rpoB Gene Research Articles

First Report of Bacterial Leaf Streak of Rice Caused by Pantoea ananatis in Guangdong Province, China.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 μL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

Pattern of Mutations in Rifampicin Resistance (rpoB) Gene and Analysis of Rifampicin Indeterminate Result in Tuberculosis Detected by Xpert MTB/RIF Assay in Kalaburagi, India

Tuberculosis (TB) is a serious disease that has been observed since ancient times. In the mid-to late-20th century, the main clinical approach to this disease involved focusing on its diagnosis, prevention, and treatment. However, in the 21st century, the focus has shifted toward the diagnosis and treatment of drug-resistant TB. With the use of the Xpert MTB/RIF assay at the frontlines in India, interpreting indeterminate results to treatment with rifampicin, an antitubercular drug, can be challenging. This is further exacerbated by a lack of knowledge regarding mutation frequency in antitubercular drug-resistant genes in this region. Among antitubercular drugs, rifampicin is the most potent and effective drug for the treatment of tuberculosis; hence, understanding the pattern of rifampicin resistance (rpoB) gene mutations will provide insights into the genetic basis of this resistance, which may help in the prevention and treatment of TB. This retrospective observational study presents sociodemographic details, sample types, Mycobacterium tuberculosis load, types of probe mutations detected, and rifampicin indeterminate results from the Xpert MTB/RIF assay. Of the 314 samples analyzed, 258 showed rifampicin resistance as detected by MTB, with 56 samples of MTB-detected rifampicin indeterminate results. Type E probe mutation (58.9%) was the most common type, while the least frequent mutation was Type C probe (1.5%). No missing probe was observed in approximately 8.9% of samples. Among the 56 rifampicin indeterminate results, the maximum Cycle threshold value did not cross 34.5 in six samples.

Drug susceptibility testing and line probe assay of first-line anti-tuberculosis drugs among presumptive tuberculosis patients attending a secondary care hospital in Bhubaneswar.

Pyrazinamide (PZA) is important for identification in multi-drug-resistant tuberculosis patients before starting therapy. PZA drug susceptibility testing (DST) is essential for the management of drug-resistant and susceptible TB patients. The degree of drug resistance among TB patients and discrepancy between DST results of the phenotype and genotype were assessed. Socio-demographic and clinical profiles of TB patients recruited in the study were documented. Sputum samples were processed for diagnosis using TrueNat Xpert MTB, TrueNat Xpert MTB Plus, and MGIT culture. Rifampicin (RIF) line probe assay (LPA) showed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100%, whereas isoniazid (INH) LPA testing showed a sensitivity of 85.7%, a specificity and PPV of 100%, and NPV of 94.8%. The gene mutation for RIF resistance was between the codon, 530-533 of rpoB gene, and that for INH resistance was at the codon, 315 of katG gene. Our findings demonstrated high prevalence of mono- and poly-drug resistance as well as pyrazinamide resistance.

Bartonella species in dromedaries and ruminants from Lower Shabelle and Benadir regions, Somalia.

