嗜水气单胞菌ATCC7966 luxS基因缺失株构建及特性研究

目的研究群体感应信号分子AI-2合成酶编码基因luxS对嗜水气单胞菌ATCC 7966生理特性及毒力因子的影响。方法利用同源重组原理,构建含有中间为卡那霉素抗性基因,两侧为luxS基因上、下游同源序列片段的基因敲除质粒,将构建好的质粒转化大肠埃希氏菌SM 10(λpir)感受态细胞,利用接合法将质粒转入嗜水气单胞菌ATCC 7966,通过抗性筛选、PCR和DNA测序确认嗜水气单胞菌luxS基因缺失突变株。比较野生株ATCC 7966和luxS基因缺失突变株生长速度、AI-2合成、胞外蛋白酶合成及生物膜形成的差异。结果嗜水气单胞菌luxS基因缺失突变株ΔluxS构建成功。与野生株相比,突变株生长周期延长,基本丧失合成AI-2和胞外蛋白酶的能力;突变株生物膜形成能力降低,生长24h时生物膜形成能力为野生株的14.5%,36h时生物膜形成能力为野生株的60.0%,突变株生物膜形成速度明显比野生株缓慢,结构更为疏松。结论 luxS基因参与调控嗜水气单胞菌的生长、AI-2合成和毒力因子表达,本研究为进一步研究luxS基因在嗜水气单胞菌致病性中发挥的作用奠定基础。     

Therapeutics and delivery vehicles for local treatment of osteomyelitis

Osteomyelitis, or the infection of the bone, presents a major complication in orthopedics and may lead to prolonged hospital visits, implant failure, and in more extreme cases, amputation of affected limbs. Typical treatment for this disease involves surgical debridement followed by long-term, systemic antibiotic administration, which contributes to the development of antibiotic-resistant bacteria and has limited ability to eradicate challenging biofilm-forming pathogens including Staphylococcus aureus—the most common cause of osteomyelitis. Local delivery of high doses of antibiotics via traditional bone cement can reduce systemic side effects of an antibiotic. Nonetheless, growing concerns over burst release (then subtherapeutic dose) of antibiotics, along with microbial colonization of the nondegradable cement biomaterial, further exacerbate antibiotic resistance and highlight the need to engineer alternative antimicrobial therapeutics and local delivery vehicles with increased efficacy against, in particular, biofilm-forming, antibiotic-resistant bacteria. Furthermore, limited guidance exists regarding both standardized formulation protocols and validated assays to predict efficacy of a therapeutic against multiple strains of bacteria. Ideally, antimicrobial strategies would be highly specific while exhibiting a broad spectrum of bactericidal activity. With a focus on S. aureus infection, this review addresses the efficacy of novel therapeutics and local delivery vehicles, as alternatives to the traditional antibiotic regimens. The aim of this review is to discuss these components with regards to long bone osteomyelitis and to encourage positive directions for future research efforts.     

Impact of environmental biofilms: Industrial components and its remediation

The growth of technology and requirements globally for various commodities has brought about new challenges. Biofilms are aggregations of microbial cells, which contaminate and spoil industrial components and environments. These microbial cells with extracellular polymeric substances colonize living and nonliving surfaces and pose a serious problem for all industries, affecting their processes, leading to a reduction of product quality and economic loss. Industries, such as medical, food, water, dairy, wine, marine, power plants are exposed to biofilm formation. Pipe blockages, waterlogging and reduction of the heat-transfer efficiency, hamper the operating system of plants. Many industries do not set up remedial measures to control biofilm formation as they are not aware of this threat. Various conventional methods to control these biofilms are adopted by industries in their regular workflow, but these are temporary solutions. This calls for further research into remediation of the biofilm and its control for industrial components. This review article addresses the problems of biofilms and proposes solutions for various industrial components. Nanotechnology promises several options, and bring about a new aspect into the industrial economy, by solving the problems of environmental biofilms.     

High Prevalence of Biofilm-Forming MRSA in the Conjunctival Flora in Chronic Dacryocystitis

Objectives: To report the microbiological spectrum of conjunctival flora and prevalence of biofilm-forming Methicillin-resistant Staphylococcus aureus (MRSA) in conjunctival flora in chronic dacryocystitis.

Design: Prospective, case-control study.

Methods: We included patients with unilateral chronic dacryocystitis, and their unaffected eyes as control. Microbiological profile and antibiotic susceptibility of the isolates was determined by standard microbiological procedures. S. aureus isolates were further evaluated for Methicillin resistance by Oxacillin resistance screening agar method and mecA polymerase chain reaction (PCR) and for biofilm synthesis by Congo red agar method, Microtitre plate (MTP) assay, and ica A and ica D PCR.

