Classification and distribution of large intestinal bacteria in nonhibernating and hibernating leopard frogs (Rana pipiens).

The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens. Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators. The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species. The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators. Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.     

The contribution of marine aggregate‐associated bacteria to the accumulation of pathogenic bacteria in oysters: an agent‐based model

Bivalves process large volumes of water, leading to their accumulation of bacteria, including potential human pathogens (e.g., vibrios). These bacteria are captured at low efficiencies when freely suspended in the water column, but they also attach to marine aggregates, which are captured with near 100% efficiency. For this reason, and because they are often enriched with heterotrophic bacteria, marine aggregates have been hypothesized to function as important transporters of bacteria into bivalves. The relative contribution of aggregates and unattached bacteria to the accumulation of these cells, however, is unknown. We developed an agent‐based model to simulate accumulation of vibrio‐type bacteria in oysters. Simulations were conducted over a realistic range of concentrations of bacteria and aggregates and incorporated the dependence of pseudofeces production on particulate matter. The model shows that the contribution of aggregate‐attached bacteria depends strongly on the unattached bacteria, which form the colonization pool for aggregates and are directly captured by the simulated oysters. The concentration of aggregates is also important, but its effect depends on the concentration of unattached bacteria. At high bacterial concentrations, aggregates contribute the majority of bacteria in the oysters. At low concentrations of unattached bacteria, aggregates have a neutral or even a slightly negative effect on bacterial accumulation. These results provide the first evidence suggesting that the concentration of aggregates could influence uptake of pathogenic bacteria in bivalves and show that the tendency of a bacterial species to remain attached to aggregates is a key factor for understanding species‐specific accumulation.     

Hydroxyapatite adherence as a means to concentrate bacteria.

Adherence to hydroxyapatite (HA) was examined as a method to concentrate bacteria from foods. Using HA at a level of 10% and suspensions of an Escherichia coli strain containing 10(9), 10(6), and 10(3) cells per ml, kinetic studies revealed that maximum adherence was attained within 5 min for all cell concentrations and that comparable log reductions (1.0 to 1.5) of cells in suspension were seen regardless of initial cell concentration. Eleven species of spoilage and pathogenic bacteria were found to adhere to HA, with seven species adhering at proportions of greater than 95%. Fluorescent viability staining revealed that cells bound to HA remained viable. There was greater than 92% adherence of indigenous bacteria to HA from three of five 1:10 dilutions of ground beef, indicating promise for the use of HA for concentrating bacteria from meat and other food samples.     

Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria

Nodules were formed in the Madeira cockroach, Leucophaea maderae, in response to injections of low doses (3 x 10(4) bacteria/insect) of three strains of Bacillus cereus and Escherichia coli K12 D31. The most pathogenic strain of bacteria used, B. cereus B1, produced the greatest cellular response, while the least pathogenic, E. coli K12 D31, injected at the same dose, caused little nodule formation. Similarly, nodules were generally found to be larger following injection of pathogenic bacteria such as B. cereus B1 than to the weak pathogen, E. coli K12 D31. There was, however, no difference in the extent of nodule formation with the four bacterial strains/species if they were heat killed prior to injection. Histologically, the nodules formed in response to all bacterial species employed were similar, with a central necrotic core enclosing cell debris and occasional bacteria, and an outer, thin sheath of plasmatocyte-like hemocytes. Possible reasons for the enhanced cellular reactivity observed in L. maderae to pathogenic bacteria are discussed.     

Antibiotic Resistance is Widespread in Urban Aquatic Environments of Rio de Janeiro,Brazil

Bacterial resistance to antibiotics has become a public health issue. Over the years, pathogenic organisms with resistance traits have been studied due to the threat they pose to human well-being. However, several studies raised awareness to the often disregarded importance of environmental bacteria as sources of resistance mechanisms. In this work, we analyze the diversity of antibiotic-resistant bacteria occurring in aquatic environments of the state of Rio de Janeiro, Brazil, that are subjected to distinct degrees of anthropogenic impacts. We access the diversity of aquatic bacteria capable of growing in increasing ampicillin concentrations through 16S rRNA gene libraries. This analysis is complemented by the characterization of antibiotic resistance profiles of isolates obtained from urban aquatic environments. We detect communities capable of tolerating antibiotic concentrations up to 600 times higher than the clinical levels. Among the resistant organisms are included potentially pathogenic species, some of them classified as multiresistant. Our results extend the knowledge of the diversity of antibiotic resistance among environmental microorganisms and provide evidence that the diversity of drug-resistant bacteria in aquatic habitats can be influenced by pollution.     