Bartonellosis, caused by bacteria of the genus Bartonella, is a zoonotic disease with several mammalian reservoir hosts. In Somalia, a country heavily reliant on livestock, zoonotic diseases pose significant public health and economic challenges. To the best of our knowledge, no study has been performed aiming to verify the occurrence of Bartonella spp. in Somalia. This study investigated the occurrence and molecular characterization of Bartonella in dromedary (Camelus dromedarius, Linnaeus, 1758), cattle, sheep, and goats from Somalia. 530 blood samples were collected from various animals (155 dromedary, 199 goat, 131 cattle, and 45 sheep) in Benadir and Lower Shabelle regions. DNA was extracted for molecular analysis, and a qPCR assay targeting the NADH dehydrogenase gamma subunit (nuoG) gene was used for Bartonella screening. Positive samples were also subjected to PCR assays targeting seven molecular markers including: nuoG, citrate synthase gene (gltA), RNA polymerase beta-subunit gene (rpoB), riboflavin synthase gene (ribC), 60 kDa heat-shock protein gene (groEL), cell division protein gene (ftsZ), and pap31 and qPCR targeting the 16-23S rRNA internal transcribed spacer (ITS) followed by Sanger sequencing, BLASTn and phylogenetic analysis. Out of 530 tested animals, 5.1% were positive for Bartonella spp. by the nuoG qPCR assay. Goats showed the highest Bartonella occurrence (17/199, 8.5%), followed by sheep (6/44, 6.8%), cattle (4/131, 3.1%), and dromedary (1/155, 1.9%). Goats, sheep, and cattle had higher odds of infection compared to dromedary. Among nuoG qPCR-positive samples, 11.1%, 14.8%, 11.1%, and 25.9% were positive in PCR assays based on nuoG, gltA, and pap31 genes, and in the qPCR based on the ITS region, respectively. On the other hand, nuoG qPCR-positive samples were negative in the PCR assays targeting the ribC, rpoB, ftsZ, and groEL genes. While Bartonella bovis sequences were detected in cattle (nuoG and ITS) and goats (gltA), Bartonella henselae ITS sequences were detected in dromedary, goat, and sheep. Phylogenetic analysis placed gltA Bartonella sequence from a goat in the same clade of B. bovis. The present study showed, for the first time, molecular evidence of Bartonella spp. in dromedary and ruminants from Somalia and B. henselae in sheep and goats globally. These findings contribute valuable insights into Bartonella spp. occurrence in Somali livestock, highlighting the need for comprehensive surveillance and control measures under the One Health approach.

Mannheimia indoligenes sp. nov., proposed for clade V organisms of Mannheimia.

A set of 25 strains belonging to clade V of Mannheimia mainly isolated from cattle was investigated and is proposed to represent Mannheimia indoligenes sp. nov. The species can be separated from the other validly published species of the genus by pheno- and genotype. Only indole separates M. indoligenes and Mannheimia varigena while two to seven characters separate M. indoligenes from other species of Mannheimia. Thirteen strains belonging to biogroups 6, 7, 8C, 9, 10, 12 and UG5 formed a monophyletic group based on 16S rRNA gene sequence comparisons with 98-100 % similarity. Eight of these strains were further included in the whole genome comparison. Digital DNA-DNA hybridization showed that the similarities between the suggested type strain M14.4T and the other strains of M. indoligenes were 62.9 % or higher. The average nucleotide identity was 95.5 % or higher between M14.4T and the other strains of the species. The rpoB gene sequence similarity was 95-100 % within M. indoligenes. MALDI-TOF allowed a clear separation from other Mannheimia species further supporting classification as a novel species and making it the diagnostic identification tool of choice for M. indoligenes. The type strain is M14.4T (=CCUG 77347T=DSM 116804T) isolated from a cattle tongue in Scotland.

Macroevolutionary Dynamics in Micro-organisms: Generalists Give Rise to Specialists Across Biomes in the Ubiquitous Bacterial Phylum Myxococcota.

Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity. However, relatively little is known about how prokaryotes successfully transition and persist across environments, and how these processes might vary between biomes and lineages. Here, we investigate biome transitions and specialization in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2,000 unique Myxococcota lineages, with the majority (77%) classified as biome specialists and with only <5% of lineages distributed across the salt barrier. Discrete character evolution models revealed that specialists in one biome rarely transitioned into specialists in another biome. Instead, evolved generalism mediated transitions between biome specialists. State-dependent diversification models found variation in speciation rates across the tree, but this variation was independent of biome association or specialization. Our findings were robust to phylogenetic uncertainty, different levels of species delineation, and different assumed amounts of unsampled diversity resulting in an incomplete phylogeny. Overall, our results are consistent with a "jack-of-all-trades" tradeoff where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists and shed light on how bacteria could transition between biomes.

Streptococcus hohhotensis sp. nov., isolated from the breast milk of a healthy woman.

