rpoB Sequences Research Articles

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for identification of Corynebacterium species.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool that has initiated a revolution in the clinical microbiology laboratory for identification of nosocomial pathogens. The efficacy of MALDI-TOF MS produced by bioMerieux, Marcy l'Etoile, France (VITEK MS) for detecting Corynebacterium remains unknown. Corynebacterium isolates were isolated from clinical specimen in a tertial teaching hospital from 2012 to 2013. All strains confirmed by rpoB sequencing were identified by API Coryne (bioMerieux), Phoenix (BD) and VITEK MS, respectively. The coincidence rate was used to evaluate the consistency and accuracy across three methods. In all, 75 Corynebacterium isolates were collected in this study. The dominant isolates were Corynebacterium striatum (58.7%), Corynebacterium jeikeium (16.0%), Corynebacterium amycolatum (5.3%), Corynebacterium urealyticum (5.3%), Corynebacterium glucuronolyticum (2.7%) and Corynebacterium minutissimum (2.7%). We found that there was no significant difference in the identification of corynebacterium to genus level by MS (100%, 75/75) or Phoenix (93.3%, 70/75) (P=0.058). However, 92.0% (69/75) strains were successfully identified to species by MS while which by Phoenix and API was 78.7% and 65.3% respectively. Compared with gene sequencing, the coincidence rate of identification by MS was significantly higher than Phoenix (P=0.036) and API (P<0.001). Compared with API Coryne (bioMerieux) and Phoenix, VITEK MS shown significant shorter detecting period and less cost. VITEK MS was a powerful tool, which could be applied in clinical laboratory, improving the diagnosis for Corynebacterium infection.

Citrobacter portucalensis sp. nov., isolated from an aquatic sample.

A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).

Proposal for the reclassification of obligately purine-fermenting bacteria Clostridium acidurici (Barker 1938) and Clostridium purinilyticum (Dürre et al. 1981) as Gottschalkia acidurici gen. nov. comb. nov. and Gottschalkiapurinilytica comb. nov. and of Eubacterium angustum (Beuscher and Andreesen 1985) as Andreesenia angusta gen. nov. comb. nov. in the family Gottschalkiaceae fam. nov.

Several strictly anaerobic bacteria that are Gram-stain-positive have the ability to use uric acid as the sole source of carbon and energy. The phylogeny of three such species, Clostridium acidurici, Clostridium purinilyticum, and Eubacterium angustum, members of the Clostridium cluster XII that ferment purines, but not most amino acids or carbohydrates, has been re-examined, taking advantage of their recently sequenced genomes. Phylogenetic analyses, based on 16S rRNA gene sequences, protein sequences of RpoB and GyrB, and on a concatenated alignment of 50 ribosomal proteins, revealed tight clustering of C. acidurici and C. purinilyticum. Eubacterium angustum showed consistent association with C. acidurici and C. purinilyticum , but differed from these two in terms of the genome size, G+C content of its chromosomal DNA and its inability to form spores. We propose reassigning C. acidurici and C. purinilyticum to the novel genus Gottschalkia as Gottschalkia acidurici gen. nov. comb. nov. (the type species of the genus) and Gottschalkia purinilytica comb. nov., respectively. Eubacterium angustum is proposed to be reclassified as Andreesenia angusta gen. nov. comb. nov. Furthermore, based on the phylogenetic data and similar metabolic properties, we propose assigning genera Gottschalkia and Andreesenia to the novel family Gottschalkiaceae. Metagenomic sequencing data indicate the widespread distibution of organisms falling within the radiation of the proposed family Gottschalkiaceae in terrestrial and aquatic habitats from upstate New York to Antarctica, most likely due to their ability to metabolize avian-produced uric acid.

Antimicrobial resistance in Mycobacterium abscessus complex isolated from patients with skin and soft tissue infections at a tertiary teaching hospital in Taiwan.

