rpoB Sequences Research Articles

Outbreak with clonally related isolates of Corynebacterium ulcerans in a group of water rats.

BackgroundThe zoonotic bacterium Corynebacterium ulcerans may be pathogenic both in humans and animals: toxigenic strains can cause diphtheria or diphtheria-like disease in humans via diphtheria toxin, while strains producing the dermonecrotic exotoxin phospholipase D may lead to caseous lymphadenitis primarily in wild animals. Diphtheria toxin-positive Corynebacterium ulcerans strains have been isolated mainly from cattle, dogs and cats.ResultsHere, we report a series of ten isolations of Corynebacterium ulcerans from a group of water rats (Hydromys chrysogaster) with ulcerative skin lesions, which were kept in a zoo. The isolates were clearly assigned to species level by biochemical identification systems, Fourier-transform infrared-spectroscopy, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and partial rpoB sequencing, respectively. All ten isolates turned out to represent the same sequence type, strongly indicating a cluster of infections by clonally-related isolates as could be demonstrated for the first time for this species using multilocus sequence typing. Unequivocal demonstration of high relatedness of the isolates could also be demonstrated by Fourier-transform infrared-spectroscopy. All isolates were lacking the diphtheria toxin encoding tox-gene, but were phospholipase D-positive.ConclusionsOur results indicate that water rats represent a suitable new host species that is prone to infection and must be regarded as a reservoir for potentially zoonotic Corynebacterium ulcerans. Furthermore, the applied methods demonstrated persistent infection as well as a very close relationship between all ten isolates.

Clinical implications of species identification in monomicrobial Aeromonas bacteremia.

BackgroundAdvances in Aeromonas taxonomy have led to the reclassification of aeromonads. Hereon, we aimed to re-evaluate the characteristics of Aeromonas bacteremia, including those of a novel species, Aeromonas dhakensis.Methodology/Principal FindingsA retrospective study of monomicrobial Aeromonas bacteremia at a medical center in southern Taiwan from 2004–2011 was conducted. Species identification was based on rpoB sequencing. Of bacteremia of 153 eligible patients, A. veronii (50 isolates, 32.7%), A. dhakensis (48, 31.4%), A. caviae (43, 28.1%), and A. hydrophila (10, 6.5%) were the principal causative species. A. dhakensis and A. veronii bacteremia were mainly community-acquired and presented as primary bacteremia, spontaneous bacterial peritonitis, or skin and soft-tissue infection, whereas A. caviae was associated with hospital-onset bacteremia. The distribution of the AmpC β-lactamase and metallo-β-lactamase genes was species-specific: bla AQU-1, bla MOX, or bla CepH was present in A. dhakensis, A. caviae, or A. hydrophila, respectively, and bla CphA was present in A. veronii, A. dhakensis, and A. hydrophila. The cefotaxime resistance rates of the A. caviae, A. dhakensis, and A. hydrophila isolates were higher than that of A. veronii (39.5%%, 25.0%, and 30% vs. 2%, respectively). A. dhakensis bacteremia was linked to the highest 14-day sepsis-related mortality rate, followed by A. hydrophila, A. veronii, and A. caviae bacteremia (25.5%, 22.2%, 14.0%, and 4.7%, respectively; P = 0.048). Multivariate analysis revealed that A. dhakensis bacteremia, active malignancies, and a Pitt bacteremia score ≥ 4 was an independent mortality risk factor.Conclusions/SignificanceCharacteristics of Aeromonas bacteremia vary between species. A. dhakensis prevalence and its associated poor outcomes suggest it an important human pathogen.

Disputed rpoB mutations can frequently cause important rifampicin resistance among new tuberculosis patients.

