Antibiotic Production by Cutaneous Brevibacterium sp.

Three isolates of a Brevibacterium species isolated from human skin produced an antibiotic active against Gram positive and Gram negative bacteria. The active compound is most probably a peptide with'a molecular weight in the range 1100–1400; there is chromatographic similarity to compounds such as tyrothricin. Production of such a compound in vivo could contribute to microbial interaction in, for example, tinea pedis.     

Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N₃), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.     

大熊猫源细菌耐药性研究进展

耐药菌和耐药基因已成为一种新型环境污染物,引发世界公共卫生问题。细菌耐药性尤其是多重耐药菌在人医临床、畜禽养殖以及环境传播等多个方面得到越来越多的关注,而关于大熊猫等野生动物的耐药性研究相对较少。大熊猫(Ailuropoda melanoleuca)是世界公认的珍稀野生动物,其种群数量易受到各种疾病的威胁,尤其是肠道细菌性疾病。随着抗菌药物在疾病预防和控制中的普遍使用,由此带来的耐药性危害日益明显。本文总结了关于大熊猫源细菌耐药的国内外研究报道,对其耐药表型、耐药基因型、耐药机制及水平传播机制等方面内容进行了综述,旨在为大熊猫源细菌耐药性的研究和防控提供依据,为临床科学用药提供理论参考,从而助力大熊猫迁地保护。     

A Novel Transport Mechanism for MOMP in Chlamydophila pneumoniae and Its Putative Role in Immune-Therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE^−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.     

Antimicrobial activity of Coniothyrium minitans and its macrolide antibiotic macrosphelide A

Aims: Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A. Methods and Results: Thirteen isolates of C. minitans were tested for ability to inhibit a number of filamentous fungi, yeasts, oomycetes and bacteria in agar based tests. Activity was found against some ascomycetes, basidiomycetes, oomycetes and Gram‐positive bacteria, but not against zygomycetes, yeasts or Gram‐negative bacteria tested. Six C. minitans isolates (Conio, Contans, IVT1, CM/AP/3118, B279/1, A1/327/1) were found to produce macrosphelide A in liquid culture and no other antibiotics were detected. On agar, macrosphelide A inhibited growth of some ascomycetes, basidiomycetes, oomycetes and all four Gram‐positive bacteria tested, including the medically important Staphylococcus aureus with a minimum inhibitory concentration of ≤500 μg ml^?1. There was no inhibition observed against the yeasts and Gram‐negative bacteria when macrosphelide A was tested at 700 μg ml^?1. Conclusions: The spectrum and level of activity of macrosphelide A produced by C. minitans against micro‐organisms are extended markedly compared to previous reports. Significance and Impact of the Study: Macrosphelide A was effective against Staph. aureus. Further study on the control of this bacterium is merited in view of the development of antibiotic resistance.     

Reservoirs of Antibiotic Resistance Genes

A potential concern about the use of antibiotics in animal husbundary is that, as antibiotic resistant bacteria move from the farm into the human diet, they may pass antibiotic resistance genes to bacteria that normally reside in a the human intestinal tract and from there to bacteria that cause human disease (reservoir hypothesis). In this article various approaches to evaluating the risk of agricultural use of antibiotics are assessed critically. In addition, the potential benefits of applying new technology and using new insights from the field of microbial ecology are explained.     

Reservoirs of antibiotic resistance genes

A potential concern about the use of antibiotics in animal husbundary is that, as antibiotic resistant bacteria move from the farm into the human diet, they may pass antibiotic resistance genes to bacteria that normally reside in a the human intestinal tract and from there to bacteria that cause human disease (reservoir hypothesis). In this article various approaches to evaluating the risk of agricultural use of antibiotics are assessed critically. In addition, the potential benefits of applying new technology and using new insights from the field of microbial ecology are explained.     

