Analysis of Leptospira isolates from mainland Portugal and the Azores islands

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.     

RAPD fingerprinting for the distinction of Prevotella intermedia sensu stricto from Prevotella nigrescens

A collection of 70 oral strains including reference strains and clinical isolates identified as Prevotella intermedia sensu lato was constituted to cover a large clinical and geographical diversity. Electrophoresis of the enzyme malate dehydrogenase allowed the identification of the 70 study strains as Prevotella intermedia sensu stricto (n= 36), Prevotella nigrescens (n= 31) and three unclassified strains. By using four primers, DNA fingerprints were generated from 20 strains as random amplified polymorphic DNA (RAPD). Matching co-migrating amplicon positions by pairwise comparison allowed the clustering of the fingerprints as two groups coincident with the P. intermedia/P. nigrescens assignment by enzyme electrophoresis of malate dehydrogenase. Our data suggest that isolates identified asP. intermedia sensu lato by conventional criteria can be speciated asP. intermedia sensu stricto or P. nigrescens by RAPD fingerprinting.     

Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.     

Molecular typing of Leptospira spp. based on putative O-antigen polymerase gene (wzy), the benefit over 16S rRNA gene sequence

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.     

Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.     

Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards

AIMS: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. METHODS AND RESULTS: A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. CONCLUSION: Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.     

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi,Japan

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates’ partial β-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

     

Leptospira-rat-human relationship in Luzon,Philippines

Leptospirosis is a zoonotic infection that is caused by the pathogenic species of Leptospira. Rats are the most important reservoirs of these organisms. Our study aimed to characterize Leptospira isolates from humans and rats and elucidate the Leptospira-rat-human relationship in Luzon, Philippines. Forty strains were isolated from humans and rats. The isolates were confirmed to be Leptospira and pathogenic through rrl- and flaB-PCR, respectively. Around 73% of the isolates were found to be lethal to hamsters. Serotyping showed that there were mainly three predominant leptospiral serogroups in the study areas namely Pyrogenes, Bataviae, and Grippotyphosa. Gyrase B gene sequence analysis showed that all the isolates belonged to Leptospira interrogans. Most had 100% similarity with serovar Manilae (15/40), serovar Losbanos (8/40), and serogroup Grippotyphosa (8/40). Strains from each group had highly identical pulsed-field gel electrophoresis patterns and were further grouped as A (Pyrogenes, 14), B (Bataviae, 8), and C (Grippotyphosa, 10). Results further revealed that similar serotypes were isolated from both humans and rats in the same areas. It is suggested that these three predominant groups with highly similar intra-group PFGE patterns may have been primarily transmitted by rats and persistently caused leptospirosis in humans particularly in the Luzon islands.     

Phylogenetic analysis of Leptospira strains of pathogenic serovars using 23S rDNA gene sequences.

The 23S ribosomal DNAs were amplified from 11 strains of Leptospira interrogans sensu lato by polymerase chain reaction (PCR) and sequenced. The PCR products of about 290-bp DNA fragments indicated more than 97% sequence similarity to each other. The phylogenetic tree based on the 23S ribosomal DNAs obtained in this study revealed that 11 strains of L. interrogans examined composed a cluster distinct to that of L. weilii and L. borgpetersenii, confirming that these strains were similar to strain Moulton of L. interrogans serovar canicola in 23S rDNA sequence.     

致病钩体表层膜蛋白新基因（Lslp）的克隆表达及免疫原性研究

克隆表达钩端螺旋体表层膜蛋白新基因Lslp并分析表达产物的免疫原性。根据前期研究得到的致病钩体新基因Lslp(GenBankAF32 5 80 7)的序列设计引物 ,在 6株致病钩体中扩增Lslp基因并测序。以BamHⅠ酶切Lslp和pGEX 1 λT ,构建重组质粒并用酶切和PCR鉴定 ,进一步在大肠杆菌中诱导表达 ,并进行免疫印迹分析 ;纯化表达产物免疫家兔 ,ELISA检测血清抗体滴度。结果显示Lslp在 6株致病钩体中均能扩增出相应片段 ,且序列同源性达到99 6 % ;构建高效原核表达重组质粒pGST LslP ,经IPTG诱导在大肠杆菌中可表达出 6 6kDGST融合蛋白 ,并能与全钩抗血清发生免疫印迹反应 ;将上述融合蛋白免疫新西兰大白兔产生 1 :5 1 2 0高滴度的IgG抗体。研究结果提示致病钩体膜蛋白新基因Lslp可在大肠杆菌进行高效表达 ,表达产物能被全钩抗血清识别 ,为研究钩体的致病机制和筛选保护性抗原提供了基础     