Greater Mymensingh area, Bangladesh. To document among new tuberculosis (TB) patients the proportions and treatment outcomes of silent, non-disputed and disputed (generally missed by rapid drug susceptibility testing [DST]) rpoB mutations, and their detection by commercial molecular assays. Retrospective analysis of rpoB sequences from randomly selected ethanol-preserved diagnostic sputum samples; comparison of sequencing with conventional DST results and standard first-line treatment outcome; retesting of samples with mutations using the Xpert MTB/RIF and GenoType MTBDRplus assays. Of 1091 samples, 5.8% failed amplification, and six contained other mycobacteria. In 2005 and 2010, respectively 2/500 (0.4%) and 11/522 (2.1%) amplicons showed non-silent mutations. At least 7/13 of these belonged to the disputed group, with 5/7 patients suffering adverse treatment outcome. One silent mutation went undetected by commercial assays. Following routine DST indications, only three cases with a non-silent mutation were eventually detected. Disputed rpoB mutations may be responsible for the majority of rifampicin (RMP) resistance among new cases, and lead to adverse outcomes of first-line treatment. Silent mutations do not necessarily cause Xpert or line-probe assay false RMP-resistant results. Molecular RMP DST could greatly simplify resistance surveillance, in addition to offering the best prospects for early and accurate individual diagnosis.

Uruburuella testudinis sp. nov., isolated from tortoise (Testudo).

A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis. In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5-100%, and of up to 98.2% with U. suis . DNA-DNA hybridization indicated the organisms represented a novel species with only 40% DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C(16 : 0) and summed feature C(16 : 1)ω7c/iso-C(15 : 0) 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624(T) ( = DSM 26510(T) = CCUG 63373(T)).

Growth inhibition and microcystin degradation effects of Acinetobacter guillouiae A2 on Microcystis aeruginosa

Strain A2 with algicidal activity against Microcystis aeruginosa was isolated and identified with the genus Acinetobacter on the basis of phenotypic tests and 16S rRNA gene analysis. It was identified with the species Acinetobactor guillouiae by partial rpoB sequence analysis. When 10% (v/v) of the bacterial culture was co-incubated with M. aeruginosa culture, algicidal efficiency reached 91.6% after 7 days. Supernatant of A2 culture showed similar algicidal activity, while the cell pellet had little activity, suggesting that Acinetobacter guillouiae A2 indirectly attacked M. aeruginosa cells by secreting an extracellular algicidal compound, which was characterized as heat-stable. A significant decrease in the microcystin (microcystin-LR) concentration was observed after 10% (v/v) addition of A2 culture. Transcription of three microcystin-related genes (mcyA, mcyD and mcyH) was also found to be inhibited. The algicidal compound 4-hydroxyphenethylamine was obtained by further isolation and purification using various chromatographic techniques. The EC50, 3d and EC50, 7d values of 4-hydroxyphenethylamine against M. aeruginosa were 22.5 and 10.3 mgL−1, respectively. These results indicate that A. guillouiae strain A2 inhibits growth of M. aeruginosa and degrades microcystin production. The identified compound, 4-hydroxyphenethylamine, has potential for development as a new algicidal formulation or product.

Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

ObjectiveA PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in ‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. ResultsThe sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. ConclusionOur findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Nontuberculous mycobacterial ocular infections--comparing the clinical and microbiological characteristics between Mycobacterium abscessus and Mycobacterium massiliense.

PurposeTo analyze the clinical characteristics of nontuberculous mycobacterial (NTM) ocular infections and the species-specific in vitro antimicrobial susceptibility.Material and MethodsIn 2000 to 2011 at the National Taiwan University Hospital, multilocus sequencing of rpoB, hsp65 and secA was used to identify NTM isolates from ocular infections. The clinical presentation and treatment outcomes were retrospectively compared between species. Broth microdilution method was used to determine the minimum inhibitory concentrations of amikacin (AMK), clarithromycin (CLA), ciprofloxacin (CPF), levofloxacin (LVF), moxifloxacin (MXF) and gatifloxacin (GAF) against all strains. The activities of antimicrobial combinations were assessed by the checkerboard titration method.ResultsA total of 24 NTM strains (13 Mycobacterium abscessus and 11 Mycobacterium massiliense) were isolated from 13 keratitis, 10 buckle infections, and 1 canaliculitis cases. Clinically, manifestations and outcomes caused by these two species were similar and surgical intervention was necessary for medically unresponsive NTM infection. Microbiologically, 100% of M. abscessus and 90.9% of M. massiliense ocular isolates were susceptible to amikacin but all were resistant to fluoroquinolones. Inducible clarithromycin resistance existed in 69.3% of M. abscessus but not in M. massiliense isolates. None of the AMK-CLA, AMK-MXF, AMK-GAF, CLA-MXF and CLA-GAF combinations showed synergistic or antagonistic effect against both species in vitro.Conclusions M. abscessus and M. massiliense are the most commonly identified species for NTM ocular infections in Taiwan. Both species were resistant to fluoroquinolones, susceptible to amikacin, and differ in clarithromycin resistance. Combined antimicrobial treatments showed no interaction in vitro but could be considered in combination with surgical interventions for eradication of this devastating ocular infection.

