Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats,captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Genetic diversity of Histoplasma capsulatum isolated from infected bats randomly captured in Mexico, Brazil, and Argentina, using the polymorphism of (GA)(n) microsatellite and its flanking regions

The genetic diversity of 47 Histoplasma capsulatum isolates from infected bats captured in Mexico, Brazil, and Argentina was studied, using sequence polymorphism of a 240-nucleotides (nt) fragment, which includes the (GA)(n) length microsatellite and its flanking regions within the HSP60 gene. Three human clinical strains were used as geographic references. Based on phylogenetic analyses of 240-nt fragments achieved, the relationships among H. capsulatum isolates were resolved using neighbour-joining and maximum parsimony methods. The tree topologies obtained by both methods were identical and highlighted two major clusters of isolates. Cluster I had three sub-clusters (Ia, Ib, and Ic), all of which contained Mexican H. capsulatum samples, while cluster II consisted of samples from Brazil and Argentina. Sub-cluster Ia included only fungal isolates from the migratory bat Tadarida brasiliensis. An average DNA mutation rate of 2.39 × 10(-9) substitutions per site per year was estimated for the 240-nt fragment for all H. capsulatum isolates. Nucleotide diversity analysis of the (GA)(n) and flanking regions from fungal isolates of each cluster and sub-cluster underscored the high similarity of cluster II (Brazil and Argentina), sub-clusters Ib, and Ic (Mexico). According to the genetic distances among isolates, a network of the 240-nt fragment was graphically represented by (GA)(n) length haplotype. This network showed an association between genetic variation and both the geographic distribution and the ecotype dispersion of H. capsulatum, which are related to the migratory behaviour of the infected bats studied.     

Isolation of Histoplasma capsulatum from tissues of bats captured in the Aguas Buenas Caves,Aguas Buenos,Puerto Rico

Of 30 bats belonging to four species, only one species, Brachyphylla cavernarum, yielded positive for Histoplasma capsulatun. Bats were collected at the Aguas Buenas Caves in Puerto Rico. The other species are: Artibeus jamaicensis, Monophyllus redmani portoricensis, and Erophylla bibombifrons; these resulted negative for the fungus.     

Keratinophilic fungi isolated from children's sandpits in the Nablus area,West Bank of Jordan

The keratinophilic fungi of 29 sandpits from kindergarten schools and public parks in the city of Nablus was analysed to evaluate their role in the epidemiology of diseases caused by these fungi. Seventy two species were recovered 28 of which were common to both kindergartens and public parks sandpits. High percentage (57.4%) of fungal isolates found had been identified as the causes of various types of mycoses. Eight species of dermatophytes and closely related fungi were recovered, of which the followings were the most commonly found species in sandpits: Chrysosporium keratinophilum (20.7%), Microsporum gypseum (17.2%), Trichophyton mentagrophytes (6.9%), and C. evolceanui (6.9%).     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. Fungi are a rich source of chitinases; however, the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin-binding domains (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.     

Elevation gradients of diversity for rodents and bats in Oaxaca, Mexico

1  This study documents patterns of rodent and bat diversity related to abiotic and biotic factors along elevational gradients in the Sierra Mazateca (640–2600 m a.s.l.) and Sierra Mixteca (700–3000 m a.s.l.) in Oaxaca, Mexico.
2  The two transects share similar faunas: 17 and 23 rodent species were captured in the sierras Mazateca and Mixteca, respectively, 14 of which occurred on both transects. Rodent species richness was similar in the wet season and the dry season along both transects. Rodent species richness peaked at 1025–1050 m in tropical semi-deciduous forest on both transects. Endemic species were restricted to high-elevation habitats.
3  Sixteen and 17 bat species were captured in the sierras Mazateca and Mixteca, respectively; 11 occurred on both transects. Bat species richness was higher in the wet season than in the dry season in the Sierra Mazateca. Bat species richness peaked at 1850 m in pine–oak forest in the Sierra Mazateca, and at 750 m and 1050 m in tropical semi-deciduous forest in the Sierra Mixteca, decreasing abruptly at higher elevations on both transects.
4  Patterns of trophic diversity of rodents and bats coincided with those of species richness on each transect. Species richness increased with increasing habitat diversity; increased with increasing rainfall and productivity; increased with increasing resource diversity; and increased in areas with high rates of speciation (rodents only).
5  The need for conservation action in Oaxaca is urgent and proponents should promote establishment of protected areas linking lowland habitats with high species richness to high-elevation habitats harbouring large numbers of endemic forms.     

Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters

Aims:  To apply and compare two PCR-based methods for typing saltwater Bdellovibrio- and-like organisms (BALOs) and to understand ecological and phylogenetic aspects of the BALOs isolated from shrimp mariculture systems.
Methods and Results:  Using double-layer agar technique, the numbers of culturable BALOs that lyse Vibrio alginolyticus were found to be 10–10³ PFU ml⁻¹ in the surface water samples. A total of 130 BALOs isolates were differentiated into five phylotypes by denaturing gradient gel electrophoresis targeting the 16S rDNA V3 region and four phylotypes by amplified rDNA restriction analysis of the Bacteriovoracaceae -specific 16S rDNA fragment respectively. Phylogenetic analysis of representative isolates showed that all of them were identified as Bacteriovorax spp., but affiliated with four different clusters in the family Bacteriovoracaceae.
Conclusions:  The two PCR-based methods both can be chosen to rapidly type the saltwater BALOs at cluster level. And the relatively large numbers of BALOs with various phylotypes recovered from the same habitats suggested that these predators might play important ecological role in shrimp mariculture environments.
Significance and Impact of the Study:  We proposed two effective methods to distinguish rapidly large numbers of BALOs isolates and our results would be helpful to understand the diversity and function of BALOs in mariculture environments.     

Histoplasma capsulatum isolated from Didelphis albiventris (Marsupialia: Didelphidae) in the State of Minas Gerais, Brazil

We report the isolation of Histoplasma capsulatum from a culture of the viscera of Didelphis albiventris, one of the marsupial species found in Brazil. To our knowledge, this is the first report of the isolation of this fungus from this mammalian species. This finding confirms the ubiquitous presence of H. capsulatum in nature.     

PCR-测序法对啮齿类动物汉坦病毒的检测和基因分型

目的探讨PCR-测序法在啮齿类动物汉坦病毒临床检测中的应用价值。方法以Genbank 7种血清亚型24株汉坦病毒代表性毒株为基础,从病毒基因S片段序列设计引物,采用邻位相连法进行系统进化分析,以该方法对浙江省近年从野生啮齿类动物中临床分离的汉坦病毒毒株进行分型鉴定。结果所建系统发育树将所分析的毒株分为5个区域,引起HFRS的四种血清型具有较稳定的拓扑结构,且能与引起HPS的血清型进行区分。11株实验毒株进行PCR扩增和测序,结果表明该引物具有高度的敏感性和特异性,其中9株浙江省分离的血清型未知毒株的系统发育分析发现其包含引起HFRS的3株HTN和1株SEO血清型,其他5株属两种未知血清型。讨论所建立的PCR-测序法具有用于临床检测汉坦病毒的价值。     

Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms

Aim: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. Methods and Results: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. Conclusion: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. Significance and Impact of the Study: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.     

Detection and characterization of diverse alpha- and betacoronaviruses from bats in China

Bats have been implicated as important reservoir hosts of alpha- and betacoronaviruses. In this study, diverse coronaviruses (CoVs) were detected in 50 of 951 (positive rate 5.3%) intestinal specimens of eight bat species collected in four provinces and the Tibet Autonomous Region of China by pan-coronavirus RT-PCR screening. Based on 400-nt RNA-dependent RNA polymerase (RdRP) sequence analysis, eight belonged to genus Alphacoronavirus and 42 to Betacoronavirus. Among the 50 positive specimens, thirteen gave rise to CoV full-length RdRP gene amplification for further sequence comparison, of which three divergent sequences (two from a unreported province) were subjected to full genome sequencing. Two complete genomes of betacoronaviruses (JTMC15 and JPDB144) and one nearly-complete genome of alphacoronavirus (JTAC2) were sequenced and their genomic organization predicted. The present study has identified additional numbers of genetically diverse bat-borne coronaviruses with a wide distribution in China. Two new species of bat CoV, identified through sequence comparison and phylogenetic analysis, are proposed.

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四川地区猪源艰难梭菌分子分型调查

[背景] 艰难梭菌是一种重要的人畜共患肠道病原菌,可引起人和多种动物抗生素相关性腹泻或假膜性肠炎。四川作为我国主要的生猪产区,还未有猪源艰难梭菌流行病学调查的相关报道,对猪源艰难梭菌的防控及保障猪肉安全带来挑战。[目的] 调查四川地区猪源艰难梭菌的感染、流行情况,并对分离出的艰难梭菌进行分子分型研究。[方法] 收集来自四川生猪主要产区6个养殖场中猪的粪便标本（n=110）,采用厌氧培养技术在艰难梭菌鉴别培养基上进行分离培养;采用PCR方法扩增艰难梭菌4个毒素基因（tcdA、tcdB、cdtA、cdtB）和7个管家基因（adk、atpA、dxr、glyA、recA、sodA、tpi）,对分离株进行毒素基因分型和多位点序列分型。[结果] 从110份样品中,经革兰氏染色镜检及PCR鉴定,共分离出20株艰难梭菌,分离率高达18.18%;毒素基因分型结果显示共获得3种毒素基因型,包括tcdA⁺tcdB⁺cdtA/cdtB⁺（n=3）、tcdA⁺tcdB⁺cdtA/cdtB^—（n=6）、tcdA^—tcdB^—cdtA/cdtB^—（n=11）;多位点序列分型结果显示获得5个ST型,包括ST11（n=3）、ST3（n=1）、ST35（n=2）、ST36（n=4）、ST109（n=10）;进化树结果显示,所有分离株聚集为2个大群,分别为3个分支和17个分支。[结论] 四川主要生猪产区猪群存在艰难梭菌感染,分离株的分子分型呈多样性,主要流行型为ST11、ST3、ST35、ST36、ST109型,并且存在ST11型高毒力菌株流行的风险。     

