Immunological Recognition of Fibronectin-Binding Proteins of Staphylococcus aureus and Staphylococcus capitis,Strain LK499

Antibodies to fibronectin-binding proteins (FnBPs) of Staphylococcus aureus, including binding domain of FnBPA, the D region, or the A-C regions of FnBPB were produced in rabbits and mice. These antibodies were used to characterize cell-associated FnBPs of S. aureus strain Cowan I, S. aureus strain U320 and a coagulase-negative Staphylococcus capitis strain LK499 as well as extracellular FnBPs in culture supernatants of the strain U320. FnBPs of S. aureus were predominantly FnBPA, while FnBPB was hardly detected on the cells or in culture supernatant of these S. aureus strains. Moreover, S. capitis strain LK499 possessed different FnBP(s) compared to S. aureus because the antibodies to S. aureus FnBPs did not recognize FnBP(s) on S. capitis.     

Emergence of vancomycin-intermediate <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> from predominant methicillin-resistant <Emphasis Type="Italic">S. aureus</Emphasis> clones in a Korean hospital

The genetic and epidemiological features of four vancomycin-intermediate Staphylococcus aureus (VISA) isolates obtained from a Korean hospital were evaluated in this study. The VISA isolates were genotyped as sequence type (ST) 5-staphylococcal cassette chromosome mec (SCCmec) II variant (n=2) and ST239-SCCmec III (n=2), which were derived from the predominant methicillin-resistant S. aureus (MRSA) clones in Korean hospitals. One VISA isolate was acquired during vancomycin treatment, whereas three VISA isolates were obtained from the patients who had not previously been exposed to glycopeptides. As VISA is likely to arise from the predominant MRSA clones and may then possibly spread between patients, the emergence of VISA should be monitored with great care in hospitals.     

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.     

The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus

Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2–5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory system VraTSR, that is present in all S. aureus strains.     

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system.     

Staphylococcal alpha‐phenol soluble modulins contribute to neutrophil lysis after phagocytosis

Staphylococcus aureus community‐acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA‐MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA‐MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α‐type and β‐type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time‐lapse microscopy. Phagocytosis of the CA‐MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increasedneutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.     

2016-2017年宁夏地区牛源金黄色葡萄球菌的全基因组分析

[目的] 了解宁夏地区奶牛乳腺炎金黄色葡萄球菌（Staphylococcus aureus,SA）代表菌株的基因组序列基本特征,进一步探究其耐药基因型、毒力及进化关系,为兽医临床防治提供理论依据。[方法] 采用纸片法对97株金黄色葡萄球菌临床分离株进行抗菌药物敏感性试验,同时进行葡萄球菌蛋白A（Staphylococcus aureus protein A,spa）分型、多位点序列分型（multilocus sequence typing,MLST）,根据分型结果选取16株代表菌株进行全基因组测序,并对获得的测序序列进行处理分析。[结果] 药敏试验结果显示97株分离株对18种抗菌药物存在不同程度的耐药,其中9株耐甲氧西林金黄色葡萄球菌（methicillin-resistantStaphylococcus aureus,MRSA）对青霉素、氨苄西林、苯唑西林、头孢噻呋、磺胺异噁唑、红霉素、庆大霉素和克林霉素等8种抗菌药物完全耐药,甲氧西林敏感金黄色葡萄球菌（methicillin-sensitiveStaphylococcus aureus,MSSA）菌株对青霉素、氨苄西林、磺胺异噁唑耐药率较高。耐药基因数据库（antibiotic resistance genes database,ARDB）注释分析显示16株代表菌株共携带21种耐药基因,其中norA、tet38、bacA、mepA的携带率较高,与药敏试验结果具有一定的相关性。毒力基因数据库（virulence factors of pathogenic bacteria,VFDB）注释分析显示所有菌株携带多种与粘附、宿主免疫逃逸、分泌、胞外酶编码、铁摄取等疾病相关的毒力基因,MRSA菌株均携带较多毒力因子,MSSA菌株携带毒力因子数目不等。基因岛预测结果显示16株代表菌株存在不同数量的基因岛且MRSA菌株携带基因岛数目及毒力基因岛较多,但耐药基因岛数目与MSSA差异不明显。SNP分析结果显示部分分离株同源性较高,同源性较高的两株MRSA的全基因组基本序列特征差异较小,携带的耐药、毒力基因情况相似。[结论] 宁夏地区牛源SA分离株耐药性情况严重且具有较高的毒力水平,本研究为家畜相关MRSA（livestock-associated MRSA,LA-MRSA）与MSSA基因组序列信息的比较分析及宁夏地区SA感染的临床防控提供参考依据。     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

