Potentiality of tricyclic compound thioridazine as an effective antibacterial and antiplasmid agent.

Thioridazine (Th), which is therapeutically used in psychiatric patients, was found to possess conspicuous antimicrobial activity when tested against 316 strains belonging to a number of Gram positive and Gram negative bacteria. Although Staphylococcus aureus, Vibrio chloerae and V. parahaemolyticus were found to be most sensitive, Th was highly bactericidal against S. aureus and bacteriostatic for vibrios and other Gram negative organisms. In the study of antiplasmid/curing effect of Th on twelve multiply antibiotic and Th resistant bacteria, it was observed that elimination of R plasmids was facilitated by choice of optimal concentration of Th. Significant elimination of single and combined antibiotic resistance occurred in E. coli and Shigella flexneri and not in S. aureus.     

Marine bacteria from Danish coastal waters show antifouling activity against the marine fouling bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas, Phaeobacter, and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida, Pseudoalteromonas tunicata, and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata, reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.     

Predatory Bacteriovorax communities ordered by various prey species

The role of predation in altering microbial communities has been studied for decades but few examples are known for bacterial predators. Bacteriovorax are halophilic prokaryotes that prey on susceptible gram-negative bacteria. We recently reported novel observations on the differential selection of Bacteriovorax phylotypes by two different prey, Vibrio parahaemolyticus and Vibrio vulnificus. However, the conclusion is restricted by the limited number of prey tested. In this study, we have conducted two independent investigations involving eight species of prey bacteria while using V. vulnificus and V. parahaemolytics as reference strains. Water samples collected from Dry Bar, Apalachicola Bay were used to establish microcosms which were respectively spiked with prey strains Vibrio cholerae, Escherichia coli or Pseudomonas putida to examine the response of native Bacteriovorax to freshwater bacteria. Indigenous Vibrio sp., Pseudoalteromonas sp., Photobacterium sp. and a clinical strain of V. vulnificus were also tested for the impact of saltwater prey on the Bacteriovorax community. At 24 hour intervals, optical density of the microcosm samples and the abundance of Bacteriovorax were measured over five days. The predominant Bacteriovorax plaques were selected and analyzed by 16S rRNA gene amplification and sequencing. In addition, the impacts of prey on predator population and bacterial community composition were investigated using culture independent denaturing gradient gel electrophoresis. Strikingly, Cluster IV was found consistently as the predominant phylotype produced by the freshwater prey. For all saltwater prey, subgroups of Bacteriovorax phylotype IX were the major predators recovered. The results suggest that prey is an important factor along with temperature, salinity and other environmental parameters in shaping Bacteriovorax communities in aquatic systems.     

Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria

Three challenge experiments were carried out on larvae of the great scallop Pecten maximus. Larvae were bath-challenged with Vibrio pectenicida and 5 strains resembling Vibrio splendidus and one Pseudoalteromonas sp. Unchallenged larvae were used as negative controls. The challenge protocol was based on the use of a multidish system, where the scallop larvae (10, 13 and 15 d post-hatching in the 3 experiments, respectively) were distributed to 2 ml wells with stagnant seawater and exposed to the bacterial cultures by bath challenge. Presence of the challenge bacteria in the wells was verified by polymerase chain reaction (PCR). A significantly increased mortality was found between 24 and 48 h in most groups challenged with V. pectenicida or V. splendidus-like strains. The exception was found in larval groups challenged with a Pseudoalteromonas sp. LT 13, in which the mortality rate fell in 2 of the 3 challenge experiments. Larvae from the challenge experiments were studied by immunohistochemistry protocol. Examinations of larval groups challenged with V. pectenicida revealed no bacterial cells, despite a high degree of positive immunostaining. In contrast, intact bacterial cells were found in larvae challenged with V. splendidus. In the case of larvae challenged with the Pseudoalteromonas sp., positive immuno-staining was limited to visible bacteria inside the digestive area and cells of the mucosa. The experiments confirm that V. splendidus and V. pectenicida are pathogenic to scallop larvae, and that the Pseudoalteromonas strain is probably not a primary pathogen, although it cannot be ruled out as a secondary pathogen.     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Polynucleotide sequence relationships among Japanese and American strains of Vibrio parahaemolyticus

