氯氰碘柳胺与环丙沙星对耐甲氧西林金黄色葡萄球菌的体外联合药敏试验分析

目的 评价氯氰碘柳胺与环丙沙星联用对60株环丙沙星均为耐药的耐甲氧西林金黄色葡萄球菌(MRSA)的体外抗菌效应.方法 采用棋盘法设计,微量肉汤稀释法测定不同浓度组合的抗菌药物对60株甲氧西林耐药的金黄色葡萄球菌(MRSA)的最低抑菌浓度,并计算部分抑菌浓度指数(FICI)判定联合效应.结果 氯氰碘柳胺与环丙沙星联合用药后,其FICI分布:FIC≤0.5为20.0％,0.5 ＜FIC≤1为66.7％,1＜FIC≤2为13.3％,FIC＞2为0.结论 体外氯氰碘柳胺与环丙沙星联用对60株耐甲氧西林金黄色葡萄球菌的作用主要为相加和协同作用.     

Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus

We examined the antiseptic susceptibilities and distribution of antiseptic-resistance genes qacA and smr in 98 isolates of methicillin-resistant Staphylococcus aureus obtained in 1992. Seventy-one strains were resistant to antiseptics. The qacA and smr genes were detected in 10 and 20 strains, respectively. The remaining 41 strains without qacA and smr were divided into two groups that exhibited low-level (n = 22) and high-level (n = 19) resistance to acriflavin. DNA cloning and sequencing suggested that norfloxacin-resistance gene norA was responsible for the high-level resistance to acriflavin. Our results indicated that four or more antiseptic-resistance genes exist in methicillin-resistant S. aureus and that antiseptic-resistant methicillin-resistant S. aureus strains without qacA and smr are widely spread in Japan.     

Resistance heterogeneity in methicillin-resistant Staphylococcus aureus

Abstract A striking feature of methicillin resistance in Staphylococcus aureus is the considerable heterogeneity of expression of resistance by cells in clonal populations: some are sensitive (or almost so), others are highly resistant, and others show intermediate resistance to the antibiotic. Subclones generally are also heterogeneous, suggesting variable inheritance or control of expression of resistance.
The degree of heterogeneity and mean resistance is influenced by environmental parameters: temperature, osmolality, pH, light, anaerobiosis, chelating agents and metal ions, and prior exposure to β-lactam antibiotics.     

Key adhesin gene in community-acquired methicillin-resistant Staphylococcus aureus

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) possessing the Panton-Valentine leukocidin (PVL) gene (luk(PV)) is associated with skin and soft tissue infections, osteomyelitis, and necrotizing pneumonia. There are geographically two types of CA-MRSA: one (sequence type ST30) that is worldwide (pandemic) and the other (sequence types, e.g., ST1, ST8 or ST80) that is continent-specific. The pandemic type, but not continent-specific type, possessed the bone sialoprotein-adhesin gene (bbp), which was associated with osteomyelitis. No recent hospital-acquired MRSA had the bbp gene, while past PVL-positive nosocomial outbreak-derived strains did possess it. The collagen-adhesin gene (cna) was associated with pandemic CA-MRSA, though with positive cases even in continent-specific CA-MRSA and PVL-negative Japanese region-specific CA-MRSA. Thus, the pandemic type is characterized by the combination of luk(PV) and bbp (and cna) genes. A specific real-time PCR assay for the bbp gene was developed, and dual assay for bbp and luk(PV) in one test tube became possible.     

Transposition of penicillinase determinants in methicillin-resistant Staphylococcus aureus

Abstract A methicillin-resistant Staphylococcus aureus (MRSA) typical of those being isolated in Australian hospitals has been studied. It contains two plasmids, one of 1.4 megadalton (MDa) and one of 18 MDa. When selection is made for resistance to nucleic acid-binding (NAB) compounds in mixed-culture transfer, two types of transcipients are obtained; those containing an 18-MDa plasmid and resistant to NAB compounds, trimethoprim and aminoglycosides such as gentamicin and kanamycin and those having a 22 MDa plasmid and the additional phenotype of penicillinase production. The penicillinase determinants on the 22-MDa plasmid have been found to transpose to the chromosome and from the chromosome to an 18-MDa plasmid similar to that found in the original isolate. Restriction enzyme analysis has shown that a 7.3-kilobase pair (kb) element is involved. This has been designated Tn 3852 .     

Detection of methicillin-resistant Staphylococcus aureus in Iberian pigs

Aims: Iberian pigs are bred in Spain for the production of high‐value dry‐cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin‐resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. Methods and Results: From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. Conclusions: Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. Significance and Impact of the Study: To our knowledge, this is the first detection of MRSA in food animals raised in free‐range conditions.     

耐甲氧西林金黄色葡萄球菌感染控制策略的新进展

耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus Aureus, MRSA)已成为一种越来越具有侵袭性和流行性的病原菌。通过染色体介导、质粒转移、基因表达调控和主动外排系统等途径,MRSA对包括万古霉素在内的多种抗生素产生了抗药性。从生态学和进化的角度来考虑,仅仅通过利用抗生素本身来解决耐药性的困境是不够的。这就迫使人们去寻找一类完全不同于化学药物治疗MRSA感染的机制。随着对免疫学和生物学认识的不断深入,基于免疫逃逸、细菌群体感应、基因调控等理论的发现,涌现了一批生物制剂抗MRSA感染的研究,相对于传统的抗生素治疗这是一个全新突破的领域。此外还有传统中草药的研发也提示其在抗MRSA方面存在积极的活力。本综述总结了生物制剂、新型策略化学药物和传统中草药治疗MRSA感染的最新进展,以寻找解决抗生素治疗困境的新线索。     