Mycobacterium abscessus complex (MABC) is the most common non-tuberculous mycobacterium that causes complicated skin and soft tissue infections (cSSTIs). The selection of antimycobacterial agents for successful treatment of such infections is a critical issue. To investigate the antimicrobial susceptibility patterns of MABC isolates from skin and soft tissue to a variety of antimycobacterial agents. Sixty-seven MABC isolates were collected and partial gene sequencing of secA1, rpoB and hsp65 was used to classify them into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMA) and M. abscessus subsp. bolletii (MBO). The MICs of 11 antimycobacterial agents for these 67 isolates were determined using a broth microdilution method and commercial Sensititre RAPMYCOI MIC plates, as recommended by CLSI. In total, 28 MAB, 38 MMA and 1 MBO were isolated from patients with cSSTIs at our hospital. Most MABC strains were resistant to ciprofloxacin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin and trimethoprim/sulfamethoxazole. In addition, most MABC strains were intermediately susceptible or resistant to cefoxitin. Eighteen of the 28 MABs and 1 MBO isolate harboured the T28 polymorphism in the erm(41) gene. Two of the 38 MMA isolates had an rrl A2059G point mutation. Most of the MABC strains were susceptible to amikacin and tigecycline. In Taiwan, amikacin, clarithromycin and tigecycline have good activity against MMA and MAB erm(41) C28 sequevar isolates, whereas amikacin and tigecycline, rather than clarithromycin, have good activity against both MBO and MAB erm(41) T28 sequevar isolates. Clinical trials are warranted to correlate these data with clinical outcomes.

Acinetobacter colistiniresistens sp. nov. (formerly genomic species 13 sensu Bouvet and Jeanjean and genomic species 14 sensu Tjernberg and Ursing), isolated from human infections and characterized by intrinsic resistance to polymyxins.

Strains of the genusAcinetobacter, classified as genomic species 13BJ/14TU have been previously associated with human infections and resistance to colistin. To clarify the taxonomy of this provisional group, we investigated 24 strains that have been isolated from humans since the 1960s in 10 countries. The genus-wide analysis of the rpoB and gyrB sequences of all strains and whole-genome sequences of strains representing different rpoB/gyrB genotypes showed that the 24 strains formed a distinct monophyletic group within the so-called haemolytic clade of the genus Acinetobacter. The distinctness of the group at the species level was supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by matrix-assisted laser desorption ionization-time-of-flight MS. The 24 strains had very similar metabolic features and could be distinguished from other members of the genus by the combination of strong haemolytic and proteolytic activities and the ability to oxidize d-glucose and grow on phenylacetate and/or l-phenylalanine. The minimum inhibitory concentrations of the 24 strains to colistin and polymyxin B ranged from 16 to 64 mgl-1 and from 4 to 32 mgl-1, respectively, so uniformly reaching the current clinical resistance breakpoint (4 mg l-1) for these drugs. Genus-wide comparison revealed that such a consistently high level of resistance to polymyxins is a unique feature among species of the genus Acinetobacter,which occur in humans. We conclude that genomic species 13BJ/14TU represents a biologically meaningful and medically relevant species, for which the name Acinetobacter colistiniresistens sp. nov. is proposed. The type strain is NIPH 2036T (=CCM 8641T=CIP 110478T=CCUG 67966T=CNCTC 7573T).

Bartonella in Rodents and Ectoparasites in the Canary Islands, Spain: New Insights into Host-Vector-Pathogen Relationships.

Bartonella genus is comprised of several species of zoonotic relevance and rodents are reservoirs for some of these Bartonella species. As there were no data about the range of Bartonella species circulating among rodents in the Canary Islands, our main aim was to overcome this lack of knowledge by targeting both the citrate synthase (gltA) and the RNA polymerase beta subunit (rpoB) genes. A total of 181 small mammals and 154 ectoparasites were obtained in three of the Canary Islands, namely Tenerife, La Palma, and Lanzarote. The overall prevalence of Bartonella DNA in rodents was 18.8%, whereas the prevalence in ectoparasites was 13.6%. Bartonella sequences closely related to the zoonotic species Bartonella elizabethae, Bartonella tribocorum, and Bartonella rochalimae were identified in rodents, whereas two different gltA haplotypes similar to B. elizabethae were also detected in fleas. Furthermore, Bartonella queenslandensis DNA was also identified in rodents. A strong host specificity was observed, since B. elizabethae DNA was only found in Mus musculus domesticus, whereas gltA and rpoB sequences closely related to the rest of Bartonella species were only identified in Rattus rattus, which is probably due to the host specificity of the arthropod species that act as vectors in these islands. Our results indicate that humans may contract Bartonella infection by contact with rodents in the Canary Islands.