A Problem of Species Determination Using Housekeeping Genes in the Streptococcus anginosus Group

For identification of three species categorized in Streptococcus anginosus group (SAG), genotyping employing multiplex PCR for detection of species-specific genes has been widely used. We wondered whether possession of the specific genes correlated to differentiation of housekeeping genes, with an attempt to obtain more information on genetic background of clinical isolates of SAG strains and to evaluate reliability of the multiplex PCR. We therefore determined nucleotide sequences of some housekeeping genes of 52 SAG isolates from healthy volunteer to investigate whether the sequences phylogenetically corresponded to the identification by the multiplex PCR. The phylogenetic trees drawn with all of the sequences of 16S rRNA, sodA, groEL and rpoB were found to clearly correlate to species identified by the multiplex PCR. Interestingly, phylogenetic tree drawn only with single housekeeping gene often did not match with the species identified by the multiplex PCR. Our analysis implies even housekeeping genes can have their sequence varieties as independent manner from presence of species-specific genes in SAG strains, and the choice of housekeeping genes for the phylogenetic analyses may result in failure of the species identification.

Rifampicin resistance among multi-resistant MRSA clinical isolates from Chennai, India, and their molecular characterization.

High-level methicillin-resistant Staphylococcus aureus (MRSA) isolates show rapid evolution of rifampicin resistance, forcing reliance upon expensive and often inferior antibiotics to manage these infections. Accordingly, this study was conducted to: 1) evaluate the level of multidrug resistance among hospital-associated MRSA isolates from Chennai, India; 2) determine their rifampicin resistance and molecular characterization; and 3) analyze the identified rpoB gene mutations for predominant high-level rifampicin resistance-associated nucleotide changes. Conventional laboratory techniques and antibiogram evaluation by disc diffusion were utilized for isolate phenotypic identification. Among the 54 isolates obtained, 74% (42) were found to be MRSA. All the isolates exhibited complete susceptibility to linezolid and vancomycin, and variable resistance to conventional antibiotics; the MAR index value calculated was 0.64. Genotypic identification of the high-level rifampicin-resistant isolate S. aureus KM05 was established through rpoB amplification and sequencing. The evolutionary relationship of KM05 to other isolates and its rpoB gene mutation status was determined to understand the genetic basis of its high rifampicin resistance. The S. aureus KM05 rpoB gene yielded ≥ 98% sequence similarity and a close phylogenetic relationship with other known reference database organisms. It also showed mutational changes in three rpoB codon positions encoding amino acids at positions 455, 481, and 529. These results have established the prevalence of rifampicin resistance among Chennai hospital MRSA isolates, and suggest that the predominant high-level resistance nucleotide changes would serve to form a basis for their diagnosis and control of rifampicin-resistant MRSA.