A novel family of non-secreted tridecaptin lipopeptide produced by Paenibacillus elgii

Bacteria from the genus Paenibacillus make a variety of antimicrobial compounds, including lipopeptides produced by a non-ribosomal synthesis mechanism (NRPS). In the present study, we show the genomic and phenotypical characterization of Paenibacillus elgii AC13 which makes three groups of small molecules: the antimicrobial pelgipeptins and two other families of peptides that have not been described in P. elgii. A family of lipopeptides with [M?+?H]⁺ 1664, 1678, 1702, and 1717 m/z was purified from the culture cell fraction. Partial characterization revealed that they are similar to tridecaptin from P. terrae. However, they present amino acid chain modifications in positions 3, 7, and 10. These new variants were named tridecaptin G1, G2, G3, and G4. Furthermore, a gene cluster was identified in P. elgii AC13 genome, revealing high similarity to the tridecaptin-NRPS gene cluster from P. terrae. Tridecaptin G1 and G2 showed in vitro antimicrobial activity against Escherichia coli, Klebsiella pneumonia (including a multidrug-resistant strain), Staphylococcus aureus, and Candida albicans. Tri G3 did not show antimicrobial activity against S. aureus and C. albicans at all tested concentrations. An intriguing feature of this family of lipopeptides is that it was only observed in the cell fraction of the P. elgii AC13 culture, which could be a result of the amino acid sequence modifications presented in these variants.

     

A Novel Gene,fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.     

A Long-Term Survey of Methicillin-Resistant Staphylococcus aureus in the Oral Cavity of Children

Methicillin-resistant Staphylococcus aureus (MRSA), an indigenous bacteria in healthy people, often causes nosocomial infections. If the host human becomes compromised, MRSA can cause a serious infection. The long-term colonization of MRSA increases this risk. The purpose of this study was to demonstrate the incidence of S. aureus and MRSA colonization in the oral cavities of healthy children, and to examine the stability of identical strains of MRSA over a long-term period. Fourteen children were examined in two stages (first stage: 1987–88, second stage: 1992–93). Five of the 14 children were negative for S. aureus in both stages, seven children were positive in both stages and two children were positive in only the second stage. The children who were colonized with S. aureus in the first stage always harbored the bacteria in the second stage. Of the seven children that were positive for S. aureus in both stages, three persisted in carrying MRSA. We compared two MRSA strains isolated from the same children in both stages by coagulase typing, antibiogram typing and DNA fingerprinting. In two children, the strains showed the same coagulase types, similar antibiograms and similar DNA fragment profiles. These data strongly suggest that identical strains of MRSA persisted in the oral cavities for more than five years, and that the oral cavity can serve as a reservoir for MRSA with the potential to cause nosocomial infections.     

Mutation in the C-Di-AMP Cyclase dacA Affects Fitness and Resistance of Methicillin Resistant Staphylococcus aureus

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.     

