Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.     

蒙古戈壁地区自然发酵乳中乳酸菌的分离鉴定

从采集自蒙古国戈壁地区的6份自然发酵乳中分离到14株乳酸菌,经过形态特征,生理生化特性,糖发酵试验和乳酸旋光性的测定,鉴定结果:乳酸球菌5株,包括Lactococcus lactissubsp.cremoris 1株,Pedio-coccus.(后缩写为Ped.).urinaeequi3株,Pediococcus.pentosaceus1株;乳杆菌9株,包括Lactobacillus.(后缩写为L.)helveticus8株,Lactobacillus.delbrueckii.subsp.bulgaricus1株。蒙古国戈壁地区自然发酵乳中的优势菌为Lactobacillus.helveticus,其次为Pediococcus.urinaeequi。     

越南原始森林粘细菌的分离与鉴定

为了探讨越南热带原始森林环境中可培养粘细菌的多样性,以原始森林的土壤、腐木及树皮样品为研究对象,采用大肠杆菌法和滤纸法从中分离纯化粘细菌,将形态鉴定与16S rRNA基因测序分析相结合,对分离到的纯培养物进行分类鉴定,确定其分类地位。结果显示,从越南16个土样和22个腐木、树皮样品中共分离到70株粘细菌,经纯化后获得了32株。这些菌株分属于6个属,分别是粘球菌属（Myxococcus）14株、珊瑚球菌属（Corallococcus）7株、侏囊菌属（Nanncystis）6株、蜂窝囊菌属（Melittangium）3株、原囊菌属（Archangium）1株、软骨霉状菌属（Chondromyces）1株。这表明粘细菌在越南原始森林环境中分布广泛,并存在较为罕见的粘细菌类群。     

Four new species of Kockovaella isolated from plant leaves collected in Vietnam

Five ballistoconidiogenous yeast strains, isolated from plant leaves at Cuc Phuong National Forest of Ninh Binh, Vietnam, were assigned to the genus Kockovaella based on morphological and chemotaxonomical characteristics. They represent four new species based on analyses of 18S rDNA sequence, sequences of internal transcribed spacer regions, and DNA-DNA reassociation experiments. Four new species, Kockovaella calophylli (1 strain), Kockovaella cucphuongensis (2 strains), Kockovaella litseae (1 strain), and Kockovaella vietnamensis (1 strain) are proposed for these strains.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

目的 介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法 所用菌种包括临床分离的未知菌株和保藏菌株共23株：未知酵母菌（5株）、真皮毛孢子菌（1株）、糠秕马拉色菌（1株）、季也蒙念珠菌（5株）、未知丝状真菌（6株）、无绿藻（1株）、烟曲霉（2株）、拟青霉菌（1株）、茎点霉（1株）。用溶细胞酶（lyticase）结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260／A280检测纯度并计算质量浓度,用真菌通用引物ITS1／ITS4扩增真菌核糖体基因（rDNA）内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果 成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论 用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。     

Relative importance of bacteriocin-like genes in antagonism of Xanthomonas perforans tomato race 3 to Xanthomonas euvesicatoria tomato race 1 strains

In a previous study, tomato race 3 (T3) strains of Xanthomonas perforans became predominant in fields containing both X. euvesicatoria and X. perforans races T1 and T3, respectively. This apparent ability to take over fields led to the discovery that there are three bacteriocin-like compounds associated with T3 strains. T3 strain 91-118 produces at least three different bacteriocin-like compounds (BCN-A, BCN-B, and BCN-C) antagonistic toward T1 strains. We determined the relative importance of the bacteriocin-like compounds by constructing the following mutant forms of a wild-type (WT) T3 strain to evaluate the antagonism to WT T1 strains: Mut-A (BCN-A-), Mut-B (BCN-B-), Mut-C (BCN-C-), Mut-AB, Mut-BC, and Mut-ABC. Although all mutant and WT T3 strains reduced the T1 populations in in planta growth room experiments, Mut-B and WT T3 were significantly more effective. Mutants expressing BCN-B and either BCN-A or BCN-C reduced T1 populations less than mutants expressing only BCN-A or BCN-C. The triple-knockout mutant Mut-ABC also had a significant competitive advantage over the T1 strain. In pairwise-inoculation field experiments where plants were coinoculated with an individual mutant or WT T3 strain and the T1 strain, the mutant strains and the WT T3 strain were reisolated from more than 70% of the lesions. WT T3 and Mut-B were the most frequently reisolated strains. In field experiments where plants were group inoculated with Mut-A, Mut-B, Mut-C, Mut-ABC, and WT T1 and T3 strains, Mut-B populations dominated all three seasons. In greenhouse and field experiments, the WT and mutant T3 strains had a selective advantage over T1 strains. Bacterial strains expressing both BCN-A and BCN-C appeared to have a competitive advantage over all other mutant and WT strains. Furthermore, BCN-B appeared to be a negative factor, with mutant T3 strains lacking BCN-B having a selective advantage in the field.     

Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.     

Ballistosporous yeasts found on the surface of plant materials collected in New Zealand

In the present study, strains from the surface of plant materials collected in New Zealand that belong to the genera Bensingtonia and Bullera are classified. One strain of Bensingtonia was assigned to Ben. ingoldii, while the remaining strain was assigned to Ben. naganoensis based on DNA-DNA reassociation experiment. Twenty-one of 28 Bullera strains were assigned to B. alba (11 strains), B. crocea (6 strains) and B. variabilis (4 strains). The remaining seven strains could not be assigned to any previously known species and were described as the new species, B. coprosmaensis (1 strain), B. hannae (1 strain), B. huiaensis (1 strain), B. mrakii (3 strains) and B. unica (1 strain).Abbreviations B Bullera - Ben Bensingtonia - Sp. Sporobolomyces - G+C guanine plus cytosine     

The correlation between phenotypes and genotypes of macrolides, lincosamides and streptogramins B resistance in Staphylococcus aureus

For 31 clinical strains of S. aureus the correlation between phenotype and genotype of resistance to macrolides, lincosamides and streptogramins B (MLSB) was established.. Phenotypes were determined on the basis of: susceptibility to erythromycin and clindamycin and the ability to an induction of the resistance (phenotypes S, susceptible; R , constitutive resistant, D, resistant after induction with erythromycin, D+, resistant after induction with erythromycin and with a presence of the small colonies inside inhibition zone between erythromycin and clindamycin discs), and on the basis of the resistance to spectinomycin (spR, resistant, spS, susceptible). Among examined S. aureus strains eight phenotypes of resistance to MLSB were recognized (the corresponding genotypes are given in brackets). Six phenotypes were typical: SspS (lack of MLS-B resistance genes), NEGspS (msrA/B, 1 strain), D+spS (ermCi, 4 strains),. DspR (ermAi, 11 strains and ermAi + msrA/B, 2 strains), RspR (ermAc, 4 strains and ermA + msrA/B,1 strain and ermA + ermC, 1 strain) and RspS (ermCc, 6 strains and ermB, 1 strain). Two rare phenotypes in two single strains were observed: SspR (ermAi, the strain with altered inducibility, inductor other than erythromycin) and DspS (ermAi, presumably mutation or lack of spc in Tn554).     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family. Received: 23 April 1997 / Accepted: 31 May 1997     

Characterization of four olive-mill-wastewater indigenous bacterial strains capable of aerobically degrading hydroxylated and methoxylated monocyclic aromatic compounds

Seven aerobic bacterial strains capable of degrading several of the monocyclic aromatic compounds occurring in the phenolic fraction of olive-mill wastewaters (OMWs) were isolated from an Italian OMW. The results of the 16S rDNA restriction analysis evidenced that these strains are distributed among four different groups. One strain of each group was taxonomically characterized by sequencing the amplified 16S rDNA, and the four strains were assigned to the genera Comamonas (strain AV1A), Ralstonia (strain AV5BG), Pseudomonas (strain AV2A) and Sphingomonas (strain AV6C). The four strains, when checked for the ability to degrade nine monocyclic aromatic compounds abundant in OMWs, were found to significantly metabolize five to eight of them, both as resting cells and growing cells. Specific enzyme analyses of the same selected strains showed: (1) the occurrence of O-demethylating activities towards four methoxylated mono-aromatic acids in three of the four studied strains (strains AV1A, AV5BG and AV6C), (2) ring-cleavage activity towards protocatechuic acid in all of the strains, and (3) a ring-cleavage activity towards catechol in strain AV6C. The isolates described here exhibit a biodegradation potential towards monocyclic aromatic compounds of OMWs that is markedly broader and higher than that displayed by other aerobic bacteria described previously. These features make them excellent candidates for removing the low-molecular-weight phenolic compounds persisting in the effluent following anaerobic digestion of OMWs.     