Genotypic and phenotypic identification of aeromonas species and carbapenem resistance in Queensland, Australia

Aims This study defines the population of Aeromonas spp. in Queensland and carbapenem resistance due to carbapenemase production, CphA. Phenotypic tests were correlated with the presence of the cphA gene. Methods Isolates from blood (22), wound (46), sterile sites (11), stool (18), eye (2) and sputum (1) were characterised by rpoB and gyrB sequencing. Disc diffusion and meropenem E-test MIC were determined. Carbapenemase production was assessed by Carba NP test and cphA by PCR. Results Isolates identified as A. dhakensis (39), A. veronii (21), A. hydrophila (20), A. caviae (14), A. jandaei (4), A. bestiarum (1), and A. sanarellii (1). Disc diffusion and E-test failed to demonstrate carbapenem resistance. Carba NP test performed with 97.4% sensitivity and 95.7% specificity. The cphA gene was detected in A. veronii (21; 100%), A. hydrophila (18; 90%), A. dhakensis (34; 87.2%), A. jandaei (2; 50%), A. bestiarum (1; 100%) and not A. caviae . Discussion Phylogenetic analysis revealed A. dhakensis caused majority of infection, an unrecognised pathogen in this region. Carbapenem resistance due to cphA was prevalent. The Carba NP was a reliable phenotypic test. Adequate speciation and detection of carbapenem resistance in Aeromonas spp. is essential to guide appropriate antimicrobial therapy.

Genotyping of New and Old <i>Proteus mirabilis</i> Isolates from Mansoura Hospitals in Egypt by <i>rpoB</i> Sequence Analysis

Sequence analysis of the RNA polymerase B subunit encoding gene (rpoB) has been proposed as a useful tool for bacterial identification. This method has been implemented to differentiate five well-defined Proteus species: P. mirabilis, P. hauseri, P. penneri, P. vulgaris, and P. myxofaciens. In this study, we evaluated the usefulness of rpoB sequencing for intraspecies discrimination of P. mirabilis. The sequence of rpoB 909 bp region was analyzed in 15 newly isolated strains and 5 of 8 years old isolates from different clinical sources. Three respective groups were obtained. The first group included 13 of the new strains showing similarity with Proteus mirabilis (ATCC 29906) strain. The second group including 3 of the old strains differs from the first group with a divergence of 0.22%. Group 3 contains only a single new strain 33. The sequence of this strain shows differences in the rpoB 909 bp region analyzed from the members of group 1 and the second group by 1.65% and 1.87% divergence respectively. According to our results, genetic differences could be detected within the P. mirabilis species. Therefore much more effort should be made to re-evaluate rpoB method and validate its usefulness as a molecular diagnostic method.

Identification of Clinical Corynebacterium striatum Strains by PCR-Restriction Analysis Using the RNA Polymerase β-subunit gene (rpob)

Corynebacterium striatum is frequently encountered in the routine clinical microbiology laboratory. It is widely disseminated in the environment and constitutes part of the normal micro-biota of the skin and mucous membrane. Identification of this species by biochemical methods remains difficult and several misidentifications have been reported previously. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available Corynebacterium rpoB sequences were analyzed by computerassisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and NlaIV clearly differentiated C. striatum from all other Corynebacterium species. This method was successfully applied for the reliable identification of 67 C. striatum clinical isolates and can be used for the timely detection of infected patients or for epidemiological studies.

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based VITEK MS System for the Identification of Acinetobacter Species from Blood Cultures: Comparison with VITEK 2 and MicroScan Systems

BackgroundAcinetobacter species are the leading cause of bloodstream infection (BSI), but their correct identification is challenging. We evaluated the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based VITEK MS (bioMérieux, France), and two automated systems, VITEK 2 (bioMérieux) and MicroScan (Siemens, USA) for identification of Acinetobacter BSI isolates.MethodsA total of 187 BSI isolates recovered at a university hospital in Korea between 2010 and 2012 were analyzed. The identification results obtained using VITEK MS and two automated systems were compared with those of rpoB sequencing.ResultsOf 187 isolates analyzed, 176 were identified to the species level by rpoB sequencing: the Acinetobacter baumannii group (ABG; 101 A. baumannii, 43 A. nosocomialis, 10 A. pittii isolates) was most commonly identified (82.4%), followed by Acinetobacter genomic species 13BJ/14TU (5.3%), A. ursingii (2.1%), A. soli (2.1%), A. bereziniae (1.1%), and A. junii (1.1%). Correct identification rates to the species group (ABG) level or the species level was comparable among the three systems (VITEK MS, 90.3%; VITEK 2, 89.2%; MicroScan, 86.9%). However, VITEK MS generated fewer misidentifications (0.6%) than VITEK 2 (10.8%) and MicroScan (13.1%) (P<0.001). In addition, VITEK MS demonstrated higher specificity (100%) for discrimination between ABG and non-ABG isolates than the other systems (both, 31.8%) (P<0.001).ConclusionsThe VITEK MS system is superior to the VITEK 2 and MicroScan systems for identification of Acinetobacter BSI isolates, with fewer misidentifications and better discrimination between the ABG and non-ABG isolates.