Polynucleotide sequence relationships between two reference Vibrio parahaemolyticus strains isolated from Japanese and American gastroenteritis patients were investigated by use of (32)P-DNA/DNA reassociation in free solution. In addition, these strains were similarly compared with 22 other strains of estuarine and marine vibrios, including 11 strains previously identified as V. parahaemolyticus (2 Japanese, 1 of unknown location, and 8 American strains obtained from diverse geographical locations and sources in North America), 3 strains of V. alginolyticus, and 8 of Vibrio spp. Deoxyribonucleic acid (DNA) from the Japanese and American gastroenteritis isolates showed high relative levels of intraspecific duplex formation (92 to 93%) when reassociated, reciprocally, at 60 C. Heterologous DNA duplexes exhibited thermal elution midpoint [Tm(e)] values comparable to those obtained from homologous duplexes (88.0) when thermally eluted from hydroxyapatite, thus indicating high base-pair complementarity. Other V. parahaemolyticus strains showed DNA homologies of 85% or greater, with correspondingly high Tm(e) values (86.0 to 88.0) for the heteroduplexes formed. DNA of two of three V. alginolyticus strains (ATCC 17749 and 166-70) was 55 to 60% homologous to reference V. parahaemolyticus DNA preparations; Vibrio sp. strain 5144 (originally classified as V. parahaemolyticus biotype 2 and subsequently as V. alginolyticus strain 5144) showed only 24 to 26% DNA homology to the same reference DNA. These data provide evidence that Vibrio sp. strain 5144 is genetically distinct from the other V. alginolyticus strains used in this study. Three bioluminescent strains thought to be closely related to V. parahaemolyticus demonstrated only 24 to 31% DNA homology to the reference V. parahaemolyticus DNA. These data firmly establish the existence in some Atlantic and Gulf Coast estuaries of organisms genetically very similar to V. parahaemolyticus, the causative agent of "shirasu" food poisoning in Japan.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

多重实时PCR检测产毒素性霍乱弧菌和副溶血弧菌

设计引物和探针,优化多重实时PCR条件,以同时检测霍乱弧菌霍乱毒素基因ctxA、副溶血弧菌种特异性基因gyrB和耐热肠毒素基因tdh。该多重实时PCR方法检测产毒素性的O1群(3株)和O139群(44株)霍乱弧菌菌株、不产毒素的O1群(12株)和O139群(6株)及非O1非O139群(7株)霍乱弧菌菌株的ctxA,阳性和阴性结果与普通PCR检测结果100%符合;检测副溶血弧菌种特异性gyrB,116株副溶血弧菌均阳性,而9株其它细菌和72株霍乱弧菌均阴性;检测tdh的阳性和阴性结果也与普通PCR结果完全一致。另外还建立了检测副溶血弧菌菌株trh1和trh2的单重实时PCR方法。     

Taxonomic composition of bacteria associated with cultivated mollusks Crassostrea lugubris and Perna viridis and with the water of the Gulf of Nha Trang Lagoon, Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力

该研究以健康香梨树的新鲜花器、当年生新生枝条、叶片和果实为试验材料,采用植物组织内生细菌分离法获得内生细菌菌株,培养菌落,并采用平板对峙法初筛和发酵液复筛对梨火疫病菌(Erwinia amylovora)、梢枯病菌(Pseudomonas syringae pv.syringae)和腐烂病菌(Valsa mali va...     

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.     

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.     

