rpoB Gene Research Articles

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh

BackgroundMulti-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear–positive patients using Line Probe Assay (LPA). MethodsA cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. ResultsThe overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30–59 years (43.8 %) and predominantly in males. ConclusionTuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Rhizobium acaciae and R. anhuiense are the dominant rhizobial symbionts of Pisum sativum L. from Yunnan-Guizhou Plateau.

The aim of this study is to investigate the diversity and geographic distribution of pea-nodulating rhizobia in the subtropical region of Yunnan Province from Yunnan-Guizhou Plateau. A total of 615 rhizobial isolates were obtained from root nodules of the trapping plants and characterized genetically and symbiotically. The isolates discriminated into 43 genotypes by PCR-RFLP of IGS DNA. Multiple locus sequence analysis based on 16S rRNA, recA, atpD, dnaK, and rpoB genes placed them into eight clusters corresponding to species R. acaciae, R. anhuiense, R. binae, R. bangladeshense, R. hidalgonense, and three suspected novel populations of Rhizobium genosp. I-III. R. acaciae was the dominant group (52.5%) followed by R. anhuiense (30.7%). The other species were minor groups. Based on nodC phylogeny, all of them were the symbiovar viciae. All the tested strains showed efficient symbiotic N2 fixation on pea plants, in which WLB27, WCB18, and WNY29 presented the best PGP effects. Some of the tested strains had better IAA production, with WCB18 as the best producer (64.556 mg/L). Their distribution was mainly affected by soil available phosphorus, available potassium, and effective nitrogen. According to the results of symbiotic effect and resistance tests, strains of WLB27, WCB18, and WNY29 were selected as candidates for creating inoculants. This suggests that the pea-nodulating rhizobia in Yunnan Province form a unique community. The results gave some novel information about the diversity, diversification, and biogeography of pea-nodulating rhizobia.

First report of bacterial leaf spot on Hami melon caused by Pseudomonas aeruginosa in China.

Hami melon (Cucumis melon var. saccharinus) is an economically important crop all over the world. It is being extensively planted in greenhouse in the southwest part of Hainan province, China. A new bacterial leaf spot was observed in a 20 hm2 Hami melon plantation in Huangliu town, Ledong county, Hainan province, in January 2022. The incidence of the disease was approximately 5%. Symptoms were irregularly shaped, brown lesions with yellow haloes on mature leaves, and first appeared as small, dark-green, water-soaking spots. Specimens from the lesion margin were disinfected by submersion in 0.1% mercuric chloride for 1 min, then soaked with 75% alcohol for 30 s, and rinsed with sterilized distilled water. The tissues were then ground in 2 ml of sterile water and allowed to stand for 5min. The supernatant was streaked onto nutrient agar (NA) and incubated for 48h at 30°C. Colonies were round, smooth, colorless, nearly transparent, bead-shaped at first, and then became lightly blue. After being cultured for days on NA at 30℃, the bacteria can turn the media brown. Yellow green pigments (pyoverdin) that fluoresce under ultraviolet light could be produced by the isolates in the Luria Broth. The bacteria were gram-negative, rod shaped with a single polar flagellum, 0.4 to 1.1 × 1.4 to 3.4 µm. Its physiological and biochemical characteristics were as follows: positive for the oxidase, aerobic, arginine dihydrolase, gelatin liquefaction, denitrification, lipase, growth at 41℃, utilization of mannitol, and production of pyocyanin tests; negative for the hydrolysis of starch, levan formation, lecithinase, growth at 4℃, growth in media supplemented with 8.5% NaCl, and utilization of maltose, xylose, and ethylene glycol tests. The 16S rRNA (1,437 bp), gyrB (1,181 bp), and rpoB genes (1,510 bp) were amplified with 27F/1492R (Zhang et al. 2016）, UP-1s/UP-2sr（Hannula M，2007）, and rpoB-F/rpoB-R (Ogier, JC. et al., 2019) primer sets respectively. One of the 5 isolates collected was sequenced. A BLASTn search of GenBank revealed that the sequence of 16S rRNA gene (OQ918303) had 99.7% identity and 98% coverage comparing with the best hit Pseudomonas aeruginosa strain DSM 50071(NR_117678.1), and both gyrB (OR261077) and rpoB (OR261078) had 99.9% identity and over 98% coverage comparing with P. aeruginosa E90 (CP044006.1). A pathogenicity test was conducted by spraying a suspension of the bacteria (108 CFU/mL) onto 10 Hami melon seedlings with two true leaves. Controls were inoculated with sterile water. All inoculated plants were maintained at 28℃ with 80 to 85% relative humidity in a greenhouse. Dark-green, water soaking spots appeared on the cotyledon and stems of treated seedlings 3-5 days after inoculation, and dark green lesions with halos were observed on the true leaves at the same time. Symptoms did not occur on the control plants. The bacteria which were re-isolated from the inoculated plants were confirmed as P. aeruginosa based on the 16S rRNA gene sequence. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause disease on a variety of plants including tomato (Zhang et al., 2021), poplar (Liu, et al., 2019), ginseng (Gao et al., 2014), tinda (Mondal et al., 2012), onion (Abd-Alla et al., 2011), tobacco (Yu et al., 2008) and sweet basil (Walker et al., 2004). As far as we know, this is the first report of P. aeruginosa causing leaf spot on Hami melon in China.. This report will contribute to the recognition and diagnosis of the new disease for the Hami melon growers.

Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium Tuberculosis Isolates.

Type II toxin-antitoxin systems such as mazEF3, vapBC3, and relJK play a role in antibiotic resistance and tolerance. Among the different known TA systems, mazEF3, vapBC3, and relJK, which are type II systems, have specific roles in drug resistance. Therefore, the aim of this study was to investigate the mutations in these genes in sensitive and resistant isolates of Mycobacterium tuberculosis. Thirty-two rifampin-resistant and 121 rifampin-sensitive M. tuberculosis isolates were collected from various regions of Iran. Lineage typing was performed using the ASO-PCR method. Mutations in the rpoB gene were analyzed in all isolates by MAS-PCR. Furthermore, mutations in the mazEF3, relJK, and vapBC3 genes of the type II toxin system were assessed through PCR sequencing. These sequences were analyzed using COBALT and SnapGene 2017, and submitted to the GenBank database. Among the 153 M. tuberculosis samples, lineages 4, 3 and 2 were the most common. Lineage 2 had the highest rate of rifampin resistance. Mutations in rpoB531 were the most frequent in resistant isolates. Examination of the toxin-antitoxin system showed that rifampin-resistant isolates belonging to lineage 3 had mutations in either the toxin or antitoxin parts of all three TA systems. A mutation in nucleotide 195 (codon 65) of mazF3 leading to an amino acid change from threonine to isoleucine was detected in all rifampin-resistant isolates. M. tuberculosis isolates belonging to lineage 2 exhibited the highest rifampin resistance in our study. Identifying the mutation in mazF3 in all rifampin-resistant isolates can highlight the significance of this mutation in the development of drug resistance in M. tuberculosis. Expanding the sample size in future studies can help develop a new method for identifying resistant isolates.

Study of some virulence genes from uropathogenic Klebsiella pneumoniae and Pseudomonas aeruginosa isolated in Wasit Province, Iraq

Abstract Background: Klebsiella pneumoniae and Pseudomonas aeruginosa are the most common causes of nosocomial urinary tract infections (UTIs), and in recent years, these bacteria have become significantly more resistant to antibiotics. Objective: The objective of the study was to determine the frequency of uropathogenic K. pneumoniae and P. aeruginosa in clinical urine samples from patients with UTIs in Wasit Province. Materials and Methods: Initially, 100 clinical urine samples were taken for this investigation from (October 1 to December 1, 2022), and the isolates were initially identified using culture, microscopic analysis, and biochemical assays. Results: The findings of the biochemical results revealed that 16/100 isolates had a 16% positive response for K. pneumonia, and 13/100 isolates had a 13% positive response for P. aeruginosa. When DNA was recovered from the isolates, the nucleic acid purity was diverse from 1.8 to 2.0, and the concentration fluctuated from 50 to 360 mg/µL. By using molecular techniques, including the 16S rRNA gene, for confirmation of the diagnosis. These findings were validated by the polymerase chain reaction method used to detect the virulence genes fimH gene, where 4/16 (25%) and mrkA gene, where result 7/16 (43.75%) were positive in recovered K. pneumoniae isolated. And RNA polymerase subunit B (rpoB) gene where results 10/13 (76.92%) were positive in recovered P. aeruginosa isolated. Conclusion: In conclusion, the outcomes of this study presented a significant increase in biofilm-forming and multidrug-resistant K. pneumoniae isolates, which also harbor a wide range of virulence genes and used rpoB as high molecular identification properties to confirm the P. aeruginosa, the most efficient antibiotics for its treatment were Impinem and Nitrofurantoin for both K. pneumonia and P. aeruginosa.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Bacterial Quality and Molecular Detection of Food Poisoning Virulence Genes Isolated from Nasser Lake Fish, Aswan, Egypt.