Human breast milk contains lactic acid bacteria (LAB), which have an important influence on the composition of the intestinal microbia of infants. In this study, one strain of an α-hemolytic species of the genus Streptococcus, IMAU99199T, isolated from the breast milk of a healthy nursing mother in Hohhot city PR China, was studied to characterise its taxonomic status using phenotypic and molecular taxonomic methods. The results indicated that it represented a member of the mitis-suis clade, pneumoniae subclade of the genus Streptococcus. It is a Gram-stain-positive, catalase-negative and oxidase-negative bacterium, and the cells are globular, paired or arranged in short chains. The results of a phylogenetic analysis of its 16S rRNA gene and two housekeeping genes (gyrB and rpoB) placed it in the genus Streptococcus. A phylogenetic tree based on 135 single-copy genes sequences indicated that IMAU99199T formed a closely related branch well separated from 'Streptococcus humanilactis' IMAU99125, 'Streptococcus bouchesdurhonensis' Marseille Q6994, Streptococcus mitis NCTC 12261T, 'Streptococcus vulneris' DM3B3, Streptococcus toyakuensis TP1632T, Streptococcus pseudopneumoniae ATCC BAA-960T and Streptococcus pneumoniae NCTC 7465T. IMAU99199T and 'S. humanilactis' IMAU99125 had the highest average nucleotide identity (93.7 %) and digital DNA-DNA hybridisation (55.3 %) values, which were below the accepted thresholds for novel species. The DNA G+C content of the draft genome of IMAU99199T was 39.8 %. The main cellular fatty acids components of IMAU99199T were C16 : 0 and C16 : 1ω7. It grew at a temperature range of 25-45 °C (the optimum growth temperature was 37 °C) and a pH range of 5.0-8.0 (the optimum growth pH was 7.0). These data indicate that strain IMAU99199T represents a novel species in the genus Streptococcus, for which the name Streptococcus hohhotensis sp. nov. is proposed. The type strain is IMAU99199T (=GDMCC 1.1874T=KCTC 21155T).

Two novel Raoultella species associated with bleeding cankers of broadleaf hosts, Raoultella scottia sp. nov. and Raoultella lignicola sp. nov.

Seventeen Gram-negative, facultatively anaerobic bacterial strains were isolated from bleeding cankers of various broadleaf hosts and oak rhizosphere soil in Great Britain. The strains were tentatively identified as belonging to the genus Raoultella based on 16S rRNA gene sequencing. Multilocus sequence analysis (MLSA), based on four protein-encoding genes (fusA, leuS, pyrG, and rpoB), separated the strains into three clusters within the Raoultella genus clade. The majority of strains clustered with the type strain of Raoultella terrigena, with the remaining strains divided into two clusters with no known type strain. Whole genome sequencing comparisons confirmed these two clusters of strains as belonging to two novel Raoultella species which can be differentiated phenotypically from their current closest phylogenetic relatives. Therefore, two novel species are proposed: Raoultella scottia sp. nov. (type strain = BAC 10a-01-01T = LMG 33072T = CCUG 77096T) and Raoultella lignicola sp. nov. (type strain = TW_WC1a.1T = LMG 33073T = CCUG 77094T).

Polymerase Chain Reaction (PCR) Profiling of Extensively Drug-Resistant (XDR) Pathogenic Bacteria in Pulmonary Tuberculosis Patients.