Island biogeography of bats in Baja California, Mexico: patterns of bat species richness in a near-shore archipelago

Aim We studied the relationship between the size and isolation of islands and bat species richness in a near‐shore archipelago to determine whether communities of vagile mammals conform to predictions of island biogeography theory. We compared patterns of species richness in two subarchipelagos to determine whether area per se or differences in habitat diversity explain variations in bat species richness. Location Islands in the Gulf of California and adjacent coastal habitats on the Baja California peninsula in northwest Mexico. Methods Presence–absence surveys for bats were conducted on 32 islands in the Gulf of California using acoustic and mist‐net surveys. We sampled for bats in coastal habitats of four regions of the Baja peninsula to characterize the source pool of potential colonizing species. We fitted a semi‐log model of species richness and multiple linear regression and used Akaike information criterion model selection to assess the possible influence of log₁₀ area, isolation, and island group (two subarchipelagos) on the species richness of bats. We compared the species richness of bats on islands with greater vegetation densities in the southern gulf (n = 20) with that on drier islands with less vegetation in the northern gulf (n = 12) to investigate the relationship between habitat diversity and the species richness of bats. Results Twelve species of bats were detected on islands in the Gulf of California, and 15 species were detected in coastal habitats on the Baja peninsula. Bat species richness was related to both area and isolation of islands, and was higher in the southern subarchipelago, which has denser vegetation. Log₁₀ area was positively related to bat species richness, which increased by one species for every 5.4‐fold increase in island area. On average, richness declined by one species per 6.25 km increase in isolation from the Baja peninsula. Main conclusions Our results demonstrate that patterns of bat species richness in a near‐shore archipelago are consistent with patterns predicted by the equilibrium theory of island biogeography. Despite their vagility, bats may be more sensitive to moderate levels of isolation than previously expected in near‐shore archipelagos. Differences in vegetation and habitat xericity appear to be associated with richness of bat communities in this desert ecosystem. Although observed patterns of species richness were consistent with those predicted by the equilibrium theory, similar relationships between species richness and size and isolation of islands may arise from patch‐use decision making by individuals (optimal foraging strategies).     

生殖道念珠菌病病原真菌的调查及药敏试验

目的了解本地区生殖道念珠菌病病原真菌构成及其体外药敏试验情况。方法采用科玛嘉念珠菌显色培养基和YBC鉴定卡对患者1164份生殖道标本的致病真菌进行分离和鉴定,并用ROSCO纸片扩散法检测分离菌株对制霉菌素、酮康唑、氟康唑、伊曲康唑、咪康唑和特比萘芬的药敏情况。结果共分离9种295株念珠菌,其中自念珠菌为85．76％,近平滑念珠菌为7．46％,光滑念珠菌为3．39％,其他念珠菌为3．39％。295株念珠菌对制霉菌素、酮康唑、氟康唑、伊曲康唑、咪康唑和特比萘芬的敏感性分别为99．66％、97．29％、89．83％、72．22％、46．44％和36．61％。结论本地区生殖道念珠菌病患者致病菌分布以白念珠菌为主,体外药敏显示制霉菌素、酮康唑和氟康唑有较好的敏感性。     

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

2016‒2017年辽宁省沙门菌耐药菌谱与分子分型

目的 分析2016‒2017年辽宁省沙门菌分离株的耐药特性与脉冲场凝胶电泳（PFGE）分子分型特征,为沙门菌引起的食源性疾病暴发、防控及抗生素使用提供参考数据。方法 对分离的54株沙门菌进行血清分型和药物敏感试验。根据PulseNet沙门菌标准PFGE分型技术,选取全部菌株进行PFGE分子分型分析,应用BioNumerics软件对菌株条带进行分析,确定菌株间的特征及相关性。结果 54株沙门菌血清型居首位的是肠炎沙门菌,占46.30％;其次是鼠伤寒沙门菌,占24.07％;共分为10个血清型。对13种抗生素的耐药分析显示多重耐药菌株为36株,占66.7％,其中耐3～5种的13株（24.1%）,耐6～8种的13株（24.1%）,耐9～11种的10株（18.5%）。54株沙门菌经聚类分析获得36种带型,相似度区间为49.7%～100.0％。结论 辽宁省沙门菌分离株多重耐药状况比较严重,相同血清型其PFGE带型相似度相对较高,同时具有较显著的优势带型特点;而且发现同一PFGE型菌株的耐药谱相对比较接近。     

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India

Aims: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)‐PCR. Methods and Results: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0·95, while ERIC‐PCR showed a DI of 0·94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. Conclusions: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC‐PCR generates useful information in the case of V. parahaemolyticus associated with seafood. Significance and Impact of the Study: The study demonstrates the usefulness of nucleic acid and protein‐based studies in understanding the relationship between various isolates from seafood.