Leptospira interrogans is recognized through DC-SIGN and induces maturation and cytokine production by human dendritic cells

Leptospirosis is a global zoonotic disease, caused by pathogenic Leptospira species including Leptospira interrogans, that causes public health and livestock problems. Pathogenesis, immune response and cellular receptors for Leptospira are not well understood. Interaction of dendritic cells (DCs) with L. interrogans serovar Autumnalis L-643 and BL-6 isolated from leptospirosis patients, and both virulent and avirulent serovar Pyrogenes 2317 strains isolated from animal were investigated. Carbohydrate analysis using lectins showed that all of these leptospires contained high mannose components as a common backbone and DC-SIGN was involved in leptospires' attachment. Interaction of the L. interrogans strains with DCs induced maturation, but had different effects on IL-10, IL-12p70 and tumor necrosis factor (TNF)-alpha production. Both virulent and avirulent Pyrogenes 2317 and Autumnalis BL-6 but not L-643 strains induced IL-12p70 and TNF-alpha production, but minimal IL-10 secretion. These data demonstrated that L. interrogans binds DC-SIGN and induces DCs maturation and cytokine production, which should provide new insights into cellular immune processes during leptospirosis.     

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

ABSTRACT: BACKGROUND: In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. RESULTS: Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. CONCLUSIONS: MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.     

Detection of Leptospiraceae by amplification of 16S ribosomal DNA

The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

Killing effect and antitoxic activity of the Leptospira interrogans toxin-antitoxin system in Escherichia coli

We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for "killer protein") and its upstream partner chpI (for "inhibitory protein"). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis, chp homologs were found in all representative pathogenic strains of L. interrogans.     

Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.     

Candida orthopsilosis fungemias in a Spanish tertiary care hospital: Incidence,epidemiology and antifungal susceptibility

BackgroundFew studies exist on prevalence of fungemia by Candida orthopsilosis, with variable results.AimsTo study the incidence, epidemiology and antifungal susceptibility of C. orthopsilosis strains isolated from fungemias over two years at a tertiary hospital.MethodsCandidemia episodes between June 2007 and June 2009 in a university hospital (Puerta del Mar, Cádiz, Spain) were studied. The strains initially identified as Candida parapsilosis were genotypically screened for C. parapsilosis sensu stricto, C. orthopsilosis and Candida metapsilosis, and their antifungal susceptibility was evaluated.ResultsIn this period 52 cases of candidemia were documented. Of the 19 strains originally identified as C. parapsilosis, 13 were confirmed as C. parapsilosis sensu stricto and 6 as C. orthopsilosis. Of the 52 isolates, the most frequent species were Candida albicans (30.8%), C. parapsilosis sensu stricto (25%), C. orthopsilosis, Candida tropicalis and Candida glabrata in equal numbers (11.5%). C. orthopsilosis isolates were susceptible to amphotericin B, caspofungin, voriconazole and fluconazole, with no significant differences in MIC values with C. parapsilosis sensu stricto. The source of isolates of C. orthopsilosis were neonates (50%) and surgery (50%), and 100% were receiving parenteral nutrition; however C. parapsilosis sensu stricto was recovered primarily from patients over 50 years (69.2%) and 46.1% were receiving parenteral nutrition.ConclusionsThese findings show that C. orthopsilosis should be considered as human pathogenic yeast and therefore its accurate identification is important. Despite our small sample size our study suggests that a displacement of some epidemiological characteristics previously attributed to C. parapsilosis to C. orthopsilosis may be possible.     

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.