Relationship of periodontal clinical parameters with bacterial composition in human dental plaque

More than 600 bacterial species have been identified in the oral cavity, but only a limited number of species show a strong association with periodontitis. The purpose of the present study was to provide a comprehensive outline of the microbiota in dental plaque related to periodontal status. Dental plaque from 90 subjects was sampled, and the subjects were clustered based on bacterial composition using the terminal restriction fragment length polymorphism of 16S rRNA genes. Here, we evaluated (1) periodontal clinical parameters between clusters; (2) the correlation of subgingival bacterial composition with supragingival bacterial composition; and (3) the association between bacterial interspecies in dental plaque using a graphical Gaussian model. Cluster 1 (C1) having high prevalence of pathogenic bacteria in subgingival plaque showed increasing values of the parameters. The values of the parameters in Cluster 2a (C2a) having high prevalence of non-pathogenic bacteria were markedly lower than those in C1. A cluster having low prevalence of non-pathogenic bacteria in supragingival plaque showed increasing values of the parameters. The bacterial patterns between subgingival plaque and supragingival plaque were significantly correlated. Chief pathogens, such as Porphyromonas gingivalis, formed a network with other pathogenic species in C1, whereas a network of non-pathogenic species, such as Rothia sp. and Lautropia sp., tended to compete with a network of pathogenic species in C2a. Periodontal status relates to non-pathogenic species as well as to pathogenic species, suggesting that the bacterial interspecies connection affects dental plaque virulence.     

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections

A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.     

Altering plant-microbe interaction through artificially manipulating bacterial quorum sensing

Many bacteria regulate diverse physiological processes in concert with their population size. Bacterial cell-to-cell communication utilizes small diffusible signal molecules, which the bacteria both produce and perceive. The bacteria couple gene expression to cell density by eliciting a response only when the signalling molecules reach a critical threshold (a point at which the population is said to be 'quorate'). The population as a whole is thus able to modify its behaviour as a single unit. Amongst Gram-negative bacteria, the quorum sensing signals most commonly used are N-acylhomoserine lactones (AHLs). It is now apparent that AHLs are used for regulating diverse behaviours in epiphytic, rhizosphere-inhabiting and plant pathogenic bacteria and that plants may produce their own metabolites that interfere with this signalling. Transgenic plants that produce high levels of AHLs or which can degrade bacterial-produced AHLs have been made. These plants have dramatically altered susceptibilities to infection by pathogenic Erwinia species. In addition, such plants will prove useful tools in determining the roles of AHL-regulated density-dependent behaviour in growth promoting, biological control and pathogenic plant-associated bacterial species.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

Fluorescent nanoparticles for multiplexed bacteria monitoring

Rapid, sensitive, and selective detection of pathogenic bacteria is extremely important for proper containment, diagnosis, and treatment of diseases like foodborne illness, sepsis, and bioterrorism. Most current bacterial detection methods are time-consuming and laborious and can detect only one bacterial pathogen at a time. We have developed a method for sensitive, multiplexed monitoring of bacterial pathogens within 30 min using multicolored FRET (fluorescence resonance energy transfer) silica NPs (nanoparticles). By varying the ratio of three tandem dyes coencapsulated into the NPs, we have synthesized NPs that emit unique colors upon excitation with a single wavelength. When these NPs were conjugated to monoclonal antibodies specific for the pathogenic bacteria species Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and then incubated with small concentrations of the bacteria, simultaneous and sensitive detection of the multiple bacterial targets was achieved.     

Ectopic gene conversions in bacterial genomes.

We characterized the gene conversions found between the duplicated genes of 75 bacterial genomes from five species groups (archaea, nonpathogenic and pathogenic firmicutes, and nonpathogenic and pathogenic proteobacteria). The number of gene conversions is positively correlated with the size of multigene families and the size of multigene families is not significantly different between pathogenic and nonpathogenic taxa. However, gene conversions occur twice as frequently in pathogenic species as in nonpathogenic species. Comparisons between closely related species also indicate a trend towards increased gene conversion in pathogenic species. Whereas the length of the conversions is positively correlated with flanking sequence similarity in all five groups, these correlations are smaller for pathogenic firmicutes and proteobacteria than for nonpathogenic firmicutes and proteobacteria. These results are consistent with our previous work on E. coli genomes and suggest that pathogenic bacteria allow recombination between more divergent gene sequences. This higher permissiveness is likely adaptive because it allows them to generate more genetic variability.     

