The Genome Sequence of a Type ST239 Methicillin-Resistant Staphylococcus aureus Isolate from a Malaysian Hospital

We report the genome sequence of a healthcare-associated MRSA type ST239 clone isolated from a patient with septicemia in Malaysia. This clone typifies the characteristics of ST239 lineage, including resistance to multiple antibiotics and antiseptics.     

ICU耐甲氧西林金黄色葡萄球菌的基因检测与感染控制

目的:研究耐甲氧西林金黄色葡萄球菌(MRSA)SCCmec基因分型情况并对其耐药谱进行分析。方法:收集临床标本522例,应用多重PCR法对MRSA进行SCCmec基因分型,采用全自动微生物鉴定药敏分析仪进行细菌的鉴定及药敏试验,部分药敏试验采用K-B法。结果:522例中分离出146例MRSA,其中10株为SCCmec I型(6.84%),29株为SCCmec II型(19.86%),103株为SCCmecⅢ型(70.55%),未分型4株(2.74%)。MRSA分离株对奎奴普汀/达福普汀、替考拉宁、复方磺胺甲恶唑、万古霉素和利奈唑胺敏感,SCC mecII型与SCC mecIII型对氯霉素的耐药率分别为27.59%和11.65%,对利福平的耐药率分别为13.79%和2.91%,存在明显的差异性(P0.05),其余均呈高水平耐药。146例MRSA患者中治愈52例,占35.62%;感染相关死亡者12例,占8.22%。结论:ICU耐甲氧西林金黄色葡萄球菌的基因检测以SCC mecIII型为主,且对抗菌药物呈多药耐药。     

Detection of Malaysian methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) clinical isolates using simplex and duplex real-time PCR

The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T _m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.     

天蚕素A-马盖宁杂合肽对耐甲氧西林金黄色葡萄球菌生理代谢影响的研究

研究天蚕素A-马盖宁杂合肽作用耐甲氧西林金黄色葡萄球菌(MRSA)后对细胞生理代谢的影响。利用全波长酶标仪测定杂合肽作用MRSA后对DNA、RNA、总蛋白、β-半乳糖苷酶、碱性磷酸酶的影响,利用溶氧电极测定杂合肽对细胞呼吸作用的影响,通过生物发光分析仪检测杂合肽作用细胞后ATP生产的变化。结果显示,杂合肽作用MRSA后对细胞DNA、RNA、总蛋白的合成能力均出现抑制作用。β-半乳糖苷酶、碱性磷酸酶的表达活性同样受到了明显的抑制。杂合肽作用细胞后呼吸作用明显下降,ATP生产能力受到抑制。杂合肽作用细胞后抑制了胞内部分生物大分子的合成能力、胞内酶的表达活性及细胞的能量代谢功能,通过胞内机制发挥了抑菌作用。     

Comparison of the Virulence of Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) was compared with that of methicillin-sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID₅₀) and to kill 50% of mice (LD₅₀), respectively. The mean ID₅₀ [log₁₀ colony forming units (CFU)] for MRSA strains was 7.1 ± 0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P < 0.001). The mean LD₅₀ (log₁₀ CFU) for MRSA strains was 9.0 ± 0.42, being 1.1 higher than that for MSSA strains (P = 0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD₅₀ for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD₅₀ being insignificant (P = 0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.     

A stochastic mathematical model of methicillin resistant Staphylococcus aureus transmission in an intensive care unit: predicting the impact of interventions

OBJECTIVES: To estimate the transmission rate of MRSA in an intensive care unit (ICU) in an 800 bed Australian teaching hospital and predict the impact of infection control interventions. METHODS: A mathematical model was developed which consisted of four compartments: colonised and uncolonised patients and contaminated and uncontaminated health-care workers (HCWs). Patient movements, MRSA acquisition and daily prevalence data were collected from an ICU over 939 days. Hand hygiene compliance and the probability of MRSA transmission from patient to HCW per discordant contact were measured during the study. Attack rate and reproduction ratio were estimated using Bayesian methods. The impact of a number of interventions on attack rate was estimated using both stochastic and deterministic versions of the model. RESULTS: The mean number of secondary cases arising from the ICU admission of colonised patients, also called the ward reproduction ratio, R(w), was estimated to be 0.50 (95% CI 0.39-0.62). The attack rate was one MRSA transmission per 160 (95% CI 130-210) uncolonised-patient days. Results were not sensitive to uncertainty in measured model parameters (hand hygiene rate and transmission probability per contact). Hand hygiene was predicted to be the most effective intervention. Decolonisation was predicted to be relatively ineffective. Increasing HCW numbers was predicted to increase MRSA transmission, in the absence of patient cohorting. The predictions of the stochastic model differed from those of the deterministic model, with lower levels of colonisation predicted by the stochastic model. CONCLUSIONS: The number of secondary cases of MRSA colonisation within the ICU in this study was below unity. Transmission of MRSA was sustained through admission of colonised patients. Stochastic model simulations give more realistic predictions in hospital ward settings than deterministic models. Increasing staff does not necessarily lead to reduced transmission of nosocomial pathogens.     