革兰氏阴性杆菌氟喹诺酮耐药株的实验研究

对８２株临床分离的氟喹诺酮药物（Ｆｌｕｏｒｏｑｕｉｎｏｌｏｎｅｓ,ＦＱＮＳ）耐药菌进行了多种抗生素敏感试验。结果显示８２株耐药菌对５种药物耐药率分别为：诺氟沙星９７．５６％、依诺沙星９６．３４％、罗美沙星９５．１２％、氧氟沙星９２．６８％、环丙沙星８７．８％。３２株菌对ＦＱＮＳ存在较普遍的交叉耐药现象,对氨苄青霉素、庆大霉素、丁胺卡那霉素及头孢菌素类的多重耐药率也较高。８２株菌中有４３株菌携带质粒（５２．４４％）。质粒消除及转化实验结果表明：质粒与ＦＱＮＳ耐药性无关。     

Esterase B1 activity variation within and among insecticide resistant, susceptible and heterozygous strains of Culex quinquefasciatus (Diptera: Culicidae)

Amplification of the esterase B1 gene of Culex quinquefasciatus Say results in high titers of an esterase enzyme that confers resistance to organophosphate insecticides. Esterase activity of individuals was measured in samples from an organophosphate resistant strain (Tem-R), a susceptible strain (S-Lb), and their reciprocal F1 progeny. Within-strain variation, as measured by coefficients of variation, was fairly consistent between sexes within strains and among strains (average, 12%). On average, individuals from the Tem-R strain had about 120 times the esterase activity of individuals from the S-Lab strain. The mean esterase activities of the F1 strains were significantly higher than the average of the Tem-R and S-Lab strain mean esterase activities, suggesting enhanced expression of the amplified esterase B1 genes in F1 individuals. Reciprocal F1 strains did not differ significantly in esterase activity or resistance, indicating that maternal effects do not influence either of these measures in these strains. The levels of esterase activity of the strains are discussed in relation to their resistance.     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

辣椒上CMV株系鉴别寄主的筛选与应用

根据“基因对基因”理论和日本小室与都凡按寄主的科属关系及被害症状划分株系的方法,研究了辣椒CMV的“基因型株系”和“致病型株系”。从373个甜、辣椒品种(系)中,筛选出一套抗性不同的差别品种,编号为:LS-8501(HR)、LS-8502(R)、LS-8503(T)、LS-8504(S)、LS-8505(HS)。用这套差别品种做“基因型”株系鉴别寄主,将59个CMV分离物划分为5个株系,命名为:CMV-P0,CMV-P1,CMV-P2,CMV-P3,CMV-P4。又从7科39种不同科属寄主值物中,筛选出一套“致病型”株系的鉴别寄主谱7种,用这套鉴别寄主将59个CMV分离物划分为5个株系群,即十字花科株系群,藜科株系群,茄科、葫芦科株系群,豆科株系群,普通黄色花叶株系群。文中比较了两种方法划分的株系致病性与辣椒病症表现型之间的关系,以及各株系的分布。还讨论了“基因型”鉴别寄主谱及“基因型”株系划分方法盼学术价值和实用性,比较了5个株系与国内外已分化的CMV株系的异同点。     

The role of strain in the response of rapidly growing young male rat bones to parathyroid hormone

Human parathyroid hormone (hPTH 1-34) stimulates an anabolic response in human and animal skeletons; however, it is unclear if the effect is strain dependent. To determine if the anabolic response to hPTH (1-34) was dependent upon strain in rats we used 2 outbred strains (Sprague Dawley, Wistar), 2 inbred strains (Fischer 344, Wistar spontaneously hypertensive:SHR), and 2 mutant strains (Zucker obese, Zucker lean) of rats. Male rats, 5 weeks of age, from each strain were treated subcutaneously with 80 microg/kg body weight hPTH (1-34) or vehicle for 12 days. The response to PTH was similar in all strains whereby PTH exerted an anabolic effect on femoral bone mass and cancellous bone histology that was independent of strain differences. Histomorphometric indices of bone volume, mineralized surface and bone formation in lumbar vertebrae increased in all PTH-treated rats. Additionally, femur bone mineral content and bone mineral density measured by dual energy X-ray absorptiometry (DEXA), and ash weight increased in all PTH-treated rats. These increases occurred regardless of strain. In summary, PTH exerted comparable anabolic effects on bone mass, bone mineral density and bone formation in all rat models tested demonstrating that the skeletal responsiveness to PTH was not dependent upon strain.