Inhibition of colony-spreading activity of Staphylococcus aureus by secretion of δ-hemolysin

Staphylococcus aureus spreads on the surface of soft agar, a phenomenon we termed "colony spreading." Here, we found that S. aureus culture supernatant inhibited colony spreading. We purified δ-hemolysin (Hld, δ-toxin), a major protein secreted from S. aureus, as a compound that inhibits colony spreading. The culture supernatants of hld-disrupted mutants had 30-fold lower colony-spreading inhibitory activity than those of the parent strain. Furthermore, hld-disrupted mutants had higher colony-spreading ability than the parent strain. These results suggest that S. aureus negatively regulates colony spreading by secreting δ-hemolysin.     

The Sortase A Substrates FnbpA, FnbpB, ClfA and ClfB Antagonize Colony Spreading of Staphylococcus aureus

Staphylococcus aureus is an important human pathogen that is renowned both for its rapid transmission within hospitals and the community, and for the formation of antibiotic resistant biofilms on medical implants. Recently, it was shown that S. aureus is able to spread over wet surfaces. This motility phenomenon is promoted by the surfactant properties of secreted phenol-soluble modulins (PSMs), which are also known to inhibit biofilm formation. The aim of the present studies was to determine whether any cell surface-associated S. aureus proteins have an impact on colony spreading. To this end, we analyzed the spreading capabilities of strains lacking non-essential components of the protein export and sorting machinery. Interestingly, our analyses reveal that the absence of sortase A (SrtA) causes a hyper-spreading phenotype. SrtA is responsible for covalent anchoring of various proteins to the staphylococcal cell wall. Accordingly, we show that the hyper-spreading phenotype of srtA mutant cells is an indirect effect that relates to the sortase substrates FnbpA, FnbpB, ClfA and ClfB. These surface-exposed staphylococcal proteins are known to promote biofilm formation, and cell-cell interactions. The hyper-spreading phenotype of srtA mutant staphylococcal cells was subsequently validated in Staphylococcus epidermidis. We conclude that cell wall-associated factors that promote a sessile lifestyle of S. aureus and S. epidermidis antagonize the colony spreading motility of these bacteria.     

金黄色葡萄球菌疫苗及其免疫治疗进展

具有多重抗生素耐药性的金黄色葡萄球菌是导致医院感染最常见的致病菌,而目前高致病性耐甲氧西林菌株(MRSA)在社区中的传播使得健康人群也面临极大威胁,因此针对金黄色葡萄球菌的疫苗和免疫治疗的研究迫在眉睫。多数的研究以金黄色葡萄球菌细胞壁锚定蛋白、荚膜多糖、外毒素等为靶点,虽然在一些临床前试验中显示出疫苗和免疫治疗对金葡菌感染具有保护性,但是目前临床试验结果却并不乐观,还没有一个经得起临床检验的疫苗。本文章从临床前试验和临床试验两个方面综述了目前关于金黄色葡萄球菌的疫苗和免疫治疗进展。     

乳酸杆菌代谢产物对引起阴道炎常见细菌的抑制作用的初步探讨

目的探讨乳酸杆菌代谢产物对临床常见引起阴道炎的大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、白色念珠菌、伤寒杆菌和肠球菌的抑菌作用。方法采用营养琼脂平板培养基定量涂菌,国际标准药敏杯给药的药敏试验法,检测乳酸杆菌代谢产物对大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、白色念珠菌、伤寒杆菌和肠球菌的抑菌环的大小。结果乳酸杆菌代谢产物对大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌和伤寒杆菌有明显的抑菌作用,对肠球菌、白色念珠菌无抑菌作用。结论在临床上可应用乳酸杆菌及其制剂调节阴道微生态平衡,治疗细菌性阴道炎。     

Colony spreading in Staphylococcus aureus

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.     

Methicillin-resistant Staphylococcus aureus: a pervasive pathogen highlights the need for new antimicrobial development

Staphylococcus aureus (S. aureus) has entered the spotlight as a globally pervasive drug-resistant pathogen. While historically associated exclusively with hospital-acquired infections in immunocompromised hosts, the methicillin-resistant form of S. aureus has been spreading throughout communities since the 1990s. Indeed, it has now become a common household term: MRSA. S. aureus has developed numerous mechanisms of virulence and strategies to evade the human immune system, including a host of surface proteins, secreted enzymes, and toxins. In hospital intensive care units, the proportion of MRSA-related S. aureus infections has increased strikingly from just 2 percent in 1974 to 64 percent in 2004. Its presence in the community has been rising similarly, posing a significant public health burden. The growing incidence of MRSA unfortunately has been met with dwindling efforts to develop new, more effective antibiotics. The continued emergence of resistant strains of bacteria such as MRSA demands an urgent revival of the search for new antibiotics.     

