associated the AMS production with the presence of these plasmids in the cells. The biochemical properties exhibited by the AMS suggested that they might be bacteriocins (Bac). The bacteriocin encoded by pRJ34 showed properties identical to those of the bacteriocins encoded by other small staphylococcal Bac plasmids. However, the bacteriocin encoded by the large plasmid pRJ35 has shown some properties which distinguish it from the other bacteriocins of Staph. aureus described so far, suggesting it may be a new member of the staphylococcal bacteriocin family.     

Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus aureus

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward Gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.     

Comparison of Purified Alpha-Toxins from Various Strains of Staphylococcus aureus

Alpha-toxin from five strains of Staphylococcus aureus, including Wood 46, was purified by isoelectric focusing. The alpha-toxins obtained from different strains were similar. The isoelectric point of the purified toxins was 8.65 +/- 0.15. Sharp concentration peaks were not always obtained. In the ultracentrifuge the alpha-toxins migrated usually as three peaks which could be dissociated with propionic acid to yield one peak. A single line of identity was obtained in immunoelectrophoresis when a heterologous antiserum was reacted with the five purified toxins. It was concluded that the widespread use of the Wood 46 strain for the production of alpha-toxin is justified.     

Opsonization of Staphylococcus aureus by Guinea Pig 7S Gamma-Globulin

Opsonization of Staphylococcus aureus to phagocytosis by guinea pig peritoneal exudate cells (PEC) was examined with various isolated serum globulins. Significant enhancement of in vitro phagocytosis was afforded by partially purified 7S_γ1-immunoglobulin. The rate of killing or the rate of ingestion of ³H-thymidine labeled bacteria by PEC was used as an index of phagocytosis. The results indicate that the immunoglobulin can mediate the adsorption of an allogeneic surface independent of a specific antigenic component.     

Antimicrobial Resistance among Enterococci from Pigs in Three European Countries

Enterococci from pigs in Denmark, Spain, and Sweden were examined for susceptibility to antimicrobial agents and copper and the presence of selected resistance genes. The greatest levels of resistance were found among isolates from Spain and Denmark compared to those from Sweden, which corresponds to the amounts of antimicrobial agents used in food animal production in those countries. Similar genes were found to encode resistance in the different countries, but the tet(L) and tet(S) genes were more frequently found among isolates from Spain. A recently identified transferable copper resistance gene was found in all copper-resistant isolates from the different countries.     

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.     

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.     

253株金黄色葡萄球菌耐药现状分析

目的 了解医院金黄色葡萄球菌临床分布情况及其对常用抗菌药物的耐药率,为临床合理使用抗菌药物提供依据.方法 回顾分析医院2010年5月至2011年4月检出的金黄色葡萄球菌,采用VITEK-AMS全自动微生物分析仪进行菌种鉴定和药敏分析.结果 共检出金黄色葡萄球菌253株,菌株的主要来源为痰130株(51.4％)、血液39株(15.4％)、创面24株(9.5％);菌株主要科室分布前3位是神内科35株(13.8％)、ICU30( 11.8％)、脑外科26株(10.3％);其中耐甲氧西林金黄色葡萄球菌( MRSA)为165株(65.2％),MRSA对多种抗菌药物耐药率＞70.0％,MSSA为88株(34.8％),对除青霉素、红霉素外的大多数抗菌药物敏感,未发现耐万古霉素菌株.结论 MRSA检出率高,耐药现状严重,应加强对金黄色葡萄球菌耐药性的监测,并根据药敏试验结果合理使用抗菌药物.     

Macrolide Resistance in Staphylococcus aureus

A heat-inducible mutant, resistant to macrolide antibiotics (Mac), was isolated from Staphylococcus aureus MS537 in which Mac-resistance was induced by subinhibitory concentrations of erythromycin (EM). After induction at 42 C, this mutant acquired a high resistance to both Mac and lincomycin (LMC). Transduction and biochemical studies revealed that spiramycin (SP)-resistance in this mutant was induced by exposure to a high temperature (42 C) or by treatment with EM in broth but not in phosphate buffer. Induction did not take place when chloramphenicol (CM) was added to the induction mixture. Ribosomes from the mutant cultured at 42 C decreased their affinity for SP and consequently polypeptide synthesis on such ribosomes was not inhibited by SP, when compared with those cultured at 30 C. From these results, it was concluded that alteration of ribosomes took place after induction by exposure at high temperatures or by EM-treatment and that the mechanism of SP-resistance after induction was accounted for by a decrease in SP-binding to ribosomes.     

Macrolide Resistance in Staphylococcus aureus

It was demonstrated that spiramycin (SP)-resistance could be related to the decrease in binding of ribosomes to SP and that the SP-binding to ribosomes was related with inhibition of polypeptide synthesis by SP in a cell-free system in staphylococcal strains. These facts were also observed in Mac (macrolide)-inducible strains, in which resistance to Mac antibiotics is enhanced by prior treatment with subinhibitory concentrations of erythromycin. From these results, it was concluded that the mechanism of resistance to Mac antibiotics is accounted for by alteration of ribosomes in staphylococcal strains and that this alteration of ribosomes is caused not only by mutation but also by induction.