Infaunal burrows are enrichment zones for Vibrio parahaemolyticus

Vibrio parahaemolyticus, a species that includes strains known to be pathogenic in humans, and other Vibrionaceae are common, naturally occurring bacteria in coastal environments. Understanding the ecology and transport of these organisms within estuarine systems is fundamental to predicting outbreaks of pathogenic strains. Infaunal burrows serve as conduits for increased transport of tidal waters and V. parahaemolyticus cells by providing large open channels from the sediment to salt marsh tidal creeks. An extensive seasonal study was conducted at the North Inlet Estuary in Georgetown, SC, to quantify Vibrionaceae and specifically V. parahaemolyticus bacteria in tidal water, fiddler crab (Uca pugilator, Uca pugnax) burrow water, and interstitial pore water. Numbers of V. parahaemolyticus bacteria were significantly higher within burrow waters (4,875 CFU ml(-1)) than in creek water (193 CFU ml(-1)) and interstitial pore water (128 CFU ml(-1)), demonstrating that infaunal burrows are sites of V. parahaemolyticus enrichment. A strong seasonal trend of increased abundances of Vibrionaceae and V. parahaemolyticus organisms during the warmer months of May through September was observed. Multilocus sequence typing (MLST) analysis of isolates presumed to be V. parahaemolyticus from creek water, pore water, and burrow water identified substantial strain-level genetic variability among V. parahaemolyticus bacteria. Analysis of carbon substrate utilization capabilities of organisms presumed to be V. parahaemolyticus also indicated physiological diversity within this clade, which helps to explain the broad distribution of these strains within the estuary. These burrows are "hot spots" of Vibrionaceae and V. parahaemolyticus cell numbers and strain diversity and represent an important microhabitat.     

1株副溶血性弧菌Bh-06的分离、鉴定及其药敏试验

从患病的南美白对虾分离出1株副溶血性弧菌Bh-06,通过革兰染色和Biolog全自动细菌鉴定仪鉴定,并对健康南美白对虾进行攻毒试验和对该菌株进行药物敏感试验。试验结果表明:Bh-06菌株为革兰阴性弧菌,通过细菌自动鉴定仪鉴定为副溶血性弧菌;该菌在培养第5小时进入对数早期,对数期一直延续到25小时;该菌在1.0×106cfu/mL浓度时可引起南美白对虾发病,而且浓度越高,病症越严重;该菌对氧哌嗪青霉素产生高度的敏感性。     

临床病原菌种类及耐药性监测

目的 探讨病原菌种类及其对抗菌药物的耐的耐药状况。方法 收集１９９８年１月－１９９９年１２月临床感染标本分离的病原菌并分析其种类,药敏试验采用Ｋｉｒｂｙ－Ｂａｕｅｒ纸片扩散法。结果 １１８２株病原菌,革兰氏阳性球菌６０４株（５１．１％）,革兰氏阴性杆菌５７８株（４８．９％）。病原菌以金黄一萄球菌、大肠埃希菌、表皮葡萄球菌、铜绿假单胞菌最多见。去甲万古霉素、阿米卡星、新霉素对革兰氏阳性球菌抗菌作用较     

用基于TaqMan探针的Real-time PCR技术定量检测副溶血弧菌

副溶血弧菌是一种引起食源性疾病的重要病原菌,传统的鉴定方法费时费力且容易出现假阴性,建立一种定量检测副溶血弧菌基因的方法尤为重要。根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的RealtimePCR方法。通过对9种细菌(12株菌株)的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌均不产生扩增曲线,证明了引物和探针具有很高的特异性。细菌纯培养物品和人工布菌的检测敏感度分别为1CFUPCR反应体系和10CFUPCR反应体系,相关系数均为0.99(r2=0.99),整个试验可在1h内完成。建立的方法可用于海产品中副溶血弧菌的快速定量检测。     

晚期肿瘤患者下呼吸道感染临床分析

目的：探讨晚期肿瘤患者下呼吸道感染的细菌学分类情况及抗生素耐药性特点。方法：采用回顾性调查107例晚期肿瘤患者并下呼吸道感染痰菌分离的病原菌并分析其特点,药敏试验采用K—B纸片法。结果：137株病原菌,G杆菌67株(48．9％)．G^ 球菌44株(32．1%),真菌26株(19．0％)。主要致病细菌依次为铜绿假单胞菌19．7％．金黄色葡萄球菌13．9％,肺炎克雷伯菌10．9％。万古霉素、新霉素、环丙沙星对G^ 球菌较敏感;环丙沙星、阿米卡星、头孢哌酮对G杆菌作用较强．其他抗菌药物对各种病原菌均呈现不同程度的耐药。结论：晚期恶性肿瘤并下呼吸道感染的病原菌多为条件致病菌,耐药率高。应加强病原菌培养及药敏监测,以指导临床科学使用抗生素。     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。