Introduction Pulmonary tuberculosis (TB) remains a global health concern, exacerbated by the emergence of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. This study employs advanced molecular techniques, specifically polymerase chain reaction (PCR) profiling, to comprehensively characterize the genetic landscape of XDR pathogenic bacteria in patients diagnosed with pulmonary TB. The objective of the study is to elucidate the genes that are associated with drug resistance in pulmonary TB strains through the application of PCR and analyze specific genetic loci that contribute to the development of resistance against multiple drugs. Materials and methods A total of 116 clinical samples suspected of TB were collected from the tertiary healthcare setting of Saveetha Medical College and Hospitals for the identification of MTB, which includes sputum (n = 35), nasal swabs (n = 17), blood (n = 44), and bronchoalveolar lavage (BAL) (n = 20). The collected specimens were processed and subjected to DNA extraction. As per the protocol, reconstitution of the DNA pellet was carried out. The reconstituted DNA was stored at -20 °C for the PCR assay. From the obtained positive sample specimens, XDR pulmonary TB specimens were focused on the targeted genes, specifically the rpoB gene for rifampicin resistance, inhA, and katG gene for thepromoter region for isoniazid resistance. Results Out of a total of 116 samples obtained, 53 tested positive for pulmonary TB, indicative of a mycobacterial infection. Among these positive cases, 43 patients underwent treatment at a tertiary healthcare facility. Subsequently, a PCR assay was performed with the extracted DNA for the target genes rpoB, inhA, and katG. Specifically, 22 sputum samples exhibited gene expression for rpoB, inhA, and katG, while nine nasal swabs showed expression of the rpoB and inhA genes. Additionally, rpoB gene expression was detected in seven blood specimens, and both rpoB and inhA genes were expressed in five BAL samples. Conclusion The swift diagnosis and efficient treatment of XDR-TB can be facilitated by employing advanced and rapid molecular tests and oral medication regimens. Utilizing both newly developed and repurposed anti-TB drugs like pretomanid, bedaquiline, linezolid, and ethionamide. Adhering to these current recommendations holds promise for managing XDR-TB effectively. Nevertheless, it is significant to conduct well-designed clinical trials and studies to further evaluate the efficacy of new agents and shorter treatment regimens, thus ensuring continuous improvement in the management of this challenging condition.

Antimicrobial resistance by mutation in the genes folp1, rpob and GYRA in patients with lepra diagnosed between 2017 and 2020 at the center of dermatological specialties-paraguay

Antimicrobial resistance by mutation in the genes folp1, rpob and GYRA in patients with lepra diagnosed between 2017 and 2020 at the center of dermatological specialties-paraguay

Use of rpoB gene phylogenetic marker-based distinction of abiotic stress tolerant and plant-growth promoting Bacillus paralicheniformis isolates from their closely related Bacillus licheniformis

Use of rpoB gene phylogenetic marker-based distinction of abiotic stress tolerant and plant-growth promoting Bacillus paralicheniformis isolates from their closely related Bacillus licheniformis

First Report of Enterobacter mori Causing Bacterial Wilt on Tomato in China.

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in China. In October 2023, a new bacterial disease was discovered on tomato plants in a 0.3-acre farm's greenhouse (35.514806N, 118.996106E) in Longshan Town, Shandong Province, China. Over 50% of the tomato plants showed symptoms of stem rot, leaf wilt, or plant death. Three diseased tomato plants were collected for pathogen isolation and purification. Two leaf samples, each about 1 cm2, were cut from the junction area of healthy and diseased parts and disinfected with 75% alcohol for 60 s, followed by 0.1% HgCl2 for 90 s, and then washed three times with sterilized H2O. The samples were subsequently ground with 1.0 mL sterilized H2O. The ground samples were diluted to 10-4, 10-5, and 10-6 and then were plated on a potato dextrose agar (PDA) plate, respectively. White mucous bacterial colonies appeared at 28℃ for 24~48 h, no fungal colony was observed. Six bacterial colonies were randomly selected for gram staining and found to be gram-negative. To further determine their species classification, fragments of the 16SrDNA, hsp60, gyrB, and rpoB genes were separately amplified using previously reported PCR conditions and with primer pairs, including 27F/1492R (Wu et al., 2023), HSP60-F /HSP60-R (Gül et al., 2023), gyrB UP-1 / gyrB UP-2r (Yamamoto et al., 1995) and rpoB CM81-F / rpoB CM32b-R (Brady et al., 2008). Sequence analysis showed that the obtained sequences of the 16SrDNA, hsp60, gyrB, and rpoB genes among these six colonies were identical and 100%, 100%, 99.31%, and 99.36% similar to those of Enterobacter mori accessions OP601841 (with a coverage of 100%), MT199160 (83%), OP676246 (100%), and MN594495 (100%), at nucleotide level, respectively. Sequences of the above four genes of 23LSFQ were submitted to GenBank under the accession numbers PP461247, PP474090, PP136037, and PP136038, respectively. We selected one of these six colonies, 23LSFQ, for further analysis. The phylogenetic tree based on the concatenated sequences of the above four genes using the maximum likelihood method with MEGAX software showed that 23LSFQ is grouped with E. mori LMG25706 (NCBI: txid980518). To determine the pathogenicity of 23LSFQ , we sprayed 23LSFQ (OD600=0.8) onto five 30-day-old healthy plants of the tomato cultivars Alisa Craig, Jinpeng NO.1, and Chaobei, respectively. These seedlings were incubated in a chamber at 28°C with a 16 h light/ 8h dark photoperiod and 60% relative humidity. The leaves of the inoculated plants became curled and wilted at two days post inoculation (dpi) and appeared necrotic at 10 dpi. The symptoms were similar to those observed in field-infected tomato plants. No symptoms were observed on the plants inoculated with water. We further sequenced the re-isolated bacteria from the symptomatic inoculated seedlings. Results showed that they belong to E. mori. The experiment was repeated three times. E. mori has been found to cause diseases on peaches (Ahmad et al., 2021), watermelons (Wu et al., 2023), Canna indica, (Zhang et al., 2023), and strawberries (Ji et al., 2023). E. cloacae has been found to cause diseases on tomatoes in Heilongjiang province (Jin et al., 2023). This is the first report of E. mori causing leaf yellowing and wilting on tomatoes in China. These results are significant for the safe production and disease control of greenhouse tomatoes.

