Isolation of hexavalent chromium-reducing Cr-tolerant facultative anaerobes from tannery effluent

Several facultative anaerobes tolerant to high levels of chromate (>400 mg/ml) were isolated from tannery effluents. These isolates displayed varying degrees of Cr(VI) reduction under aerobic and anaerobic conditions at room temperature (24+/-2 degrees C). Interestingly, eight isolates were efficient in reducing 70% Cr(VI) anaerobically. This includes 5 isolates of genus Aerococcus, two isolates of Micrococcus and single isolate of genus Aeromonas. These isolates were subjected to further characterization for possible use in Cr(VI) detoxification of industrial wastes. This is the first report of Aerococcus sp. capable of Cr(VI) reduction >70% anaerobically. These bacteria were further checked for tolerance to a variety of other heavy metals. Our study indicates the possible use of these bacteria in environmental clean up.     

Isolation and characterisation of bacterial colonies from seeds and in vitro cultures of Fraxinus spp. from Italian sites

Culturable bacteria were isolated from seeds, embryos and contaminated in vitro cultures of ash (Fraxinus excelsior L., F. ornus L. and F. angustifolia L.) and were identified using morphological and molecular analyses. Fourteen morphologically distinct isolates were recovered from seeds of Fraxinus spp. 16S rDNA sequencing categorised these isolates into ten separate genera. Three strains isolated from contaminated in vitro cultures, Pantoea agglomerans, Staphylococcus succinus and Aerococcus viridans, were used for comparative analysis with isolates from seeds. Antibiotic sensitivity testing of the isolated contaminants, including phytotoxicity of antibiotics on in vitro cultures of ash, was also investigated. Phytotoxic effects on explants immersed in ampicillin or cultured on medium containing ampicillin were negligible, however tetracycline, either alone or in combination with other antibiotics, had phytotoxic effects. We conclude that ampicillin is a suitable antibiotic to limit the growth of contaminating bacteria during the in vitro culture of ash.     

Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Genetic characterization of the lobster pathogen Aerococcus viridans var. homari by 16S rRNA gene sequence and RAPD

A combination of 16S rRNA sequencing and random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity within Aerococcus viridans var. homari, the causative agent of gaffkemia in lobsters. A collection of 7 A. viridans var. homari strains and 2 avirulent A. viridans-like cocci isolated from homarid lobsters harvested from different regions on the Atlantic Coast of North America were analyzed. The isolates are separated geographically and temporally between the years 1947 and 2000. Sequencing of 16S rRNA genes confirmed the inclusion of all 9 isolates in the monophyletic A. viridans clade (99.8 to 100% similarity). RAPD analysis revealed that the 9 A. viridans var. homari isolates could be separated into 2 distinct subtypes. Subtype 1 included the 7 pathogenic lobster isolates and constituted a homogeneous group regardless of their geographical, temporal or virulence differences. Subtype 2 contained the 2 avirulent A. viridans-like cocci that had distinct RAPD patterns and clustered separately with the non-marine A. viridans. RAPD analysis represented a useful method for determining molecular subtyping for the intraspecific classification and epidemiological investigations of A. viridans var. homari.     

抗菌和细胞毒活性海洋细菌的筛选及其次生代谢基因证据

从不同海域的海水、海泥和海洋生物中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析和多聚酮合酶(PKSⅠ型)、非核糖体肽合成酶(NRPS)的筛选。结果显示,在分离到的346株海洋细菌中,42株细菌具有抗菌活性,12株具有细胞毒活性。对12株具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析,它们分别属于Agrobacterium,Pseudoalteromons,Bacillus,Paracoccus,Rheinheimera,Aerococcus,Exiguobacterium和Alteromonas8个属。对这12株具有细胞毒活性的细菌菌株进行进一步的PKS和NRPS筛选,得到了4株含有PKSⅠ型的KS结构域或NPRS的A结构域的海洋细菌,为从聚酮和非核糖体肽等生物合成途径去深入研究其次生代谢产物提供了基因学的证据。     

Serological cross-reactivity of environmental isolates of Enterobacter, Escherichia, Stenotrophomonas, and Aerococcus with Shigella spp.-specific antisera