Screening and selection of novel animal probiotics isolated from bovine chyme

Probiotics, gut-colonizing microorganisms capable of conferring a number of health benefits to their hosts, are highly desirable as animal feed supplements. Members of the Gram-positive genus Bacillus are often utilized as probiotics, since endospores formed by those bacteria render them highly resistant to environmental extremes and therefore capable of surviving gastrointestinal tract conditions. In this study, 84 distinct bacterial colonies were obtained from bovine chyme and 29 isolates were determined as Bacillus species. These isolates were principally screened for their antimicrobial activity against a group of two Gram-positive and four Gram-negative bacteria, including known human and animal pathogens such as Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Seven strains displaying strong antimicrobial activity against the test cohort were further evaluated for other properties desirable from animal probiotics, including high spore-forming capacity and adhesiveness, resistance to pH extremes and ability to form biofilms. The isolates were found to resist simulated gastrointestinal conditions and most of the antibiotics tested. In addition, plasmid presence was checked and cytotoxicity tests were performed to evaluate the potential risks of antibiotic resistance transfer and unintended pathogenic effects on host, respectively. We propose that the bacterial isolates are suitable for use as animal probiotics.     

The Pharmaco –, Population and Evolutionary Dynamics of Multi-drug Therapy: Experiments with S. aureus and E. coli and Computer Simulations

There are both pharmacodynamic and evolutionary reasons to use multiple rather than single antibiotics to treat bacterial infections; in combination antibiotics can be more effective in killing target bacteria as well as in preventing the emergence of resistance. Nevertheless, with few exceptions like tuberculosis, combination therapy is rarely used for bacterial infections. One reason for this is a relative dearth of the pharmaco-, population- and evolutionary dynamic information needed for the rational design of multi-drug treatment protocols. Here, we use in vitro pharmacodynamic experiments, mathematical models and computer simulations to explore the relative efficacies of different two-drug regimens in clearing bacterial infections and the conditions under which multi-drug therapy will prevent the ascent of resistance. We estimate the parameters and explore the fit of Hill functions to compare the pharmacodynamics of antibiotics of four different classes individually and in pairs during cidal experiments with pathogenic strains of Staphylococcus aureus and Escherichia coli. We also consider the relative efficacy of these antibiotics and antibiotic pairs in reducing the level of phenotypically resistant but genetically susceptible, persister, subpopulations. Our results provide compelling support for the proposition that the nature and form of the interactions between drugs of different classes, synergy, antagonism, suppression and additivity, has to be determined empirically and cannot be inferred from what is known about the pharmacodynamics or mode of action of these drugs individually. Monte Carlo simulations of within-host treatment incorporating these pharmacodynamic results and clinically relevant refuge subpopulations of bacteria indicate that: (i) the form of drug-drug interactions can profoundly affect the rate at which infections are cleared, (ii) two-drug therapy can prevent treatment failure even when bacteria resistant to single drugs are present at the onset of therapy, and (iii) this evolutionary virtue of two-drug therapy is manifest even when the antibiotics suppress each other''s activity.     

广州儿童医院重症监护病房感染病原菌的分布及耐药性分析

目的探讨儿科重症监护病房（PICU）感染病原菌的分布及耐药情况,为临床合理选用抗菌药提供参考。方法对广州市儿童医院PICU病房2003年11月-2005年10月各类感染标本所分离的病原菌的分布及耐药性进行回顾性分析。结果共检出295株病原菌,其中革兰阴性杆菌213株（72．2％）,主要为铜绿假单胞菌、不动杆菌等非发酵菌;革兰阳性球菌58株（19．7％）,主要为葡萄球菌;真菌24株（8．1％）。药敏结果提示铜绿假单胞菌及不动杆菌对亚胺培南、头孢哌酮／舒巴坦、环丙沙星及阿米卡星较为敏感,铜绿假单胞菌对头孢噻肟耐药率较高,而不动杆菌对头孢哌酮、氨曲南、庆大霉素耐药严藿。肠杆菌科细菌对氨苄西林、氨苄西林／舒巴坦、哌拉西林及多种头孢菌素耐药率较高而对亚胺培南、头孢哌酮／舒巴坦、阿米卡星等较敏感。葡萄球菌对青霉素、红霉素严重耐药,但对万古霉素、替考拉宁及阿米卡星敏感性高。结论铜绿假单胞菌等非发酵菌已成为PICU病房感染的主要病原菌。根据病原菌种类及药敏结果合理应用抗菌药是有效控制危重病患儿感染和减少耐药菌株产生的重要手段。     

Current status and emerging role of glutathione in food grade lactic acid bacteria

ABSTRACT: Lactic acid bacteria (LAB) have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms.     