Use of RAPD to investigate the epidemiology of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> infection in Malaysian hospitals

Summary Randomly ampli.ed polymorphic DNA (RAPD) with four different decamer oligonucleotide primers was performed on 50 clinical Staphylococcus aureus isolates obtained from di.erent hospitals in Malaysia. All the four primers generated polymorphisms in all 50 isolates of S. aureus studied, revealing DNA markers with sizes ranging from 100 to 7000 bp. The dendrogram generated from the RAPD analysis revealed two major groups (Groups I-II) with three clusters each in one group. S. aureus strains isolated from the same hospital were found to be genetically closely related and most of them were placed in the same cluster. In addition RAPD di.erentiated between MRSA and non-MRSA based on the clustering, where all MRSA and non-MRSA were placed in their respective clusters. The RAPD analysis showed that there could be four to .ve clones of S. aureus spreading around Malaysia, of which two clones may be MRSA. The overall genetic distances ranged from 0.088235 to 0.954545 among the isolates. This technique was found to be a simple and e.ective method for epidemiological investigation. Because of these e.cient features, this technique may have more general application for the study of S. aureus infections in hospitals and the community.     

金黄色葡萄球菌肠毒素检测的研究进展

金黄色葡萄球菌(Staphylococcus aureus)是一种重要的人畜共患致病菌,广泛存在于自然界中,其产生的肠毒素(staphylococcal enterotoxins,SE)可通过污染食物而导致食物中毒。目前,随着人们对食品安全重视度的加深,国际上已将肠毒素的检测列入食品检验法规,因此建立灵敏、快速的肠毒素检测方法,是食品安全检测的一项重要研究内容。     

Effects of Staphylococcus aureus lysozyme on human fibroblasts.

The purified lysozyme excreted by Staphylococcus aureus strains promotes elongation and spreading on plastic surfaces and stimulates DNA synthesis of human fibroblasts (WI38). This enzyme also raises twofold the saturation density level of cultures of these cells. It is suggested that the primitive and main effect of lysozyme on fibroblasts is the triggering of morphogenesis.     

Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus in the food industry

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.     

CYCLOHEXIMIDE AS A MEDIA AMENDMENT FOR ENUMERATING BACTERIAL POPULATIONS IN ANIMAL FEEDS

The present study was designed to evaluate cycloheximide as a potential media amendment for differential bacterial and fungal enumeration of animal feeds. The objectives of this study were to determine the effect of cycloheximide on bacterial growth rates and to evaluate its efficacy for the reduction of indigenous spreading fungi when enumerating bacterial populations in three types of feeds and after short or long-term storage. Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens were grown in tryptic soy broth containing cycloheximide to determine its effect on bacterial specific growth rates. Growth rates of B. cereus and S. aureus were significantly decreased by the addition of 600 and 1000 mg/L cycloheximide respectively, but other pure cultures were not significantly influenced by cycloheximide addition. Intrinsic bacterial populations from feed were not significantly affected by cycloheximide additions at concentrations from 10 to 300 mg/L, but the indigenous spreading molds from feeds were significantly decreased by these cycloheximide concentrations and were decreased below detection levels by 300 mg/L of cycloheximide. The addition of 300 mg/L of cycloheximide effectively eliminates fungal growth for accurate enumeration of bacterial populations in feeds.     

LukM/LukF'-PV is the most active Staphylococcus aureus leukotoxin on bovine neutrophils