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Antibacterial activity of some salt marsh halophytes and mangrove plants against methicillin resistant Staphylococcus aureus

The antibacterial activity of aqueous and methanol extracts of leaves/shoots of five salt marsh halophytes and six mangroves was studied against methicillin resistant, clinical isolates of Staphylococcus aureus. There was a clear comparability between the salt marsh halophytes and mangroves in their antibacterial action. The mangrove plants possessed higher antibacterial potency than the salt marsh halophytes. The highest activity was recorded with the methanol extract of Excoecaria agallocha followed by the methanol extracts of Aegiceras corniculatum, Lumnitzera racemosa and Ceriops decandra. The minimum inhibitory concentration (MIC) values ranged from 0.125 to 4 mg/mL and 1 to 16 mg/mL for methanol and aqueous extracts, respectively. Further separation of active principle from the potent mangrove plant will be useful for the control of drug resistant strains of S. aureus.     

Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

PMA结合ddPCR检测食品中金黄色葡萄球菌的研究

研究了将叠氮溴化丙锭(PMA)与微滴式数字PCR(ddPCR)技术相结合,用于金黄色葡萄球菌活菌的检测。结果表明,强烈光照15 min,可以使PMA与死菌DNA共价交联,同时钝化游离的PMA;可以有效抑制金黄色葡萄球菌死菌DNAPCR扩增的PMA终浓度为2.0μg/m L;不抑制活菌DNA扩增的PMA最高浓度是5.0μg/m L。在不同死、活菌比例下,PMA-ddPCR可以定量检测活菌,避免了死菌DNA的干扰,本方法的检出限为10 copy/20μL。利用PMA-ddPCR检测人工污染鸡肉样品,最低可检出102cfu/m L的金黄色葡萄球菌。表明PMA-ddPCR方法的灵敏度高。     

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.     

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 °C for 15 min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, α-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4 Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.     

Multiplex PCR for simultaneous detection of enterococcal genes vanA and vanB and staphylococcal genes mecA, ileS-2 and femB

The experimental transfer of the vanA gene cluster from Enterococcus faecalis to Staphylococcus aureus has raised fears about the occurrence of such genetic transfer in clinical isolates of methicillin-resistant staphylococci. Recently, infections by a S. aureus strain carrying the enterococcal vancomycin resistance vanA gene cluster were reported. The possible emergence and dissemination of these strains is a serious health threat and makes optimization of prevention strategies and fast detection methods absolutely necessary. In the present study, we developed a PCR protocol for simultaneous detection of enterococcal vanA and vanB genes , the staphylococcal methicillin and mupirocin resistance markers mecA and ileS-2, and identification of S. aureus. As no vancomycin-resistant S. aureus isolates were available for our study, we used mixtures of enterococcal and staphylococcal colonies that harbored the different resistance markers to show that these genes could be detected simultaneously. This protocol could be used to facilitate the detection and identification of predictable S. aureus or methicillin-resistant strains carrying vanA or vanB.     

耐甲氧西林金黄色葡萄球菌的两种新SCCmec型别及其抗药性研究

为探明本地区耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus)的耐药性、流行病学分布状况及携带的葡萄球菌染色体mec盒(SCCmec)型别,用K-B琼脂扩散法、E-test和多位点PCR,对临床分离的金黄色葡萄球菌菌株进行了SCCmec分型及耐药性测定。结果发现了两种新的SCCmec型别,新1型含Ⅱ型的mecA上游特异性位点B和位于mecA内的M位点以及Ⅲ型的下游位点F,缺乏Ⅱ型上游位点C和下游位点D、G;新2型含Ⅰ、Ⅱ型的上游特异性位点A、B和两个Ⅲ型的下游位点F、H,同样缺乏位点C、D、G,可能分别为原有Ⅱ型和Ⅰ、Ⅱ型与Ⅲ型的基因重组株;且携带有新SCCmec型别的MRSA菌株,其流行病学分布特点及抗药性也与国外已报导的菌株不同,多分自门诊病人,且耐药性高,抗药谱广,值得引起高度重视和关注。     

Structural and Enzymatic Analysis of TarM Glycosyltransferase from Staphylococcus aureus Reveals an Oligomeric Protein Specific for the Glycosylation of Wall Teichoic Acid

Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5′-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism.     

Synergistic effects of the combination of galangin with gentamicin against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antimicrobial killing activity toward methicillin-resistant Staphylococcus aureus (MRSA) has been a serious emerging global issue. New effective antimicrobials and/or new approaches to settle this issue are urgently needed. The oriental herb, Alpinia officinarum, has been used in Korea for several hundreds of years to treat various infectious diseases. As it is well known, one of the active constituents of Alpinia officinarum is galangin. Against the 17 strains, the minimum inhibitory concentrations (MICs) of galangin (GAL) were in the range of 62.5 ~ 125 microg/ml, and the MICs of gentamicin (GEN) ranged from 1.9 microg/ml to 2,000 microg/ml. The fractional inhibitory concentrations (FICs) of GAL, in combination with GEN, against 3 test strains were 0.4, 3.9, and 250 microg/ml, and were all 15.62 microg/ml in GEN. The FIC index showed marked synergism in the value range of 0.19 to 0.25. By determining time-kill curves, also confirmed the low synergism of the GAL and GEN combination against 4 h, 8 h, 12 h, and 24 h cultured MRSA. The time-kill study results indicated a low synergistic effect against 3 test strains. Thus, the mixture of GAL and GEN could lead to the development of new combination antibiotics against MRSA infection.     

PCR Detection of Oxytetracycline Resistance Genes otr(A) and otr(B) in Tetracycline-Resistant Streptomycete Isolates from Diverse Habitats

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc^R) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc^R streptomycetes originated from bulk and rhizosphere soil. Fewer Tc^R streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc^R streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.