The microbial analysis of fish is critical for ensuring overall health. Uncooked fish can serve as a conduit for transmitting several types of microbes; the current investigation sought to assess the bacterial levels in various kinds of fish from Nasser Lake, Aswan, Egypt, considered the chief source of potable water in Egypt. Two hundred and fifty fish samples, including 50 of each Oreochromis niloticus, Sander lucioperca, Lates niloticus, Clarias gariepinus, and Mormyrus kannume, from Nasser Lake, Aswan, Egypt, were collected to detect the bacterial load, isolation, and identification of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio parahaemolyticus and their virulence genes. The findings revealed that Oreochromis niloticus and Clarias gariepinus exhibited higher bacterial loads than other fish species. Incidences of bacterial contamination among examined fishes were 28.8%, 20.4%, and 16% for Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio parahaemolyticus, respectively. Additionally, PCR analysis detected the presence of aerA (60%) and Act (40%) genes in A. hydrophila, rpoB (70%) and LasB (30%) genes in P. aeruginosa, and ToxR (70%) and tdh (50%) genes in V. parahaemolyticus. The study suggested that the bacterial contamination levels in Oreochromis niloticus and Clarias gariepinus could be notably more significant than in other species that could potentially be harmful to the consumers, especially considering the identification of particular bacteria known to cause foodborne illnesses. Further recommendations emphasized that regular monitoring and assessments are required to preserve their quality.

A rapid field-ready electrical biosensor consisting of bismuthine-derived Au island decorated BiOCl nanosheets for Raphidiopsis raciborskii detection in freshwater.

Cyanobacteria play an essential role in nutrient cycling in aquatic ecosystems. However, certain species adversely affect the environment and human health by causing harmful cyanobacterial algal blooms (cyanoHABs) and producing cyanotoxins. To address this issue, continuous cyanoHAB monitoring has been considered; however, a gold standard has not yet been established. In this study, we aimed to develop a dual DNA-targeting capacitive-type biosensor for rapid field-ready monitoring of Raphidiopsis raciborskii, a causative species of cyanoHAB. To enhance the sensing signal, a plate-like Au-BiOCl nanocomposite was synthesized using a spontaneous carbonation process without additional additives. The alternating-current electrothermal flow (ACEF) technique was applied to enable rapid DNA and probe binding within 10 min. The limits of detection (LODs) for R. raciborskii RubisCO large subunit (rbcL) and RNA polymerase beta subunit (rpoB) genes diluted in deionized (DI) water were 4.89 × 10-17 and 3.89 × 10-17 M, respectively. Furthermore, the LODs of R. raciborskii rbcl and rpoB diluted in freshwater containing HAB were 2.55 × 10-16 and 3.84 × 10-16 M, respectively, demonstrating the field-ready applicability of the device. The fabricated cyanobacterial DNA-sensing platform enabled powerful species-specific detection using a small sample volume and low target concentration without a nucleic acid amplification step, dramatically reducing the detection time. This study has considerable implications for detecting HABs, early warning systems, and species-specific environmental monitoring technology.

Enhanced detection of rifampicin and isoniazid resistance in mycobacterium tuberculosis using AuNP-qPCR: a rapid and accurate method.

To evaluate the resistance of Mycobacterium tuberculosis to Rifampicin (RIF) and Isoniazid (INH) using enhanced qPCR methodologies. This study compared the detection of drug-resistant mutations in the rpoB and katG genes using AuNP-qPCR and No-AuNP-qPCR. Calibration curves were constructed to correlate the amount of template with the Ct values for resistant strains. The AuNP-qPCR method demonstrated high efficacy in detecting RIF resistance with an area under the curve (AUC) of 0.951, sensitivity of 97.92%, specificity of 87.5%, and overall accuracy of 95.31%. Similarly, INH resistance detection by AuNP-qPCR showed an AUC of 0.981, sensitivity of 98.08%, specificity of 94.44%, and accuracy of 97.14%. Comparatively, No-AuNP-qPCR yielded lower performance metrics for RIF resistance (AUC: 0.867, sensitivity: 91.67%, specificity: 75%, accuracy: 87.5%) and INH resistance (AUC: 0.882, sensitivity: 88.46%, specificity: 83.33%, accuracy: 87.14%). AuNP-qPCR exhibits over traditional qPCR methods, making it a promising tool for rapid and precise detection of drug resistance in Mycobacterium tuberculosis. This method's robust performance underscores its potential to improve diagnostic protocols and contribute to more effective management of tuberculosis treatment.