Diverse novel mesorhizobia nodulate New Zealand native Sophora species

Forty eight rhizobial isolates from New Zealand (NZ) native Sophora spp. growing in natural ecosystems were characterised. Thirty eight isolates across five groups showed greatest similarity to Mesorhizobium ciceri LMG 14989T with respect to their 16S rRNA and concatenated recA, glnll and rpoB sequences. Seven isolates had a 16S rRNA sequence identical to M. amorphae ATCC 19665T but showed greatest similarity to M. septentrionale LMG 23930T on their concatenated recA, glnll and rpoB sequences. All isolates grouped closely together for their nifH, nodA and nodC sequences, clearly separate from all other rhizobia in the GenBank database. None of the type strains closest to the Sophora isolates based on 16S rRNA sequence similarity nodulated Sophora microphylla but they all nodulated their original host. Twenty one Sophora isolates selected from the different 16S rRNA groupings produced N2-fixing nodules on three Sophora spp. but none nodulated any host of the type strains for the related species. DNA hybridisations indicated that these isolates belong to novel Mesorhizobium spp. that nodulate NZ native Sophora species.

Arcobacter ebronensis sp. nov. and Arcobacter aquimarinus sp. nov., two new species isolated from marine environment

Two strains recovered from mussels (F128-2T) and sea water (W63T) were characterized as Arcobacter sp., but they could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR) and rpoB gene analysis. The 16S rRNA gene sequence similarity to the type strains of all Arcobacter species ranged from 92.2% to 96.7% with strain F128-2T, and from 94.1% to 99.4% with strain W63T, the most similar being A. bivalviorum (CECT 7835T) and A. defluvii (CECT 7697T), respectively. The phylogenetic analyses of 16S rRNA, and the concatenated sequences of gyrB, gyrA, rpoB, atpA and hsp60 genes confirmed that strains F128-2T and W63T belonged to two new lineages within the genus Arcobacter. Moreover, both strains showed differential phenotypic characteristics and MALDI-TOF mass spectra from all other Arcobacter species. Therefore, it has been demonstrated the existence of two new Arcobacter species and the proposed names are Arcobacter ebronensis (type strain F128-2T=CECT 8441T=LMG 27922T), and Arcobacter aquimarinus (type strain W63T=CECT 8442T=LMG 27923T).

VibrioBase: A MALDI-TOF MS database for fast identification of Vibrio spp. that are potentially pathogenic in humans

Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments.

Bisgaardia miroungae sp. nov., a new member of the family Pasteurellaceae isolated from the oral cavity of northern elephant seals (Mirounga angustirostris), and emended description of the genus Bisgaardia.

A total of 17 bacterial isolates from northern elephant seals, tentatively classified within the family Pasteurellaceae, were further characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group, closely related to the genus Bisgaardia within the family Pasteurellaceae. The rpoB gene sequence similarity was 97.2-100 % within the group and 16S rRNA gene sequence comparisons showed 99.2-99.8 % similarity within the group. According to 16S rRNA gene sequence analysis, the most closely related species with a validly published name was Bisgaardia hudsonensis with 96.9 % similarity and the most closely related species based on rpoB sequence comparison was Bisgaardia genomospecies 1 with an rpoB sequence similarity of 90.9 %. All the isolates investigated exhibited the phenotypic characteristics of the family Pasteurellaceae. However, these isolates could be separated from existing species of the genus Bisgaardia by the following characteristics: ability to grow at 42 °C, and acid production from lactose, melibiose, raffinose and l-rhamnose, but not from d-mannitol or trehalose. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the strains should be classified as representatives of a novel species within the genus Bisgaardia: Bisgaardia miroungae sp. nov. The type strain, Wildatric(T) ( = CCUG 65148(T) = DSM 28141(T)), was isolated from the oral cavity of a wild northern elephant seal at The Marine Mammal Center, California, USA in 2011. To include the novel species, the description of the genus Bisgaardia has been emended.