Molecular Prevalence, Genetic Diversity, and Tissue Tropism of Bartonella Species in Small Mammals from Yunnan Province, China.

Bartonella is an intracellular parasitic zoonotic pathogen that can infect animals and cause a variety of human diseases. This study investigates Bartonella prevalence in small mammals in Yunnan Province, China, focusing on tissue tropism. A total of 333 small mammals were sampled from thirteen species, three orders, four families, and four genera in Heqing and Gongshan Counties. Conventional PCR and real-time quantitative PCR (qPCR) were utilized for detection and quantification, followed by bioinformatic analysis of obtained DNA sequences. Results show a 31.5% detection rate, varying across species. Notably, Apodemus chevrieri, Eothenomys eleusis, Niviventer fulvescens, Rattus tanezumi, Episoriculus leucops, Anourosorex squamipes, and Ochotona Thibetana exhibited infection rates of 44.4%, 27.7%, 100.0%, 6.3%, 60.0%, 23.5%, and 22.2%, respectively. Genetic analysis identified thirty, ten, and five strains based on ssrA, rpoB, and gltA genes, with nucleotide identities ranging from 92.1% to 100.0%. Bartonella strains were assigned to B. grahamii, B. rochalimae, B. sendai, B. koshimizu, B. phoceensis, B. taylorii, and a new species identified in Episoriculus leucops (GS136). Analysis of the different tissues naturally infected by Bartonella species revealed varied copy numbers across different tissues, with the highest load in spleen tissue. These findings underscore Bartonella's diverse species and host range in Yunnan Province, highlighting the presence of extensive tissue tropism in Bartonella species naturally infecting small mammalian tissues.

Carbapenemase genes in clinical and environmental isolates of Acinetobacter spp. from Quito, Ecuador.

Carbapenem-resistant Acinetobacter spp. is associated with nosocomial infections in intensive care unit patients, resulting in high mortality. Although Acinetobacter spp. represent a serious public health problem worldwide, there are a few studies related to the presence of carbapenemases in health care facilities and other environmental settings in Ecuador. The main aim of this study was to characterize the carbapenem-resistant Acinetobacter spp. isolates obtained from four hospitals (52) and from five rivers (27) close to Quito. We used the disc diffusion and EDTA sinergy tests to determine the antimicrobial susceptibility and the production of metallo β-lactamases, respectively. We carried out a multiplex PCR of gyrB gene and the sequencing of partial rpoB gene to bacterial species identification. We performed molecular screening of nine carbapenem-resistant genes (blaSPM, blaSIM, blaGIM, blaGES, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, and blaOXA-143) by multiplex PCR, followed by identification using sequencing of blaOXA genes. Our findings showed that carbapenem-resistant A. baumannii were the main species found in health care facilities and rivers. Most of the clinical isolates came from respiratory tract samples and harbored blaOXA-23, blaOXA-366, blaOXA-72, blaOXA-65, blaOXA-70, and blaOXA-143-like genes. The river isolates harbored only the blaOXA-51 and probably blaOXA-259 genes. We concluded that the most predominant type of carbapenem genes among isolates were both blaOXA-23 and blaOXA-65 among A. baumannii clinical isolates.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone

ObjectivesWe evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. MethodsFTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. ResultsOf the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DiscussionFTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Description of Corynebacterium hiratae sp. nov. isolated from a human tissue bone a novel member of Corynebacterium Genus.