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.     

Bacterial evolution and the cost of antibiotic resistance.

Bacteria clearly benefit from the possession of an antibiotic resistance gene when the corresponding antibiotic is present. But do resistant bacteria suffer a cost of resistance (i.e., a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put resistance genes into naive bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments (in vitro and in vivo) show that the cost of antibiotic resistance can be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.     

Bacterial outer membrane constriction

The outer membrane of Gram‐negative bacteria is a crucial permeability barrier allowing the cells to survive a myriad of toxic compounds, including many antibiotics. This innate form of antibiotic resistance is compounded by the evolution of more active mechanisms of resistance such as efflux pumps, reducing the already limited number of clinically relevant treatments for Gram‐negative pathogens. During cell division Gram‐negative bacteria must coordinate constriction of the outer membrane in conjunction with other crucial layers of the cell envelope, the peptidoglycan cell wall and the inner membrane. Coordination is crucial in maintaining structural integrity of the envelope, and represents a highly vulnerable time for the cell as any failure can be fatal, if not least disadvantageous. However, the molecular mechanisms of cell division and how the biogenesis of the three layers is synchronised during constriction remain largely unknown. Perturbations of the outer membrane have been shown to increase the effectiveness of antibiotics in vitro, and so with improved understanding of this process we may be able to exploit this vulnerability and improve the effectiveness of antibiotic treatments. In this review the current knowledge of how Gram‐negative bacteria facilitate constriction of their outer membranes during cell division will be discussed.     

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

Background

Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.

Methods

Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.

Results

In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI ?92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI ?57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.

Conclusions

In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.     

Increasing Awareness about Antibiotic Use and Resistance: A Hands-On Project for High School Students

Background

Health-promoting education is essential to foster an informed society able to make decisions about socio-scientific issues based on scientifically sustained criteria. Antibiotic resistance is currently a major public health issue. Considering that irrational antibiotic use has been associated with the development and widespread of antibiotic resistant bacteria, educational interventions to promote prudent antibiotic consumption are required.

Methodology/Principal Findings

This study focuses on the outcomes of an interventional program implemented at the University of Porto, Portugal, to promote awareness about antibiotic resistance at high school levels (15–17 year old). The project Microbiology recipes: antibiotics à la carte articulates a set of wet and dry lab activities designed to promote the participants’ understanding of concepts and processes underlying antibiotics’ production and activity, such as the notion of mechanisms of action of antibiotics. Following a mix-method approach based on a pre−/post design, the effectiveness of this project was assessed by gathering data from surveys, direct observation and analysis of artifacts of 42 high school students (aged 15 and 16 years). The results indicate that the participants developed a more comprehensive picture of antibiotic resistance. The project was shown to promote more sophisticated conceptualizations of bacteria and antibiotics, increased awareness about the perils of antibiotic resistance, and enhanced consciousness towards measures that can be undertaken to mitigate the problem. The participants regarded their experiences as enjoyable and useful, and believed that the project contributed to improve their understanding and raise their interest about the issues discussed. Furthermore, there were also improvements in their procedural skills concerning the laboratory techniques performed.

Conclusions/Significance

This study evidences the possibility of increasing high school students’ awareness about the consequences of antibiotic resistance and the importance of judicious antibiotic use. The findings inform about the educational benefits of incorporating hands-on activities in science education programs.     

‘Inverted’ analogs of the antibiotic gramicidin S with an improved biological profile

A series of gramicidin S derivatives 4–15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.