Characterization of Corynebacterium diphtheriae, C. confusum and C. amycolatum isolated from sows with genitourinary infection

Porcine Corynebacterium infection is still poorly studied, even though the pig has been described as an asymptomatic carrier of Corynebacterium species, including the zoonotic species C. ulcerans, C. confusum and C. amycolatum. Here we present the identification, molecular and antimicrobial susceptibility characterization of coryneform bacteria isolated from sows with urinary tract infection. C. diphtheriae, C. confusum and C. amycolatum were isolated from sows with urinary infection and metritis. Corynebacterium species were identified by MALDI-TOF MS and confirmed by 16S rRNA and rpoB sequencing. All porcine C. diphtheriae strains were further characterized as non-toxigenic (tox−). SE-AFLP genotyping was also performed and enabled not only Corynebacterium species differentiation but also the assessment of C. amycolatum genetic heterogeneity. All studied porcine Corynebacterium strains presented alarming resistance profiles with high MIC values for macrolides/lincosamide, tetracyclines and quinolones, which can be related with high usage in both veterinary and human medicine. Isolation of zoonotic Corynebacterium species from commercial swine is important for assessing the potential zoonotic risk for farmers and further implication for both human and animal treatment.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria

BackgroundThe differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. MethodsA total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. ResultsThe sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15–100%, p<0.0001) and 97.8% (95% CI 91.86–99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56–100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529–534, 524–529, and 514–520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). ConclusionsThe entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48–72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples.

Phylogenetic analysis of Mycobacterium massiliense strains having recombinant rpoB gene laterally transferred from Mycobacterium abscessus.

Recent multi locus sequence typing (MLST) and genome based studies indicate that lateral gene transfer (LGT) events in the rpoB gene are prevalent between Mycobacterium abscessus complex strains. To check the prevalence of the M. massiliense strains subject to rpoB LGT (Rec-mas), we applied rpoB typing (711 bp) to 106 Korean strains of M. massiliense infection that had already been identified by hsp65 sequence analysis (603 bp). The analysis indicated 6 smooth strains in M. massiliense Type I (10.0%, 6/60) genotypes but no strains in M. massiliense Type II genotypes (0%, 0/46), showing a discrepancy between the 2 typing methods. Further MLST analysis based on the partial sequencing of seven housekeeping genes, argH, cya, glpK, gnd, murC, pta and purH, as well as erm(41) PCR proved that these 6 Rec-mas strains consisted of two distinct genotypes belonging to M. massiliense and not M. abscessus. The complete rpoB sequencing analysis showed that these 6 Rec-mas strains have an identical hybrid rpoB gene, of which a 478 bp partial rpoB fragment may be laterally transferred from M. abscessus. Notably, five of the 6 Rec-mas strains showed complete identical sequences in a total of nine genes, including the seven MLST genes, hsp65, and rpoB, suggesting their clonal propagation in South Korea. In conclusion, we identified 6 M. massiliense smooth strains of 2 phylogenetically distinct genotypes with a specific hybrid rpoB gene laterally transferred from M. abscessus from Korean patients. Their clinical relevance and bacteriological traits remain to be elucidated.

