Prevalence estimation of Tropheryma whipplei in duodenal biopsy tissues of Koreans.

Whipple's disease caused by Tropheryma whipplei is difficult to diagnose because of a broad spectrum of manifestations and non-specific clinical signs. In the current global era, the incidence of duodenal infection/inflammation caused by T. whipplei in Korea may has been underestimated. Here we estimated the prevalence of T. whipplei in duodenal biopsy tissues of Koreans using real-time PCRs (RT-PCRs). A total of 252 duodenal biopsy tissues were collected from Korean patients who underwent esophagogastroduodenoscopy and duodenal biopsy. DNA extracted from the duodenal biopsy tissues was analyzed using three RT-PCRs targeting T. whipplei-specific regions of the 16S-23S rRNA intergenic spacer, hsp65, and Dig15 in parallel. In the samples positive in RT-PCRs, direct sequencing was performed for each RT-PCR target. The prevalence of T. whipplei was estimated based on the RT-PCR and sequencing results. Among the analyzed samples, T. whipplei was not detected. The prevalence of T. whipplei in duodenal biopsy tissues of Koreans was estimated to be less than 0.4%. This is the first study to attempt to detect T. whipplei in duodenal biopsy tissues of Koreans and estimate its prevalence. Our findings infer that while T. whipplei carriers exist in Korea, the incidence of duodenal infection/inflammation caused by T. whipplei is extremely rare.

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

BACKGROUND: This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. METHODS AND RESULTS: A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. CONCLUSIONS: The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

The occurrence of Affixifilum gen. nov. and Neolyngbya (Oscillatoriaceae) in South Florida (USA), with the description of A. floridanum sp. nov. and N. biscaynensis sp. nov.

South Florida (USA) has a subtropical to tropical climate with an extensive and diverse coastline that supports the growth of benthic cyanobacterial mats (BCMs). These BCMs are widespread and potentially house numerous bioactive compounds; however, the extent of the cyanobacterial diversity within these mats remains largely unknown. To elucidate this diversity, BCMs from select locations in South Florida were sampled and isolated into unicyanobacterial cultures for morphological and molecular studies. Phylogenetic relationships of isolated taxa were assessed using the markers 16S rRNA and 16S-23S rRNA ITS by both maximum likelihood and Bayesian inference. We propose Affixifilum gen. nov. based on morphological characteristics and the 16S rRNA phylogeny. Two species are included: Affixifilum granulosum comb nov. (=Neolyngbya granulosa) found in Brazil and Florida (USA) and A. floridanum sp. nov. Several other features, including pair-wise distance of 16S rRNA and 16S-23S rRNA ITS, 16S-23S rRNA ITS secondary structure, morphology, and ecology, provide support for Affixifilum. We also propose the transfer of Lyngbya regalis to Neolyngbya as N. regalis comb. nov. and include the description of one novel species, N. biscaynensis sp. nov.

Development of an Illumina-based analysis method to study bradyrhizobial population structure-case study on nitrogen-fixing rhizobia associating with cowpea or peanut.

Bradyrhizobia are Gram-negative soil bacteria that regroup a growing number of species. They are widespread in nature and recovered from various biomes that may be explained by a high genetic diversity in this genus. Among the numerous metabolic properties they can harbor, the nitrogen fixation resulting from the association with plants among which important crop legumes (soya bean, peanut, cowpea …) is of great interest, notably in a context of sustainable development. Metabarcoding is widely applied to study biodiversity from complex microbial communities. Here, we demonstrate that using a new species-specific and highly polymorphic 16S-23S rRNA intergenic spacer barcode, we could rapidly estimate the diversity of bradyrhizobial populations that associate with cowpea and peanut plants, two crop legumes of major interest in Senegal. Application of the method on indigenous bradyrhizobia associated with peanut and cowpea grown in soils collected in the center of the peanut basin shows that Bradyrhizobium vignae is a dominant symbiont. We also showed that the two plant species associate with distinct community profiles and that strains introduced by inoculation significantly modified the population structure with these two plants suggesting that application of elite strains as inoculants may well ensure optimized symbiotic performance. This approach may further be used to study the diversity of bradyrhizobia from contrasting agro-eco-climatic zones, to test whether the plant genotype influences the association outputs as well as to estimate the competitiveness for nodule occupancy and the fate of elite strains inoculated in the field.Key points• An amplicon sequencing approach targeting the Bradyrhizobium genus was developed.• Diversity of cowpea and peanut bradyrhizobia from cultivated soils was identified.• The method is well suited to test the competitiveness of defined Bradyrhizobium inoculants.