Virulence genes and antimicrobial resistance of Bacillus cereus in prepackaged pastries sold in Taizhou from 2020 to 2022

To investigate the virulence genes and antimicrobial resistance of Bacillus cereus from the pre-packaged pastries in Taizhou city. 500 pre-packaged patries were collected in taizhou city market. 97 Bacillus cereus strains were detected from them by GB 4789.14-2014 method and identified with 4 houseking genes, then 13 virulence genes were detected by polymerase chain reaction(PCR)method and the antimicrobial resistance of Bacillus cereus to 19 antibiotics was detected by paper diffusion method. The result showed that the contamination rate of Bacillus cereus was 19.4% in 500 pre-packaged pastries. The detection rate of four housekeeping genes groEL, gyr B, rpoB and Vrr were 100%, 94.8%, 97.9% and 96.9%, respectively, and 89.7% at the same time. The virulence gene test result showed that the detection rate of nheABC, entFM, bceT, cytK and hblABCD were 91.8%, 88.7%, 61.9%, 51.6% and 25.8%, emetic virulence genes had the lowest detection rate, ces and EMl were 4.1%, cer was 5.2%. 97 Bacillus cereus strains show different degrees of drug resistance to 14 antimicrobials, the resistance rates to penicillin, ampicillin, cefotaxime and cotrimoxazole were higher than 95%, but they were completely sensitive to streptomycin, vancomycin and chloramphenicol. There is a risk of contamination by diarrhea-type Bacillus cereus and vomiting-type Bacillus cereus in prepackaged pastries in Taizhou. The isolated and identified Bacillus cereus has multiple-drug resistance.

Mutations And Antibiotic Resistance In Mycobacterium Tuberculosis

Mycobacterium tuberculosis (MTB) is a bacterium known to target, infect, and destroy lung cells as well as connective tissues within the body. This bacterium is prevalent worldwide and has infected over a quarter of the current world population, becoming one of the most successful pathogens in history. Due to its extreme transmission rates as an airborne pathogen, MTB strains have been treated with antibiotics such as rifampicin and isoniazid, which inhibit bacterial infection in the human body. These first-round drugs remained as successful mechanisms for slowing and killing the pathogen, notably through rifampicin's inhibition of RNA-polymerase and isoniazid’s ability to halt the formation of the bacterial cell wall. However, TB has proven a threat due to the recent discovery of multi-drug-resistant tuberculosis strains, rendering these first-round drugs ineffective. The major objectives of the present study were to 1) review the most recent published literature on TB, 2) examine the role of mutations on antibiotic resistance in TB strains and 3) share our synthesis on the successes and challenges of TB treatment worldwide. Our research was guided through data available in the NCBI GenBank, and a review of literature. To accomplish these objectives, we reviewed relevant literature on Mycobacterium tuberculosis to collect pathophysiological data, trends in TB mutations, and present-day applications of how this disease is continually prevalent worldwide. We collected antibiotic responsive rpoB gene sequences from the National Library of Medicine’s GenBank to assess mutations specific strains of TB from four countries. We found that random mutations caused the evolution of TB strains with effective antibiotic resistance and the selective nature of the medications encourages these antibiotic-resistant genes. New medications, like bedaquiline, take considerable research but effectively find new druggable targets against these resistant mycobacteria. However, MDR TB still remains a considerable threat despite some newly developed drugs.

First Report of Pantoea agglomerans Causing Leaf Blight on Schisandra chinensis in China.