Results: We found 95 patients with unilateral chronic dacryocystitis. Aerobic Gram-positive isolates (74.2%, n = 72) were more than Gram-negative (25.7%, n = 25) or anaerobic isolates (20.5%, n = 25). S. aureus was most common (46.4%, n = 45), followed by Pseudomonas aeruginosa (10.3%, n = 10). Gram-positive isolates showed highest sensitivity to Linezolid (100%) and higher generation fluoroquinolones. Gram-negative isolates showed good sensitivity (>90%) to all tested antibiotics. S. aureus isolates showed MRSA prevalence as 93.5% and 96.9% by Oxacillin resistance screening agar method and mecA PCR, respectively. Biofilm formation was found in 71.8% MRSA isolates by MTP assay and 58.1% MRSA isolates were resistant to ≥3 classes of antibiotics.

Conclusions: Gram-positive organisms, specifically S. aureus, are the major etiological agent in chronic dacryocystitis. There is high prevalence of MRSA in these isolates and concurrent biofilm formation.     

Salivary Epithelial Cells as Model to Study Immune Response Against Cutaneous Pathogens

The human skin not only provides passive protection as a physical barrier against external injury, but also mediates active surveillance via epidermal cell surface receptors that recognize and respond to potential invaders. Primary keratinocytes and immortalized cell lines, the commonly used sources to investigate immune responses of cutaneous epithelium are often difficult to obtain and/or potentially exhibit changes in cellular genetic make‐up. Here we investigated the possibility of using salivary epithelial cells (SEC) to evaluate the host response to cutaneous microbes. Elevated secretion of IFN‐γ and IL‐12 was observed in the SEC stimulated with Staphylococcus aureus, a transient pathogen of the skin, as mono species biofilm as compared to SEC stimulated with a commensal microbe, the Staphylococcus epidermidis. Co‐culture of the SEC with both microbes as dual species biofilm elicited maximum cytokine response. Stimulation with S. aureus alone but not with S. epidermidis alone induced maximum toll‐like receptor‐2 (TLR‐2) expression in the SEC. Exposure to dual species biofilm induced a sustained upregulation of TLR‐2 in the SEC for up to an hour. The data support novel application of the SEC as efficient biospecimen that may be used to investigate personalized response to cutaneous microflora.     

The potential for sodium hexametaphosphate (SHMP) found in common children drinks to limit acid production in the oral biofilm

OBJECTIVE: Sodium hexametaphosphate (SHMP) is a widely used industrial preservative commonly found in children's drinks. In this paper we examined the effect of SHMP incorporated into children's drinks on acid production by the oral biofilm by monitoring salivary concentrations of lactic acid. MATERIALS AND METHOD: Twelve healthy adult subjects with an average age 36 years (range 26-54 years) consumed 10 ml from four children's beverages (Coca Cola and three types of Sunny Delight supplemented with SHMP) and a standard solution of sucrose. Saliva was collected at intervals following exposure of the oral biofilm to the drinks and the clearance of carbohydrates and the appearance of lactate was measured using standard enzymatic techniques. RESULTS: All the carbohydrates derived from the drinks were cleared from saliva within 15 min of consumption. Comparison of two drinks [Sunny D Normal and Sunny C] with the same carbohydrate, but different SHMP concentrations suggested that SHMP in these beverages had no significant effect on acid production. CONCLUSIONS: In this clinical study the role of SHMP, incorporated in common beverages, did not inhibit acid production from carbohydrates.     

Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation

During dental plaque formation, the interaction of different organisms is important in the development of complex communities. Fusobacterium nucleatum is considered a 'bridge-organism' that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and possibly by making the environment conducive for oxygen intolerant anaerobes. These studies were carried out to determine whether coaggregation between F. nucleatum and Tannerella forsythia is important in the formation of mixed species biofilms. Further, the role of BspA protein, a surface adhesin of T. forsythia, in coaggregation and biofilm formation was investigated. The results showed the development of synergistic mixed biofilms of F. nucleatum and T. forsythia when these bacteria were cocultured. The BspA protein was not involved in biofilm formation. Though BspA plays a role in coaggregation with F. nucleatum, presumably other adhesins are also involved. The synergistic biofilm formation between the two species was dependent on cell-cell contact and soluble components of the bacteria were not required. This study demonstrates that there is a positive synergy between F. nucleatum and T. forsythia in the development of mixed biofilms and that the cell-cell interaction is essential for this phenomenon.     

Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm

As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota, which is often considered a health asset, studies of the oral commensal microbiota have been largely limited to their implication in oral conditions such as dental caries and periodontal disease. Less emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign or pathogenic bacteria. In this study, we used salivary microbiota derived from healthy human subjects to investigate protective effects against colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing or pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into the salivary microbial community during biofilm formation. Furthermore, in saliva medium supplemented with sucrose, the oral microbiota inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign or pathogenic bacterial species, such as P. aeruginosa.