Species-specific Identification of Vibrio sp. based on 16S-23S rRNA gene internal transcribed spacer.

AIMS: To explore a prokaryotic species-specific DNA marker, 16S-23S rRNA gene internal transcribed spacer (ITS) sequence for identification and classification of Vibrio. METHODS AND RESULTS: Five hundred and seventy four ITS sequences from 60 Vibrio strains were collected, then the primary and secondary structures of ITS sequence were analysed. The ITS was divided into several subunits, and the species-specificity of these subunits were evaluated by blast. The variable subunit of ITS showed high species-specificity. A protocol to identify a Vibrio species based on ITS analysis was developed and verified. Both the specificity and sensitivity were 100%. The phylogeny analysis of Vibrio based on ITS showed that ITS devised a better classification than 16S rDNA. Finally, an identification method of Vibrio based on ITS sequencing in food samples was developed and evaluated. The results of ITS sequencing were (100%) consistent with the results identified by ISO standard. CONCLUSIONS: Vibrio could be accurately identified at the species level by using the ITS sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that the ITS can be considered as a significant DNA marker for identification and classification of Vibrio species, and it posed a new path to screen the Vibrio in food sample.

Molecular characterization and toxin quantification of Microcystis panniformis: A microcystin producer in Lake Taihu, China.

Microcystis panniformis is a bloom forming species with flat panniform-like colonies. This species was recently found in Lake Taihu, China. To specifically characterize M. panniformis based on isolated strains, morphological examination on colonial transition and genetic examination are needed. Three M. panniformis strains isolated from a water bloom sample in Lake Taihu were characterized by molecular analysis and toxin quantification. Phylogenetic analysis based on both 16S rRNA gene and internal transcribed spacer (ITS) between 16S and 23S rRNA genes were performed and compared to facilitate easy identification of the species. Relatively high similarities (98%-99%) were shown in 16S rDNA sequences between the strains of M. panniformis and those of other Microcystis species, whereas the similarities for ITS sequences were 88%-95%. In the phylogenetic tree based on the 16S rDNA sequences, the M. panniformis and M. aeruginosa strains were intermixed together with no clear division, whereas all of the M. panniformis strains were clustered together in a single clade based on the ITS sequences based phylogenyetic tree. The mcyE gene was detected in all three strains, and microcystin was determined by high-performance liquid chromatography. The molecular detection and toxin production of M. panniformis strains are of great significance for the environmental risk assessment of Microcystis blooms.

Phenotypic characterization and genetic diversity of Flavobacterium columnare isolated from red tilapia, Oreochromis sp., in Thailand.

Flavobacterium columnare is the aetiological agent of columnaris disease and severely affects various freshwater aquaculture fish species worldwide. The objectives of this study were to determine the phenotypic characteristics and genetic variability among F. columnare isolates isolated from red tilapia in Thailand. Forty-four F. columnare isolates were recovered from diseased fish in different geographical locations. The isolates exhibited homologous phenotypic characteristics but exhibited genetic diversity. One isolate was assigned to genomovar I, and the remainder were assigned to genomovar II, indicating the coexistence of these genomovars but predominance of genomovar II. Phylogenetic analysis of the 16S-23S ISR sequences revealed that a subset of the Thai isolates (n = 25) contained a smaller intergenic spacer region (ISR) (523-537 bp) and formed a unique ISR phylogenetic group. Phylogenetic analysis of the 16S rRNA gene supported the unique cluster of Thai isolates. This is the first description of the phenotypic and molecular characteristics of F. columnare isolated from red tilapia in Thailand as well as five isolates of F. columnare derived from other fish species including Nile tilapia, koi carp and striped catfish.

Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina.

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively.

Application of LAMP assay for detection of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in ICU admitted sepsis patients: A rapid and cost-effective diagnostic tool.