Corynebacterium spp. are widely disseminated in the environment, and they are part of the skin and mucosal microbiota of animals and humans. Reports of human infections by Corynebacterium spp. have increased considerably in recent years and the appearance of multidrug resistant isolates around the world has drawn attention. To describe a new species of Corynebacterium from human tissue bone is described after being misidentified using available methods. For taxonomic analyses, phylogenetic analysis of 16S rRNA and rpoB genes, in silico DNA-DNA hybridization, average nucleotide and amino acid identity, multilocus sequence analysis, and phylogenetic analysis based on the complete genome were used. Genomic taxonomic analyzes revealed values of in silico DNA-DNA hybridization, average nucleotide and amino acids identity below the values necessary for species characterization between the analyzed isolates and the closest phylogenetic relative Corynebacterium aurimucosum DSM 44532T. Genomic taxonomic analyzes indicate that the isolates analyzed comprise a new species of the Corynebacterium genus, which we propose to name Corynebacterium hiratae sp. nov. with isolate 332T (= CBAS 826T = CCBH 35,014T) as the type strain.

Convergent genetic adaptation of Escherichia coli in minimal media leads to pleiotropic divergence.

Adaptation in an environment can either be beneficial, neutral or disadvantageous in another. To test the genetic basis of pleiotropic behaviour, we evolved six lines of E. coli independently in environments where glucose and galactose were the sole carbon sources, for 300 generations. All six lines in each environment exhibit convergent adaptation in the environment in which they were evolved. However, pleiotropic behaviour was observed in several environmental contexts, including other carbon environments. Genome sequencing reveals that mutations in global regulators rpoB and rpoC cause this pleiotropy. We report three new alleles of the rpoB gene, and one new allele of the rpoC gene. The novel rpoB alleles confer resistance to Rifampicin, and alter motility. Our results show how single nucleotide changes in the process of adaptation in minimal media can lead to wide-scale pleiotropy, resulting in changes in traits that are not under direct selection.

Clavibacter tessellarius causing bacterial mosaic of wheat establishes in the Old World

AbstractThe genus Clavibacter (family Microbacteriaceae) includes gram‐positive actinobacterial species infecting diverse agricultural crops such as alfalfa, maize, pepper, potato, tomato and wheat. Clavibacter tessellarius, causing bacterial mosaic of wheat, was reported for the first time in 1976 in Nebraska (United States). Since then, the pathogen was thought to be restricted to North America (Canada and the United States). While bacterial strains suspected to be C. tessellarius have occasionally been isolated from wheat samples outside North America, occurrence of the pathogen in the Old World has not yet been confirmed by the plant protection authorities. In spring 2020 (January to May) dozens of wheat fields with young plants showing leaf chlorosis and mild mosaic symptoms were surveyed and sampled in southern Iran. Among 192 surveyed wheat fields, gram‐positive, orange‐pigmented actinobacterial strains were isolated from samples of 53 fields. Altogether, 61 bacterial strains were identified as C. tessellarius based on the phenotypic features, PCR‐based molecular tests and pathogenicity assays. Multilocus sequence analysis of five housekeeping genes (atpD, dnaK, gyrB, ppk and rpoB) using 10 representative strains from diverse geographic areas in southern Iran confirmed the phylogenetic status of the strains within C. tessellarius. Our data indicate that the bacterial mosaic pathogen occurs in four southern Iranian provinces including Bushehr, Fars, Kerman and Khuzestan. Thus, occurrence of C. tessellarius outside North America is confirmed. Potential risks associated with the emergence of the pathogen in the Old World, and the need for strict quarantine measures in the area, is further discussed.