Using protocols designed for the isolation of Shigella from environmental freshwater samples from different regions of Bangladesh, 11 bacterial strains giving rise to Shigella-like colonies on selective agar plates and showing serological cross-reaction with Shigella-specific antisera were isolated. Phylogenetic analyses revealed that three of the isolates were most closely related to Escherichia coli, four to Enterobacter sp., two to Stenotrophomonas, and two isolates belonged to the Gram-positive genus Aerococcus. The isolates cross-reacted with six different serotypes of Shigella and were, in each case, highly type-specific. Two of the isolates belonging to the Enterobacter and Escherichia genera gave extremely strong cross-reactivity with Shigella dysenteriae and Shigella boydii antisera, respectively. The Aerococcus isolates gave relatively weak but significant cross-reactions with S. dysenteriae. Western blot analysis revealed that a number of antigens from the isolates cross-react with Shigella spp. The results indicate that important Shigella spp. surface antigens are shared by a number of environmental bacteria, which have implications for the use of serological methods in attempts for the detection and recovery of Shigella from aquatic environments.     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

A taxonomic survey of lactic acid bacteria isolated from wheat (Triticum durum) kernels and non-conventional flours

In order to explore the correspondence between raw material- and mature sourdough-lactic acid bacterial (LAB) communities, 59 Italian wheat (Triticum durum) grain samples, one bran and six non-conventional flour samples were analyzed through a culture-dependent approach. The highest cell count by an agar medium specific for LAB was 2.16 log CFU/g. From about 2300 presumptive LAB (Gram-positive and catalase-negative) colonies collected, a total of 356 isolates were subjected to identification by a genetic polyphasic strategy consisting of RAPD-PCR analysis, partial 16S rRNA gene sequencing, species-specific and multiplex PCRs. The isolates were recognized as 137 strains belonging to Aerococcus, Enterococcus, Lactobacillus, Lactococcus and Pediococcus genera and a phylogram based on partial 16S rRNA genes was constructed. The species most frequently found were Enterococcus faecium, Enterococcus mundtii and Lactobacillus graminis, which are not generally reported to be typical in mature sourdoughs.     

眼部浅绿色气球菌感染4例报道

目的 提高对眼部气球菌感染的认识.方法 报告4例眼部气球菌感染病例并复习文献,对其发病因素、临床症状、诊断、及治疗和预后进行分析.结果 4例眼部感染气球菌患者为中年或老年人,基础疾病为青光眼、糖尿病(2例)及甲状腺功能亢进症,均经病原菌的分离鉴定后确诊为浅绿色气球菌感染,且对左氧氟沙星、万古霉素等常用药敏感,4例患者中除1例青光眼患者用药效果不理想外,其余均经1～2周的治疗后痊愈.结论 对眼部感染患者,应加强浅绿色气球菌的分离与鉴定.     

Phylogenetic analysis of antimicrobial lactic acid bacteria from farmed seabass Dicentrarchus labrax

The use of lactic acid bacteria (LAB) in the prevention or reduction of fish diseases is receiving increasing attention. In the present study, 47 LAB strains were isolated from farmed seabass ( Dicentrarchus labrax ) and were phenotypically and phylogenetically analysed by 16S rDNA and randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). Their antimicrobial effect was tested in vitro against a wide variety of pathogenic and spoilage bacteria. Most of the strains isolated were enterococci belonging to the following species: Enterococcus faecium (59%), Enterococcus faecalis (21%), Enterococcus sanguinicola (4 strains), Enterococcus mundtii (1 strain), Enterococcus pseudoavium (1 strain), and Lactococcus lactis (1 strain). An Aerococcus viridans strain was also isolated. The survey of their antimicrobial susceptibility showed that all isolates were sensitive to vancomycin and exhibited resistance to between 4 and 10 other antibiotics relevant for therapy in human and animal medicine. Different patterns of resistance were noted for skin and intestines isolates. More than 69% (32 strains) of the isolates inhibited the growth of the majority of pathogenic and spoilage bacteria tested, including Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Carnobacterium sp. To our knowledge, this is the first report of bioactive enterococcal species isolated from seabass that could potentially inhibit the undesirable bacteria found in food systems.     

The effect of Aerococcus viridans--the basis of the new therapeutic-prophylactic preparation M-bacterin--on the biological properties of Staphylococcus aureus

Representatives of the normal microflora from genus Aerococcus, in particular strain Aerococcus viridans 167 isolated from breast milk are studied for their effect on biological properties of Staphylococcus aureus in vitro and in vivo. It is established that the number of viable cells of the staphylococcus cultivated in the presence of antagonists in the beef-extract agar decreases progressively with each following passage, the population dying after the seventh-eight passage. Electronograms fix deep changes in the cell ultrastructure. A degree of changes in biological properties depends on the duration of the antagonist action. The results obtained reveal one of the mechanisms of the antagonistic action of aerococci-antagonists producing hydrogen peroxide.     