Carbapenem-resistant significant members of Acinetobacter calcoaceticus-Acinetobacter baumannii (CR-SM-ACB) complex have emerged as an important cause of sepsis, especially in ICUs. This study demonstrates the application of loop-mediated-isothermal-amplification (LAMP) assay for detection of CR-SM-ACB-complex from patients with sepsis. Whole-blood and culture-broths(CB) collected from patients with culture-positive sepsis were subjected to LAMP and compared with PCR, and RealAmp. Vitek-2 system and conventional PCR results were used as confirmatory references. The sensitivity and specificity of LAMP(97 % & 100 %) and RealAmp(100 % & 100 %) for detection of CR-SM-ACB-complex from CB were better than PCR(87 % & 100 %). Diagnostic accuracy of LAMP, RealAmp, and PCR for detection of SM-ACB-complex from CB was 98.5 %, 100 %, and 88.5 % respectively. Turnaround time of Culture, LAMP, PCR, and RealAmp was 28-53, 6-20, 9-23, and 6-20hours, respectively. LAMP is a simple, inexpensive tool that can be applied directly to positive CB and may be customized to detect emerging pathogens and locally-prevalent resistance genes and to optimize antimicrobial use.

The Surveillance of Borrelia Species in Camelus dromedarius and Associated Ticks: The First Detection of Borrelia miyamotoi in Egypt.

Tick-borne diseases (TBDs) are emerging and re-emerging infections that have a worldwide impact on human and animal health. Lyme borreliosis (LB) is a severe zoonotic disease caused by the spirochete Borrelia burgdorferi sensu lato (s.l.) transmitted to humans by the bite of infected Ixodes ticks. Borrelia miyamotoi is a spirochete that causes relapsing fever (RF) and is genetically related to Borrelia burgdorferi s.l. However, there have been no reports of B. miyamotoi in Egypt, and the data on LB in camels is scarce. Thus, the present study was conducted to screen and genetically identify Borrelia spp. and B. miyamotoi in Egyptian camels and associated ticks using polymerase chain reaction (PCR). METHODS: A total of 133 blood samples and 1596 adult hard ticks were collected from Camelus dromedaries at Cairo and Giza slaughterhouses in Egypt. Tick species were identified by examining their morphology and sequencing the cytochrome C oxidase subunit 1 (cox1) gene. Borrelia spp. was detected using nested PCR on the IGS (16S-23S) gene, and positive samples were genotyped using 16S rRNA and glpQ spp. genes specific for Borrelia burgdorferi and Borrelia miyamotoi, respectively. The positive PCR products were sequenced and analyzed by phylogenetic tree. RESULTS: Analysis of the cox1 gene sequence revealed that the adult ticks belonged to three genera; Hyalomma (H), Amblyomma (Am), and Rhipicephalus (R), as well as 12 species, including H. dromedarii, H. marginatum, H. excavatum, H. anatolicum, R. annulatus, R. pulchellus, Am. testudinarium, Am. hebraeum, Am. lipidium, Am. variegatum, Am. cohaerens and Am. gemma. Borrelia spp. was found in 8.3% (11/133) of the camel blood samples and 1.3% (21/1596) of the ticks, respectively. Sequencing of the IGS (16S-23S) gene found that B. afzelii, detected from H. dromedarii and H. marginatum, and B. crocidurae, which belongs to the RF group, was detected from one blood sample. B. burgdorferi and B. miyamotoi were discovered in the blood samples and tick species. Phylogenetic analysis of the glpQ gene showed that the B. miyamotoi in this study was of the Asian and European types. CONCLUSIONS: These results suggest that the camels can be infected by Lyme borrelia and other Borrelia bacteria species. This study also provides the first insight into the presence of Borrelia miyamotoi and B. afzelii DNA in camels and associated ticks in Egypt.

Quantification of Marine Picocyanobacteria on Water Column Particles and in Sediments Using Real-Time PCR Reveals Their Role in Carbon Export.