138 Use of rpoB sequence clustering for species-level identification of the Elizabethkingia genus from UK CF patients, and an investigation of associated antimicrobial susceptibility

138 Use of rpoB sequence clustering for species-level identification of the Elizabethkingia genus from UK CF patients, and an investigation of associated antimicrobial susceptibility

Pseudomonas versuta sp. nov., isolated from Antarctic soil

In this study we analysed three bacterial strains coded L10.10T, A4R1.5 and A4R1.12, isolated in the course of a study of quorum-quenching bacteria occurring in Antarctic soil. The 16S rRNA gene sequence was identical in the three strains and showed 99.7% pairwise similarity with respect to the closest related species Pseudomonas weihenstephanensis WS4993T. Therefore, the three strains were classified within the genus Pseudomonas. Analysis of housekeeping genes (rpoB, rpoD and gyrB) sequences showed similarities of 84-95% with respect to the closest related species of Pseudomonas, confirming its phylogenetic affiliation. The ANI values were less than 86% to the closest related species type strains. The respiratory quinone is Q9. The major fatty acids are C16:0, C16:1 ω7c/ C16:1 ω6c in summed feature 3 and C18:1 ω7c / C18:1 ω6c in summed feature 8. The strains are oxidase- and catalase-positive. Growth occurs at 4–30°C, and at pH 4.0–10. The DNA G+C content is 58.2–58.3mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains L10.10T, A4R1.5 and A4R1.12 into a novel species of Pseudomonas, for which the name P. versuta sp. nov. is proposed. The type strain is L10.10T (LMG 29628T, DSM 101070T).

Clonal outbreaks of [Pasteurella] pneumotropica biovar Heyl in two mouse colonies

The aim of this study was to document the pathogenic role of biovar Heyl of [ Pasteurella] pneumotropica in mouse colonies. Fifty-three isolates associated with mastitis and orbital, cutaneous and vaginal abscesses as well as isolates from the nose and vagina of healthy mice were investigated. According to phenotypic characteristics and rpoB sequencing, the isolates were identified as [ P.] pneumotropica biovar Heyl. Pulsed-field gel electrophoresis (PFGE) revealed five closely related profiles separated by only one to four fragments. The outbreak strains diverged from epidemiologically unrelated strains with the same rpoB sequence type, as shown by the PFGE profiles. The investigation documented that members of biovar Heyl of [ P.] pneumotropica caused disease outbreaks in mouse colonies since the clonality indicated a primary role of [ P.] pneumotropica biovar Heyl in the infections observed.

Molecular identification and distribution of non‐tuberculous mycobacteria isolated from clinical specimens by PCR‐sequencing method in West of Iran

Infections by non-tuberculous mycobacteria (NTM) has rapidly increased in recent years, due to high infection rates related to the populations at risk like immunocompromised individuals, patients predisposed by prior pulmonary. The aim of this study was to investigate the presence of NTM in clinical samples and genetic diversity using 16S rRNA and rpoB sequence analysis. A cross-sectional study was conducted on 45 diverse isolates collected from sputum in 2 years 2014-2015 using standard decontamination procedure. All mycobacterial isolates were grown on LJ medium and also conventional tests for preliminary identification of mycobacteria rely on traits and then DNA extraction. PCR was performed, and sequencing of 16S rRNA and rpoB genes was applied for NTM strains identification. A total of 45 isolates collected, 37 samples (83%) were evaluated as NTM. All NTM strains using molecular methods by sequencing 16S rRNA and rpoB gene were identified, by this way 12 different species have been identified which sequencing of rpoB was able to identify all species. The major species obtained were Mycobacterium simiae (22%), M. fortuitum (19%), and M. abscessus (13%). The results of our study showed that the patients were infected by a wide range of atypical mycobacteria. It was concluded that 16S rRNA gene sequencing coupled with rpoB marker is a high discriminatory power in identification of NTM. The presence of various species in clinical samples in Iran emphasizes the use of molecular method like sequence analysis of genes is necessary for reliable identification.