女性2型糖尿病患者尿路感染病原菌的分布及耐药性监测

目的探讨女性2型糖尿病患者尿路感染病原菌的种类及耐药性,为临床合理用药提供科学依据。方法对2009年1月至2011年1月在绍兴市人民医院住院的女性2型糖尿病患者尿路感染病原菌的分布及耐药性进行回顾性分析。结果分离的112株病原菌,以大肠埃希菌为主,占58.9%,其次为肺炎克雷伯菌和真菌,分别为10.7%和8.9%;其中产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌株检出率为40.9%,产ESBLs的肺炎克雷伯菌株检出率为8.3%;大肠埃希菌对碳青霉烯类、头霉素类、丁胺卡那和呋喃妥因的耐药率较低,而肺炎克雷伯菌对多种抗菌药物具有较好的敏感性。结论女性2型糖尿病患者易患尿路感染,病原菌以大肠埃希菌为主,且对多种抗菌药物的耐药率较高,临床应根据药敏结果合理选用抗菌药物。     

太子参连作对根际土壤微生物的影响

采用微生物培养法和末端限制性片断长度多态性(T-RFLP)技术分析连作对太子参根际土壤微生物多样性的影响。结果表明:连作导致太子参根际土壤细菌和好气性自生固氮菌数量极显著下降,相反,真菌、放线菌、厌气性纤维素分解菌数量极显著增加,而硝化细菌数量变化不显著。T-RFLP分析显示:与太子参-水稻-太子参轮作的土壤相比,太子参连作的土壤细菌种(属)略有减少,其中致病菌和病原菌种(属)增多,并出现一些具拮抗功能的链霉菌属(种);真菌种(属)则表现出上升的趋势,但未检索到与植物致病相关的真菌种(属)。     

头孢类抗生素对6种G^-致病菌的效价分析

目的：调查头孢类抗生素对G^-致病菌的效价情况,为头孢类抗生素的合理使用提供临床资料。方法：从我院三代常用头孢类抗生素中随机选取7种共21种抗生素,采用平板培养纸片扩散K-B法检测对我院常见的G^-致病菌（铜绿假单胞菌、大肠埃希氏菌、鲍曼不动杆菌、产酸克雷伯菌、肺炎克雷伯菌和阴沟肠杆菌）的效价情况。结果采用阳性（＋）、阴性（＋）、可变（＋／-）表示。结果：第一代头孢类抗生素的效价最低,第二代头孢类抗生素的效价整体居中,第三代头孢类抗生素的效价整体最好,但也有少数种类的效价低于第二代。结论：三代抗生素的对这六种常见致病菌的效价与代数成正比,但第三代头孢类抗生素的效价仍不够乐观。     

The effect of cigarette smoke on adherence of respiratory pathogens to buccal epithelial cells

Smoking is associated with an increased risk of respiratory tract infection in adults. In children, exposure to cigarette smoke is a risk factor for respiratory tract infection and bacterial meningitis: Active smoking and passive exposure to cigarette smoke is also associated with carriage of some potentially pathogenic species of bacteria in both adults and children. The aims of the study were to determine the effect of active smoking on: (1) bacterial binding to epithelial cells; (2) expression of host cell antigens that act as receptors for some species; and (3) the effects of passive exposure to water-soluble components of cigarette smoke on bacterial binding. Flow cytometry was used to assess binding to buccal epithelial cells of the following species labelled with fluorescein isothiocyanate: Neisseria meningitidis, Neisseria lactamica, Streptococcus pneumoniae, Bordetella pertussis, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus. Flow cytometry was also used to assess expression of host cell antigens which have been identified as bacterial receptors. For each species, binding to cells of smokers was significantly higher than to cells of non-smokers; however, expression of host cell antigens was similar on epithelial cells of both groups. Non-dilute cigarette smoke extract reduced binding of bacteria to epithelial cells, but dilutions between 1 in 10 and 1 in 320 enhanced binding. We conclude that smokers might be more densely colonised by a variety of potentially pathogenic bacteria. The enhanced bacterial binding to epithelial cells of smokers is not related to enhanced expression of host cell antigens that can act as receptors for some species, but possibly to components in the smoke that alter charge or other properties of the epithelial cell surface. Passive coating of mucosal surfaces with components of cigarette smoke might enhance binding of potentially pathogenic bacteria.     