Phylogenetically Diverse Cultivable Fungal Community and Polyketide Synthase (PKS), Non-ribosomal Peptide Synthase (NRPS) Genes Associated with the South China Sea Sponges

Compared with sponge-associated bacteria, the phylogenetic diversity of fungi in sponge and the association of sponge fungi remain largely unknown. Meanwhile, no detection of polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) genes in sponge-associated fungi has been attempted. In this study, diverse and novel cultivable fungi including 10 genera (Aspergillus, Ascomycete, Fusarium, Isaria, Penicillium, Plectosphaerella, Pseudonectria, Simplicillium, Trichoderma, and Volutella) in four orders (Eurotiales, Hypocreales, Microascales, and Phyllachorales) of phylum Ascomycota were isolated from 10 species marine sponges in the South China Sea. Eurotiales and Hypocreales fungi were suggested as sponge generalists. The predominant isolates were Penicillium and Aspergillus in Eurotiales followed by Volutella in Hypocreales. Based on the conserved Beta-ketosynthase of PKS and A domain of NRPS, 15 polyketide synthases, and four non-ribosomal peptides synthesis genes, including non-reducing and reducing PKSs and hybrid PKS–NRPS, were detected in these fungal isolates. A lateral gene transfer event was indicated in the comparison between the phylogenetic diversity of 18S rRNA genes and β-ketoacyl synthase domain sequences. Some fungi, especially those with PKS or NRPS genes, showed antimicrobial activity against P. fluorescens, S. aureus and B. subtilis. It was the first time to investigate PKS and NRPS genes in sponge-associated fungi. Based on the detected antibiotics biosynthesis-related PKS and NRPS genes and antimicrobial activity, the potential ecological role of sponge-associated fungi in the chemical defense for sponge host was suggested. This study extended our knowledge of sponge-associated fungal phylogenetic diversity and their potential roles in the chemical defense.     

Isolation of methicillin-resistant Staphylococcus aureus from caprine mastitis cases

There is no report on isolation of methicillin-resistant Staphylococcus aureus (MRSA) strains from mastitis infection in goats. This study reports two MRSA strains that were isolated from caprine mastitis. A total of 42 Staphylococcus aureus (S. aureus) strains collected from caprine mastitis cases between 2008 and 2009 were examined. Two (4.8%) out of 42 S. aureus strains were identified as MRSA by Kirby–Bauer disc diffusion and mecA polymerase chain reaction (PCR) methods. Based on the coa gene polymorphism, the goat strains were grouped into 6 types. By using rapid amplified polymorphic DNA (RAPD) assay, 10 different patterns were obtained from 42 S. aureus strains, and strains were located in 6 sub-groups. A total of 71% (n = 30) of the strains were clustered in one main group and placed 4 sub-groups by RAPD assay. The two MRSA strains produced identical patterns and distinguished from other S. aureus strains by RAPD method. This paper is the first report of MRSA isolation from caprine clinical mastitis cases.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Typing of Staphylococcal Cassette Chromosome <Emphasis Type="Italic">mec</Emphasis> Encoding Methicillin Resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Strains Isolated at the Bone Marrow Transplant Centre of Tunisia

Staphylococcal Cassette Chromosome mec (SCCmec) is a mobile genetic element that carries the gene mecA mediating the methicillin resistance in staphylococci. It is composed of mec and ccr gene complexes. Six SCCmec types have been defined so far. SCCmec typing of 13 methicillin-resistant Staphylococcus aureus (MRSA) out of 72 (18%) non redundant S. aureus strains recovered in 1998–2007 at the Bone Marrow Transplant Centre of Tunis was carried out. The isolates were identified by conventional methods. Antibiotic susceptibility was determined by oxacillin and cefoxitin disks and oxacillin MIC by E-test. Methicillin resistance was detected by mecA PCR. The SCCmec complex types were determined by PCR. The epidemiology of MRSA has been investigated by PFGE. Among 13 mecA positive strains, 12 were resistant to oxacillin (MIC = 3 to >256 μg/μl) and to cefoxitin and one strain was pre-resistant: susceptible to oxacillin (MIC = 0.19 μg/μl) and to cefoxitin. Hospital-acquired MRSA (HA-MRSA) strains had essentially SCCmec type IV (nine strains) or III (two strains) or I (one strain). One strain shown to carry ccrAB1 and ccrAB2 genes in combination with class B mec. Seven of 13 MRSA strains isolated from 2000 to 2006 were classified with major similarity group A harbored SCCmec type IV.