Antimicrobial susceptibility testing of rapidly growing mycobacteria isolated in Japan

BackgroundDifficult-to-treat infections caused by rapidly growing mycobacteria (RGM) are increasingly observed in clinical settings. However, studies on antimicrobial susceptibilities and effective treatments against RGM in Japan are limited.MethodsWe conducted susceptibility testing of potential antimicrobial agents, including tigecycline and tebipenem, against RGM. Clinical RGM isolates were collected from a university hospital in Japan between December 2010 and August 2013. They were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the sequencing of 16S rRNA, rpoB, and hsp65 genes. The samples were utilized for susceptibility testing using 16 antimicrobials, with frozen broth microdilution panels.ResultsForty-two isolates were obtained: 13, Mycobacterium abscessus complex; 12, Mycobacterium chelonae; 9, Mycobacterium fortuitum; and 8, M. fortuitum group species other than M. fortuitum. Different antimicrobial susceptibility patterns were observed between RGM species. Clarithromycin-susceptible strain rates were determined to be 0, 62, and 100% for M. fortuitum, M. abscessus complex, and M. chelonae, respectively. M. abscessus complex (100%) and >80% M. chelonae isolates were non-susceptible, while 100% M. fortuitum group isolates were susceptible to moxifloxacin. Linezolid showed good activity against 77% M. abscessus complex, 89% M. fortuitum, and 100% M. chelonae isolates. Regardless of species, all tested isolates were inhibited by tigecycline at very low minimal inhibitory concentrations (MICs) of ≤0.5 μg/mL. MICs of tebipenem, an oral carbapenem, were ≤4 μg/mL against all M. fortuitum group isolates.ConclusionsOur study demonstrates the importance of correct identification and antimicrobial susceptibility testing, including the testing of potential new agents, in the management of RGM infections.

Carbapenem-resistance and pathogenicity of bovine Acinetobacter indicus-like isolates.

The objective of this study was to characterize blaOXA-23 harbouring Acinetobacter indicus-like strains from cattle including genomic and phylogenetic analyses, antimicrobial susceptibility testing and evaluation of pathogenicity in vitro and in vivo. Nasal and rectal swabs (n = 45) from cattle in Germany were screened for carbapenem-non-susceptible Acinetobacter spp. Thereby, two carbapenem resistant Acinetobacter spp. from the nasal cavities of two calves could be isolated. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified these isolates as A. indicus-like. A phylogenetic tree based on partial rpoB sequences indicated closest relation of the two bovine isolates to the A. indicus type strain A648T and human clinical A. indicus isolates, while whole genome comparison revealed considerable intraspecies diversity. High mimimum inhibitory concentrations were observed for carbapenems and other antibiotics including fluoroquinolones and gentamicin. Whole genome sequencing and PCR mapping revealed that both isolates harboured blaOXA-23 localized on the chromosome and surrounded by interrupted Tn2008 transposon structures. Since the pathogenic potential of A. indicus is unknown, pathogenicity was assessed employing the Galleria (G.) mellonella infection model and an in vitro cytotoxicity assay using A549 human lung epithelial cells. Pathogenicity in vivo (G. mellonella killing assay) and in vitro (cytotoxicity assay) of the two A. indicus-like isolates was lower compared to A. baumannii ATCC 17978 and similar to A. lwoffii ATCC 15309. The reduced pathogenicity of A. indicus compared to A. baumannii correlated with the absence of important virulence genes encoding like phospholipase C1+C2, acinetobactin outer membrane protein BauA, RND-type efflux system proteins AdeRS and AdeAB or the trimeric autotransporter adhesin Ata. The emergence of carbapenem-resistant A. indicus-like strains from cattle carrying blaOXA-23 on transposable elements and revealing genetic relatedness to isolates from human clinical sources requires further investigations regarding the pathogenic potential, genomic characteristics, zoonotic risk and putative additional sources of this new Acinetobacter species.