Comparative analysis and validation of different assays for glycopeptide susceptibility among methicillin-resistant Staphylococcus aureus strains

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding <50 CFU (representing <10(-6) of the plated inoculum) on either BHIA containing 2 mg/l of vancomycin (V2-BHIA) or 5 mg/l of teicoplanin (T5-BHIA) was considered as fully susceptible to vancomycin or teicoplanin, respectively. The proportion of MRSA isolates yielding>50 CFU on either V2-BHIA or T5-BHIA significantly (P<0.01) increased from 7/94 (7.4 %) or 8/94 (8.5%) in period A to 16/95 (16.8 %) or 14/95 (14.7%) in period B, respectively. Vancomycin Etests MICs on MHA were of lower sensitivity (<30% and<65%), but high specificity (100% and 99%) on periods A and B isolates, respectively, compared to those on V2-BHIA. Vancomycin Etests MICs on BHIA were of a higher sensitivity (57% and 81%) but lower specificity (91% and 65%) compared to those on V2-BHIA, on periods A and B isolates, respectively, reflecting an unexpectedly high number of false positive isolates on period B isolates (28/95). Screening of decreased susceptibility to vancomycin or teicoplanin on V2-BHIA or T5-BHIA, respectively, may represent simple low-cost alternatives to Etest MICs, minimising the risk of missing potential GISA isolates.     

Antimicrobial susceptibility and clonal relatedness between community- and hospital-acquired methicillin-resistant Staphylococcus aureus from blood cultures

We compared the antimicrobial resistance and clonal relationships among the community-acquired (CA) and hospital-acquired (HA) methicillin-resistant Staphylococcus aureus (MRSA) strains that were isolated from blood cultures in a university hospital over a 4-year period. A total of 131 MRSA isolates, including 28 CA-MRSA and 103 HA-MRSA strains, were identified; antimicrobial susceptibility testing indicated that the CA-MRSA isolates were more susceptible to erythromycin (21% vs 6%; P=0.02), clindamycin (46% vs 12%; P=0.01), ciprofloxacin (43% vs 11%; P=0.01), and gentamicin (43% vs 6%; P=0.01) than were the HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) typing and antimicrobial resistance profiles separated the 20 CA-MRSA isolates into 14 and 10 different patterns, respectively, and the 53 HA-MRSA isolates were separated into 24 and 7 different patterns, respectively. Twenty-one (40%) of the 53 HA-MRSA isolates belonged to two predominant PFGE types, and most of them showed multi-drug resistant patterns. Four (20%) of the 20 CA-MRSA and 10 (19%) of the 53 HA-MRSA isolates fell into two common PFGE patterns, and each of them showed the same multi-drug resistant pattern. This study suggests that, although the CA-MRSA blood isolates showed diverse PFGE and antimicrobial resistance patterns, some of these isolates may have originated from the HA-MRSA strains.     

Presence of methicillin-resistant Staphylococcus aureus in pigs in Peru

We report the first detection of methicillin-resistant Staphylococcus aureus isolates in pigs in Peru. The isolates belong to a livestock-associated lineage previously reported in North America and Europe, CC398, and a highly virulent USA300-like ST8-IV variant, which is the predominant community-associated lineage in Latin America.     

Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.     

Mapping the protein interaction network in methicillin-resistant Staphylococcus aureus

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.     

Typing of methicillin-resistant Staphylococcus aureus with IS256

A simple one-step procedure is described for specifically amplifying and labelling insertion element IS256 which is associated with the gentamicin-resistance transposon Tn4001. The product has been used to probe DNA digests of methicillin-resistant Staphylococcus aureus. The resulting restriction fragment length polymorphisms were found to be able to distinguish isolates which were indistinguishable by other typing methods. The probe also hybridised with methicillin-resistant Staphylococcus aureus which were isolated before the emergence of gentamicin resistance, demonstrating its usefulness in typing species other than those that are gentamicin-resistant.     

和厚朴酚抑制耐甲氧西林金黄色葡萄球菌生物被膜形成

【目的】研究和厚朴酚(HNK)抑制MRSA生物被膜(BF)形成的作用机制。【方法】使用TTC法测定了HNK对供试菌株BF的形成和成熟BF的抑制作用;刚果红平板法定性检测了HNK对PIA合成的影响;分光光度法测定了HNK对供试菌株eDNA释放量的影响;RT-PCR技术检测了HNK对供试菌株icaA、cidA以及agrA基因表达量的影响。【结果】HNK对MRSA 41573 BF的形成和成熟BF均有较强的抑制作用,其中,HNK抑制MRSA 41573 BF形成的MIC和MBC分别为10μg/mL和20μg/mL;抑制成熟BF的MIC和MBC分别为50μg/mL和100μg/mL。当用亚抑菌浓度的HNK与万古霉素联合作用后,可显著提高成熟BF对万古霉素的敏感性。HNK能显著抑制PIA的合成,且呈浓度剂量依赖。HNK能抑制供试菌株eDNA的释放量,其中1/8 MIC的HNK作用供试菌株16 h后,与对照组相比,e DNA的释放量降低了28.3%。HNK可抑制供试菌株BF形成的相关基因,其中1/2 MIC的HNK作用供试菌株16 h后,与对照相比,icaA的表达量降低了59.1%,cidA的表达量降低了56%,agrA的表达量降低了72.3%。【结论】HNK能显著抑制MRSA 41573 BF的形成,其作用机制主要是通过抑制icaA和cidA基因表达量,影响PIA和eDNA的合成,进而抑制BF的形成。此外HNK也可通过调控细菌的QS系统影响BF的形成。     

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL^-1, including 15.7% with an MIC of 8 μg mL^-1 and 2% with an MIC of 16 μg mL^-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.