The Use of Streptococcus faecalis as an Indicator of the Hygienic Condition of Meat Preserves and the Possibility of Interference by Aerococcus viridans

Summary: Few arguments may be put forward in favour of the use of Escherichia coli as an indicator of the hygienic condition of preserved meat products. In products of this type E. coli occurs only rarely. The proposal, which is supported by logical considerations and practical experience, to replace this organism by Streptococcus faecalis as an indicator of possible faecal contamination is strongly upheld by the authors. However, it is necessary to stress that the ubiquitous micro-organism, Aerococcus viridans , occurs frequently in preserved meat products and, as it strongly resembles Strep. faecalis , may cause difficulties in the hygienic evaluation of such foods. The morphological and biological characteristics found suitable for differentiating these two organisms are described. Results supporting the view that Aerococcus, Pediococcus and Gaffkya be considered as distinct genera are also given.     

Microbiological examination of sebeel water.

Water samples from clay storage jugs ("zeers") located in homes and at public watering stands ("sebeels") at streets, mosques, and schools were examined. Coliforms, fecal coliforms, and fecal streptococci were detected in 100, 69, 88, and 91.56% of the samples, respectively. The general microbiology of the water and some factors affecting microbial load were studied. The predominant bacterial genera of sebeel water were found to be Staphylococcus. Aerococcus, Micrococcus, Streptococcus, Bacillus, Listeria, Lactobacillus, and Arthrobacter. A simple modification of zeer construction was suggested to help improve sanitation.     

猪源绿色气球菌的分离鉴定与药敏特性研究

目的鉴定研究2006年和2008年本实验室分别从广东清远和广西北流两个猪场发病小猪的膝关节积液中分离到2株呈四联状或成对排列,在山羊血琼脂平板上能形成明显的α-溶血圈的G+球菌。方法采用小白鼠致病性试验、细菌常规生理生化鉴定、药敏试验、耐药基因检测及16S rRNA序列分析等方法对上述分离的2株细菌进行鉴定及药敏特性研究。结果确定这2株G+球菌为正常情况下非致病性绿色气球菌(登录号分别为:GQ161096;GQ161097)。系统发育分析表明,两分离株与绿色气球菌15MS(登录号:EU075039.1)同源性分别高达99.4%和98.7%。药敏试验及耐药基因检测发现,这2株绿色气球菌除了对新霉素、氟哌酸、恩诺沙星、菌必治和环丙沙星等5种药物呈高敏外,对先锋V等13种抗菌素均不敏感。耐药基因检测结果表明,GXBL-1可检出6个耐药基因,GDQY-1则可检出多达10个耐药基因。结论从这2株分离株耐药谱检测结果可以看出,正常菌株同样携带多重耐药基因,存在着通过质粒转座子和整合子将耐药基因转移给敏感菌,导致耐药性传播的可能,应引起足够重视。     

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.     

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.     

Molecular and Phenotypic Characterization of Aerococcus viridans Associated with Subclinical Bovine Mastitis

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.     

可可西里碱性土壤样品细菌的分离和生物学特性

可可西里是位于青海玉树藏族自治州的自然环境保护区,由于当地的恶劣气候特点,其土壤微生物多样性很少被研究.采用5种分离培养基对来源于可可西里的12个盐碱土壤样品进行选择性分离,共分离得到5株细菌.其中4株菌分别属于游动微菌属(Planomicrobium)、库克菌属(Kocuria)、气球菌属(Aerococcus)和芽...     

白星花金龟成虫肠道细菌的分离鉴定

目的自星花金龟是我国北方常见腐食性昆虫,为了利用其腐食性行为转化处理有机垃圾、畜禽粪便及农作物秸杆等农业有机废弃物资源,并探索其中肠消化的微生态机制,而对其肠道细菌进行系统研究。方法从白星花金龟成虫肠道中无菌操作分离获得5株细菌,对其菌体形态、培养性状、染色反应、生理生化反应等进行鉴定及数量测定。结果白星花金龟成虫肠道的5个细菌菌株分别属于气球菌属（Aerococcus）、短状杆菌属（Brachybacterium）、棍状杆菌属（Clavibacter）、李斯特菌属（Listeria）、皮杆菌属（Dermabacter）,以皮杆菌属和棍状杆菌属数量最多,分别为1．3×10^7和1．0×10^7;其次为短状杆菌属和气球菌属,分别为8×10^6和5×10^6;李斯特菌属数量最少,为7×10^5。结论各菌株之间细菌数量存在明显差异,以皮杆菌属细菌数量最多,分离未获得真菌和放线菌。