Molecular Detection ofCandidatusBartonella mayotimonensis in North American Bats

Candidatus Bartonella mayotimonensis was detected in 2010 from an aortic valve sample of a patient with endocarditis from Iowa, the United States of America. The environmental source of the potentially new endocarditis-causing Bartonella remained elusive. We set out to study the prevalence and diversity of bat-associated Bartonella in North America. During 2015, mist nets and harp traps were used to capture 92 bats belonging to two species: little brown myotis (Myotis lucifugus Le Conte 1831, n = 73) and the gray myotis (M. grisescens A.H. Howell 1909, n = 19) in Kentucky, Michigan, Pennsylvania, and Tennessee. DNA preparations of peripheral blood samples from bats were subjected to a three-marker (gltA, rpoB, and intergenic spacer region [ISR]) multilocus sequence analysis. Sequence-verified gltA-positive PCR amplicons were obtained from nine samples. Three sequences were 99.7-100% identical with the gltA sequence of the Iowa endocarditis patient strain. Analysis of rpoB and ISR sequences demonstrated that one little brown myotis sample from the Upper Peninsula of Michigan contained Bartonella DNA, with 100% sequence identity with the Iowa endocarditis patient strain DNA. It appears possible that bats are a reservoir of Candidatus Bartonella mayotimonensis in North America.

Streptococcus himalayensis sp. nov., isolated from the respiratory tract of Marmota himalayana.

Five strains of Gram-positive-staining, catalase-negative, coccus-shaped, chain-forming organisms isolated separately from the respiratory tracts of five Marmota himalayana animals in the Qinghai-Tibet Plateau of China were subjected to phenotypic and molecular taxonomic analyses. Comparative analysis of the 16S rRNA gene indicated that these singular organisms represent a new member of the genus Streptococcus, being phylogenetically closest to Streptococcus marmotae DSM 101995T (98.4 % similarity). The groEL, sodA and rpoB sequence analysis showed interspecies similarity values between HTS2T and Streptococcus. marmotae DSM 101995T, its closest phylogenetic relative based on 16S rRNA gene sequences, of 98.2, 78.8 and 93.7 %, respectively. A whole-genome phylogenetic tree built from 82 core genes of genomes from 16 species of the genus Streptococcus validated that HTS2T forms a distinct subline and exhibits specific phylogenetic affinity with S. marmotae. In silico DNA-DNA hybridization of HTS2T showed an estimated DNA reassociation value of 40.5 % with Streptococcus. marmotae DSM 101995T. On the basis of their phenotypic characteristics and phylogenetic findings, it is proposed that the five isolates be classified as representatives of a novel species of the genus Streptococcus, Streptococcus himalayensis sp. nov. The type strain is HTS2T (=DSM 101997T=CGMCC 1.15533T). The genome of Streptococcus himalayensis sp. nov. strain HTS2T contains 2195 genes with a size of 2 275 471 bp and a mean DNA G+C content of 41.3 mol%.

The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential

This study aimed to characterize the species, antimicrobial resistance and dispersion of CRISPR systems in staphylococci isolated from the oropharynx of domestic cats in Brazil. Staphylococcus strains (n=75) were identified by MALDI-TOF and sequencing of rpoB and tuf genes. Antimicrobial susceptibility was assessed by disk diffusion method and PCR to investigate the presence of antimicrobial-resistance genes usually present in mobile genetic elements (plasmids), in addition to plasmid extraction. CRISPR − genetic arrangements that give the bacteria the ability to resist the entry of exogenous DNA − were investigated by the presence of the essential protein Cas1 gene. A great diversity of Staphylococcus species (n=13) was identified. The presence of understudied species, like S. nepalensis and S. pettenkoferi reveals that more than one identification method may be necessary to achieve conclusive results. At least 56% of the strains contain plamids, being 99% resistant to at least one of the eight tested antimicrobials and 12% multidrug resistant. CRISPR were rare among the studied strains, consistent with their putative role as gene reservoirs. Moreover, herein we describe for the first time their existence in Staphylococcus lentus, to which the system must confer additional adaptive advantage. Prevalence of resistance among staphylococci against antimicrobials used in veterinary and human clinical practice and the zoonotic risk highlight the need of better antimicrobial management practices, as staphylococci may transfer resistance genes among themselves, including to virulent species, like S. aureus.