Pest rodents as the essential elements of Mycobacterium bovis controlling programs

IntroductionAs a rule in any Bovine tuberculosis (BTB) control program, recognition of infection source is of great importance. Hence in this study, isolation and evaluation of Mycobacterial genomic patterns obtained from rodents of infected farms by RFLP method was conducted. Material and methodFifteen mice submitted from infected cattle farms with BTB that were collected in four provinces of IRAN were used in this study. Culture of Mycobacterium and RFLP–PGRS and RFLP–DR analysis of bovine isolates from the same areas were performed in the usual manner. Following the detection of different patterns between mice and cattle isolates, identification by PCR of IS6110 and RV0577, RD typing and sequencing of 16SrRNA and rpoB was conducted. ResultThree isolates were obtained out of the 15 mice samples cultured from 2 out of the 4 separate provinces. RFLP analysis with PGRS and DR probes identified 2 different patterns from 5 cattle isolates and 1 single pattern from 2 mice isolates located at Ahwaz province, and 2 different patterns from 9 bovine isolates and 1 pattern from 1 mouse isolates located at West Azerbaijan were identified.Two mice isolates from Ahwaz province were negative at the above PCRs and sequencing of 16SrRNA and rpoB revealed that these isolates are Mycobacterium kansasii. Another mouse identified as belonging to the Mycobacterium tuberculosis complex by PCRs and RD typing revealed that this isolate must be Mycobacterium microti. DiscussionThis study was unable to track tangible evidence of tuberculosis transmission by mice. Hence to prove this hypothesis, further studies are advised. However, it was found that mice are potentially a reservoir of zoonotic pathogens, and therefore its importance in this regard must be considered as an effective element of any controlling program.

Leptospira mayottensis sp. nov., a pathogenic species of the genus Leptospira isolated from humans.

A group of strains representing species of the genus Leptospira, isolated from patients with leptospirosis in Mayotte (Indian Ocean), were previously found to be considerably divergent from other known species of the genus Leptospira. This was inferred from sequence analysis of rrs (16S rRNA) and other genetic loci and suggests that they belong to a novel species. Two strains from each serogroup currently identified within this novel species were studied. Spirochaete, aerobic, motile, helix-shaped strains grew well at 30-37 °C, but not at 13 °C or in the presence of 8-azaguanine. Draft genomes of the strains were also analysed to study the DNA relatedness with other species of the genus Leptospira. The new isolates formed a distinct clade, which was most closely related to Leptospira borgpetersenii, in multilocus sequence analysis using concatenated sequences of the genes rpoB, recA, fusA, gyrB, leuS and sucA. Analysis of average nucleotide identity and genome-to-genome distances, which have recently been proposed as reliable substitutes for classical DNA-DNA hybridization, further confirmed that these isolates should be classified as representatives of a novel species. The G+C content of the genomic DNA was 39.5 mol%. These isolates are considered to represent a novel species, for which the name Leptospira mayottensis sp. nov. is proposed, with 200901116(T) ( = CIP 110703(T) = DSM 28999(T)) as the type strain.

Acinetobacter gandensis sp. nov. isolated from horse and cattle.

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467(T) showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467(T) showed the highest similarities with 'Acinetobacter bohemicus' ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467(T) were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467(T) and UG 60716 showed a DNA-DNA relatedness of 84 % with each other and a DNA-DNA relatedness with A. schindleri LMG 19576(T) of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467(T) was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467(T) ( = ANC 4275(T) = LMG 27960(T) = DSM 28097(T)).