Genotypic characterization of bacteria cultured from duck faeces

AIMS: To characterize the bacterial composition of mallard duck faeces and determine if novel bacterial species are present that could be utilized as potential indicators of avian faecal contamination. METHODS AND RESULTS: Combined samples of fresh faeces from four ducks were serially diluted and plated onto six different media selected to allow the growth of a range of organisms at 42 degrees C under three atmospheric conditions: aerobic, microaerophilic and anaerobic. Forty-seven morphologically dissimilar isolates were purified and partial sequencing of the16S rRNA indicated at least 31 bacterial species. Twenty of these could be identified to the species level including pathogenic species of Bacillus, Campylobacter, Clostridium and Streptococcus. Other species identified included: Enterococcus, Escherichia, Megamonas, Cellulosimicrobium, Neisseria, Staphylococcus and Veillonella. Potentially novel species, which could represent bacteria specific to avian fauna included Bacillus, Corynebacterium, Macrococcus and Peptostreptococcus, while four isolates had <97% similarity to known bacterial species in the available databases. CONCLUSION: A survey of the natural microflora of the mallard duck and its hybrid with the grey duck identified both bacteria that are potentially human pathogenic and putative novel bacteria species as determined by 16S rRNA sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides further evidence that duck faeces is a potential human health hazard, and has identified bacteria potentially useful for distinguishing duck faeces from other faecal sources.     

Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system

A rapid bead assay for detecting pathogenic bacteria with a simple microfluidic chip-based system was developed. Five oligonucleotide probes corresponding to the 16S rRNA of the targeted bacteria were coupled covalently to fluorescent beads. Four species of bacteria (Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, Yersinia enterocolitica, and Bacillus cereus) were used as representative food-borne pathogenic bacteria. The RNAs extracted from pure cultures of these microorganisms were fluorescently labeled and hybridized to the oligonucleotide probes-immobilized fluorescent beads (Bead assay). The duplexes of RNAs and the probes-immobilized beads were analyzed with the commercially available microfluidic chip-based system. This bead assay provided results within 3 h following RNA extraction from bacterial cells.     

Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.     

Identification of new promising plant-based lead compounds for inhibition of prokaryotic replicative DNA polymerases: combination of in silico and in vitro studies

Abstract

Emerging widespread bacterial resistance to current antibiotics with traditional targets is one of the major global concerns. Therefore, so many investigations are exploring the potential of other druggable macromolecules of bacteria such as replication machinery components that are not addressed by previous antibiotics. DNA polymerase is the major part of this machine. However, a few studies have been done on it so far. In this respect, we report the discovery of four new plant-based leads against DNA polymerase (pol) IIIC (three leads) and pol IIIE (one lead) of Gram-positive and negative bacteria by combining a sequentially constrained high-throughput virtual screenings on Traditional Chinese Medicine Database with in vitro assays. The compounds displayed relatively good levels of inhibitory effect. They were active against their designated targets at micromolar concentrations. The IC₅₀ values for them are ranged from 25 to 111?μM. In addition, they showed minimum inhibitory concentrations in the range of 8–128?μg/mL against five representatives of pathogenic bacteria species. However, they were inactive against Pseudomonas aeruginosa. Given these results, these leads hold promise for future modification and optimization to be more effective in lower concentrations and also against most of the important bacterial species.

Communicated by Ramaswamy H. Sarma     

Antagonistic activity of a natural fungal population towards pathogenic bacteria. An in vitro study.

In the present work, we performed in vitro testing of 33 species of fungi of the subdivision Deuteromycotina isolated from water and sediment of the Kolubara River for antagonistic action towards 11 species of pathogenic bacteria. Of gram-negative bacteria, the species most sensitive to metabolic fluid of the fungi were Proteus mirabilis, Salmonella enteritidis, and Shigella sonnei, while the most resistant were Klebsiella pneumoniae and Salmonella typhimurium. Of gram-positive bacteria, the most sensitive species was Staphylococcus aureus, while the most resistant was Enterococcus faecalis. Of the tested fungi, Penicillium canescens, P. simplicissimum, P. thomii, Aspergillus fumigatus, A. ochraceus, and Fusarium culmorum exerted inhibitory action on the greatest number of species of pathogenic bacteria, while Verticillium lateritium, V. tenerum, Phoma humicola, and Botrytis cinerea had an inhibiting effect on the least number of species.