Kribbella soli sp. nov., isolated from soil.

A novel actinobacterial strain, designated FMN22T, was isolated from soil and characterised using a polyphasic approach. Strain FMN22T showed high 16S rRNA gene sequence similarity to Kribbella karoonensis Q41T (99.3%), Kribbella shirazensis UTMC 693T (99.0%), Kribbella aluminosa HKI 0478T (98.9%) and Kribbella hippodromi S1.4T (98.6%). Phylogenetic analysis using the 16S rRNA and concatenated gene (gyrB, rpoB, relA, recA and atpD) sequences showed that strain FMN22T is closely related to the type strains of K. karoonensis DSM 17344T, K. shirazensis UTMC 693T, K. aluminosa HKI 0478T, K. hippodromi S1.4T, Kribbella jejuensis HD9T and Kribbella solani DSA1T. Based on concatenated gene genetic distances analysis, strain FMN22T is distinct from all Kribbella type strains. DNA-DNA hybridization experiments with closely related type strains, were found to be 59.2±2.4, 54.8±2.1, 16.4±2.3 and 38.6±2.5%, relatedness to K. karoonensis DSM 17344T, K. shirazensis DSM 45490T, K. aluminosa DSM 18824T and K. jejuensis DSM 17305T, respectively. The cell wall peptidoglycan contained LL-diaminopimelic acid, and whole cell sugars were glucose, mannose and ribose. The predominant menaquinone was MK-9(H4). The major phospholipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. Major fatty acids are anteiso-C15:0 and iso-C16:0. These chemotaxonomic traits are in good agreement with those known for representatives of the genus Kribbella. A combination of DNA-DNA hybridization results and phenotypic properties demonstrated that strain FMN22T can be clearly distinguished from all close phylogenetic relatives. Therefore, strain FMN22T (=DSM 27132T=KCTC 29219T) is considered to be the type strain of a novel species of the genus Kribbella, for which the name Kribbella soli is proposed.

Emergence of Rare Species of Nontuberculous Mycobacteria as Potential Pathogens in Saudi Arabian Clinical Setting.

BackgroundClinical relevance of nontuberculous mycobacteria (NTM) is increasing worldwide including in Saudi Arabia. A high species diversity of NTM’s has been noticed in a recent study. However, the identification in diagnostic laboratories is mostly limited to common species. The impact of NTM species diversity on clinical outcome is so far neglected in most of the clinical settings.Methodology/Principal FindingsDuring April 2014 to September 2015, a nationwide collection of suspected NTM clinical isolates with clinical and demographical data were carried out. Primary identification was performed by commercial line probe assays. Isolates identified up to Mycobacterium species level by line probe assays only were included and subjected to sequencing of 16S rRNA, rpoB, hsp65 and 16S-23S ITS region genes. The sequence data were subjected to BLAST analysis in GenBank and Ez-Taxon databases. Male Saudi nationals were dominated in the study population and falling majorly into the 46–59 years age group. Pulmonary cases were 59.3% with a surprising clinical relevance of 75% based on American Thoracic Society guidelines. Among the 40.7% extra-pulmonary cases, 50% of them were skin infections. The identification revealed 16 species and all of them are reporting for the first time in Saudi Arabia. The major species obtained were Mycobacterium monascence (18.5%), M. cosmeticum (11.1%), M. kubicae (11.1%), M. duvalli (7.4%), M.terrae (7.4%) and M. triplex (7.4%). This is the first report on clinical relevance of M. kubicae, M. tusciae, M.yongonense, M. arupense and M.iranicum causing pulmonary disease and M. monascence, M. duvalli, M. perigrinum, M. insubricum, M. holsaticum and M. kyorinense causing various extra-pulmonary diseases in Saudi Arabia. Ascites caused by M. monascence and cecum infection by M. holsaticum were the rarest incidents.Conclusions/SignificanceTo the first time in the country, clinical significance of various rare NTM’s are well explored and the finding warrants a new threat to the Saudi Arabian clinical settings.