Picocyanobacteria are the most abundant primary producers in the ocean and play a fundamental role in marine carbon cycling. Quantification of picocyanobacteria on sinking particles and in sediments is essential to understanding their contribution to the biological carbon pump. We designed a primer set targeting the 16S-23S rRNA internal transcribed spacer (ITS) sequence of cyanobacteria and established a quantitative PCR (qPCR) method for quantifying the ITS sequence abundance. High-throughput sequencing confirmed that this primer set can cover broad diversities of marine picocyanobacteria and avoid amplification of other marine cyanobacteria such as Trichodesmium and Crocosphaera. Amplification efficiencies were slightly different when seven marine Synechococcus and Prochlorococcus strains were assayed. The qPCR results were comparable with flow cytometry for water samples. Using this method, we found that, in the dark ocean, picocyanobacterial ITS sequence abundances were 10 to 100 copies/mL in the size fraction of 0.2 to 3 µm, which were 1 to 3 orders of magnitude more abundant than on the >3-µm particles. We also found that picocyanobacterial ITS abundance in sediment ranged from 105 to 107 copies/g along two nearshore-to-offshore transects in the northern South China Sea. These results further explain the important role of picocyanobacteria in carbon export. Collectively, we provide a qPCR method quantifying the total abundance of marine picocyanobacteria on water column particles and in sediments. Moreover, this newly designed primer set can be also applied to investigate the community of picocyanobacteria via high-throughput sequencing. IMPORTANCE Picocyanobacteria are the most abundant primary producers in the ocean. However, quantification of picocyanobacteria on the sinking particles and in sediments remains challenging using flow cytometry or epifluorescence microscopy. Here, we developed a real-time PCR method to quantify picocyanobacteria using a newly designed primer set specifically targeting the 16S-23S rRNA ITS sequence of cyanobacteria. We showed that in the dark ocean, picocyanobacteria are 1 to 3 orders of magnitude more abundant in small particles (0.2 to 3 µm) than in larger particles (>3 µm). This result supports the important role of direct sinking free-living picocyanobacteria cells in the carbon export to deep ocean. We also found that the picocyanobacterial ITS sequence abundance were 105 to 107 copies per gram in sediments, suggesting significant accumulation of sinking picocyanobacteria in the benthic ecosystem. This qPCR method can be used to quantify the contribution of picocyanobacteria to the biological carbon pump.

2-Methylisoborneol (2-MIB) Excretion by Pseudanabaena yagii under Low Temperature.

Outbreaks of 2-methylisoborneol (2-MIB) contamination in drinking water sources cause inconvenient odor issues in the water distribution system. In this study, microscopy-based isolation with physiological and molecular phylogenetic characterization were performed to investigate and characterize the 2-MIB odor producers that caused an odor problem in the freshwater system of the North Han River in the autumn of 2018. A benthic cyanobacterium was isolated from 2-MIB odor-issue freshwater samples and was found to be phylogenetically affiliated with Pseudanabaena yagii (99.66% sequence similarity), which was recorded in South Korea for the first time. The 2-MIB synthesis gene sequences from the odor-issue freshwater samples showed 100% similarity with those in the P. yagii strains. Protein sequences of 2-MIB synthase observed in the genome of the isolated strain showed structural and functional characteristics similar to those observed in other Pseudanabaena species. The 2-MIB production rate increased slowly during mat formation on the vessel wall; however, it rapidly increased after the temperature dropped. The 2-MIB gene was continuously expressed regardless of the temperature changes. These results suggest that the 2-MIB odor issue in the North Han River might be caused by the release of 2-MIB from the mat-forming P. yagii species in a low-temperature freshwater environment.

Bartonella Infections in Cats and Cat Fleas in Lithuania.

Bartonella are vector-borne parasitic bacteria that cause zoonotic infections in humans. One of the most common infections is cat-scratch disease caused by Bartonella henselae and Bartonella clarridgeiae. Cats are the major reservoir for these two species of bacteria, while cat fleas are vectors for the transmission of infection agents among cats. The aim of the present study was to investigate the presence of Bartonella infections in stray and pet cats and in cat fleas in Lithuania. Blood samples were taken from 163 cats presented in pet clinics and animal shelters. A total of 102 fleas representing two species, Ctenocephalides felis and Ctenocephalides canis, were collected from 12 owned cats that live both outdoors and indoors. Bartonella DNA in samples was detected using a nested PCR targeting the 16S-23S rRNA intergenic spacer (ITS) region. Bartonella DNA was detected in 4.9% (8/163) of the cats and 29.4% (30/102) of the fleas. Sequence analysis of the ITS region showed that the cats and fleas were infected with B. henselae, B. clarridgeiae and Bartonella sp., closely related to B. schoenbuchensis. This study is the first report on the prevalence and molecular characterization of Bartonella spp. in cats and cat fleas in Lithuania.