Staphylococcus aureus is a ubiquitous pathogen causing infections in humans and domestic animals. It is often associated with bovine mastitis. Among secreted virulence factors, the leukotoxins constitute a family of toxins composed of two distinct subunits (class S and F proteins) which induce first Ca2+ influx and subsequent pore formation that allows ethidium entry. As mastitis-causing isolates harbor the genes of at least two, and often three leukotoxins, we compared the biological activities of the purified leukotoxins whose genes are found in mastitis-causing isolates on bovine polymorphonuclear neutrophils (PMN): spreading on a solid support, calcium influx and ethidium entry. In the spreading assay, the homologous pair LukM/LukF'-PV was the most active leukotoxin. Within each class, either S or F, subunits were interchangeable and generated leukotoxins with different specific activity. LukM was also very active when associated with heterologous F subunits. A similar ranking of homologous pairs was also found in the ethidium entry assay: LukM/LukF'-PV > HlgA/HlgB > HlgC/HlgB > LukE/LukD = LukEv/LukDv. In the Ca2+ flux assay, LukM/F'-PV was the most active pair, but gamma-hemolysin (Hlg) was also very efficient. LukEv/Dv was more active (twofold) than LukE/D in the spreading assay, but the two variants showed similar activities in the other two assays. Supposing that spreading and ethidium entry (pore formation) reflect toxic activities on bovine PMN, and Ca2+ influx cell activation, LukM/F'-PV was by far the most cytotoxic leukotoxin, but it was closely followed by gamma-hemolysin for PMN activation. These results suggest that LukM/F'-PV may constitute a particular virulence attribute of mastitis-causing S. aureus strains.     

区级医院重症监护病区耐甲氧西林金黄色葡萄球菌的调查和分析

目的通过对区级医院重症监护病区耐甲氧西林金黄色葡萄球菌(MRSA)感染的流行初期进行调查和分析,以防止MRSA的暴发流行以及能够采取有效治疗措施。方法对2006年12月至2007年1月区级医院重症监护病区患者、医务人员及物体上送检标本中被MRSA感染的病例进行调查和分析。结果从重症监护病区患者、工作人员及物体上采集标本MRSA的感染阳性率分别为25%~47.6%、0%和20%。结论此次有局部暴发趋势的MRSA感染由于发现及时、措施得当被控制住。医院必须加强监督抗生素的合理用药和耐药性的检测,严格医务人员洗手及实行消毒隔离措施,切断传播途径,防止MRSA感染在医院的发生和流行。     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

The rise and fall of bacterial clones: Streptococcus pneumoniae

Globally spreading bacterial strains belong to clonal types that have the capacity to colonize, spread and cause disease in the community. Recent comparative genomic analyses of well-defined clinical isolates have led to the identification of bacterial properties that are required for the successful spread of bacterial clones. In this Review, we discuss the evolution of bacterial clones, the importance of recombination versus mutations for evolution of clones, common methods used to study clonal relationships among bacteria, factors that may contribute to the clonal spread of bacteria and the potential relevance of bacterial clones to clinical disease. We focus on the common pathogen Streptococcus pneumoniae, although other bacteria are also briefly discussed, such as Helicobacter pylori, Staphylococcus aureus and Mycobacterium tuberculosis.     

Sporadic methicillin resistance in community acquired Staphylococcus aureus in Mozambique

This study reports the drug resistance and clonal relationship of 24 Staphylococcus aureus community acquired isolates from patients attending Maputo Central Hospital, Mozambique, during one year (2002-2003). All the isolates produced beta-lactamase, six strains were resistant to tetracycline alone, three were resistant to erythromycin alone and one was resistant to trimethoprim-sulfamethoxazole; 11 were susceptible to all other drugs tested. Only one strain showed a multiple resistance pattern, including methicillin resistance. To investigate the clonal relationships we applied the ERIC AP-PCR and the SmaI PFGE RFLP methods. Overlapping drug resistances with these two molecular profiles, no significant correlation was obtained. The emergence of methicillin resistance in a multiple resistant strain is of great concern for resistance spreading surveillance in Mozambique.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in staphylococci

Although mobile genetic elements have a crucial role in spreading pathogenicity-determining genes among bacterial populations, environmental and genetic factors involved in the horizontal transfer of these genes are largely unknown. Here we show that SaPIbov1, a Staphylococcus aureus pathogenicity island that belongs to the growing family of these elements that are found in many strains, is induced to excise and replicate after SOS induction of at least three different temperate phages, 80alpha, phi11 and phi147, and is then packaged into phage-like particles and transferred at high frequency. SOS induction by commonly used fluoroquinolone antibiotics, such as ciprofloxacin, also results in replication and high-frequency transfer of this element, as well as of SaPI1, the prototypical island of S. aureus, suggesting that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors. Although the strains containing these prophages do not normally contain SaPIs, we have found that RF122-1, the original SaPIbov1-containing clinical isolate, contains a putative second pathogenicity island that is replicated after SOS induction, by antibiotic treatment, of the prophage(s) present in the strain. Although SaPIbov1 is not induced to replicate after SOS induction in this strain, it is transferred by the antibiotic-activated phages. We conclude that SOS induction by therapeutic agents can promote the spread of staphylococcal virulence genes.     

An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.