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus-baumannii Complex and Associated Carbapenem Resistance.

Background: Acinetobacter calcoaceticus-baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S-23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/µl and 1 ng/µl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species. Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

Molecular detection, identification, and sequence analysis of 'Candidatus Liberibacter asiaticus' associated with Huanglongbing disease of citrus in North India.

Huanglongbing (HLB, Citrus greening), caused by a phloem-limited fastidious gram-negative bacterium, "Candidatus Liberibacter spp.", is one of the devastating diseases of citrus worldwide. The pathogen belongs to the alpha-proteobacteria group and is classified on the basis of its geographical origin and 16S rRNA sequence diversity. Although the disease has been reported from all citrus growing states of India, the status and the molecular variability among the isolates from the Northern part of the country is unknown. A total of five different HLB isolates originating from Northern India showing variable symptoms were studied. The genomic regions of four different genes, i.e., 16S rRNA, intergenic 16S/23S rRNA spacer region, rplA-rplJ, and CLIBASIA_01645 were amplified by PCR, sequenced, and variations in these sequences were assessed. Analysis of 16S rRNA clearly indicated that all five isolates fit in to 'Candidatus Liberibacter asiaticus' (CLas) group. However, 16S/23S rRNA intergenic spacer region-based analysis failed to segregate these isolates beyond species level. Sequence analysis of rplA-rplJ gene and CLIBASIA_01645 loci also confirmed the existence of diversity among the 'CLas' in the surveyed areas. Further, 16S rRNA and rplA-rplJ-based SNP analysis revealed that some isolates segregated into three new lineages, two on the basis of 16Sr (16Sr-XV and 16Sr-XVI), and one based on ß-rp (rp-IV), respectively. A tandem repeat number (TRN) at CLIBASIA_01645 region were TRN = 5, 6 and 13; with TRN = 6 being common in three 'CLas' isolates. Overall, the study demonstrated that all examined five HLB isolates belonged to 'CLas' group. However, these isolates showed distinct sequence variability in three out of four genomic regions. The results provide a robust framework for understanding differences in pathogenicity among different HLB isolates as it is plausibly related to their genomic variation, and evolutionary history.

Improvement and evaluation of loop-mediated isothermal amplification combined with chromatographic flow dipstick assays for Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a major food-borne pathogen, is a gram-negative rod-shaped halophilic bacterium which inhabits marine environments throughout the world. It can pose a threat to humans after the consumption of raw or undercooked seafood. Fast detection is crucial for hindering and controlling V. parahaemolyticus infection. Compared with traditional methods, loop-mediated isothermal amplification (LAMP) is a simple, rapid and versatile method. It can be performed at one temperature without the need for cycling. As a new method in recent years, LAMP combined with a chromatographic flow dipstick (LFD) meets the needs of point-of-care testing without the need for special instruments. It avoids the limitations of LAMP, reduces detection time and increases detection accuracy. Our previous studies have suggested that the optimized LFD method can improve the sensitivity of LAMP detection and shorten the isothermal amplification time during the detection process. In the present study, two LAMP assays were improved to LFD methods, and a LFD targeting 16S23S rRNA gene internal transcribed spacer (ITS) of V. parahaemolyticus was developed. The lower limit for tlh, toxR, ITS LFD assays were detected as 3.1 × 100, 3.1 × 101, and 3.1 × 100 CFU respectively, whether in pure cultures or artificially contaminated food samples. The shortest amplification times at the limit of each assay were determined as 20 min, 35 min and 25 min. A heating block was used to perform two (tlh and ITS) LFD assays to detect 20 food samples. Compared to a standard method (GB 4789.7-2013 National Food Safety Standards, Food Microbiology Inspection, Vibrio parahaemolyticus test), tlh and ITS LFD assays showed more MPN (most probable number) results than that of culture. It demonstrated that the improved LFD technology can provide a simple and rapid detection method with high sensitivity and specificity for detection of V. parahaemolyticus.

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation.

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S-23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S-23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S-23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region.

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the ß subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.