Schisandra chinensis (Turcz.) Baill is a perennial liana, which is widely cultivated and used in China. In August 2022, Schisandra chinensis leaves with small light brown spots were found on plants growing in Fusong (127°28'E, 42°33'N) of China. There was 15% disease incidence and 50% disease severity of Schisandra chinensis in 2-ha fields of S. chinensis. As the disease progressed, the spots become darker and form round or irregular concentric circles. Leaves with brown spot symptoms were collected from the field. Leaf pieces (5 mm × 5 mm) were excised from lesion margins, surface disinfected with 75% ethanol for 1 min, followed by 1.5% sodium hypochlorite for 3 min, and incubated on Luria Bertani (LB) solid medium at 28°C for 24 hours. Eight cultures were isolated, and representative single colony (XWWZH) was selected from the pure cultures according to colony characteristics for observation The purified colonies were round, yellow, and slimy, cells were straight rod-shaped (0.40 to 0.52 × 1.12 to 1.69 µm) were observed. The isolate was Gram negative. It was positive for methyl red reaction, lysine decarboxylase reaction, gelatin hydrolysis reactionand sucrose utilization. It was negative for indole reaction and produced H2S. The bacterium was preliminarily identified as Pantoea agglomerans based on morphological and biochemical tests (Baird et al. 2007). The 16S rDNA and a portion of rpoB of strain XWWZH were amplified and sequenced. The sequences were submitted to GenBank. (Accession OP763753 and OQ813505, respectively). Phylogenetic trees were constructed based on the 16S rDNA and rpoB gene sequences. The sequences of strain XWWZH clustered with strains P. agglomerans deposited in GenBank. The pathogenicity was verified with non-wounded S. chinensis seedlings by punching holes with sterile needles and injecting a solution of 1 × 108 CFU/ml solution. Sterile ddH2O was injected in the control experiment. The inoculated seedlings were incubated in a greenhouse at 25°C with a relative humidity of 65 to 70%. Five to eight days after inoculation, inoculated leaves, exhibited symptoms which were morphologically identical to those of the originally infected leaves whereas control plants remained asymptomatic. The pathogenicity assays were repeated twice with the same results. The re-isolated pathogen had the same morphology and DNA sequences as the original isolate obtained from the field samples, completing Koch's postulates. Strains of P. agglomerans have been reported to severely infect many plants (Ren et al.2008; Lee et al. 2010; Yang et al. 2011; Guo et al. 2019; Gao et al, 2022), but to the best of our knowledge, this is the first report of a strain of P. agglomerans causing leaf blight on Schisandra chinensis in China. The identification of leaf blight caused by P. agglomerans will enable farmers to prevent and manage it ahead of time to reduce losses.

Identification and Characterization of Arcanobacterium canis from Companion Animals in Germany and The United Kingdom

Arcanobacterium canis is a novel species of the Arcanobacterium most closely related to A. haemolyticum. This study aims to characterize two A. canis isolates recovered from companion animals, specifically the claw of a cat and a vaginal swab from a dog. This study used real-time PCR to characterize A. canis isolated from companion animals. Two isolates of A. canis were recovered from purulent material from the claw of an 11-year-old cat in Germany and a vaginal swab of a dog in the United Kingdom. The samples were characterized phenotypically and genotypically. Both isolates were analyzed using culture methods, biochemical analysis, MALDI-TOF MS, real-time PCR amplification and sequencing of the 16S rRNA gene, and rpoB, gap, and tuf genes. The findings showed that the isolates P5197-15 and M214-96-1 obtained from companion animals were successfully characterized and confirmed to species level by real-time PCR amplification and sequencing of the 16S rRNA gene, as well as the genes of rpoB, gap, and tuf. This study seeks to comprehensively understand the characteristics of A. canis isolates obtained from companion animals. Such knowledge is essential for accurate diagnosis, treatment, and control of infections caused by this pathogen in veterinary medicine. Additionally, it contributes to the broader understanding of the genetic diversity and characteristics of A. canis, which can have implications for public health and animal well-being.

Selection and Characterization of Antagonistic Bacteria as a High Potential Control Agent for Xanthomonas oryzae pv. oryzae Causing Bacterial Leaf Blight Disease in Purple Rice

Bacterial leaf blight disease is one of the most serious diseases in rice caused by Xanthomonas oryzae pv. oryzae. Purple rice is one of the rice varieties that has been infected by this pathogen. Thus, screening and selecting highly efficient antagonistic bacteria for controlling the pathogen is necessary for disease control to reduce epidemics and crop loss. Forty-eight isolates of antagonistic bacteria were isolated from rice rhizosphere soil. The highly effective antagonistic bacteria were selected by dual culture method. The results showed bacteria strains KY16 and KY17 were the most effective antagonists that were able to inhibit X. oryzae pv. oryzae. The inhibition growth zone of antagonists were approximately 12.70 mm and 11.95 mm, respectively. The morphological and some biochemical characteristics of antagonistic bacteria belong to Bacillus sp. Additionally, the antagonists KY16 and KY17 were identified as Bacillus siamensis based on 16S rRNA, gyrA, and rpoB gene sequences. Antimicrobial biosynthesis genes of the antagonistic bacteria were also examined. The results revealed that iturin A and surfactin biosynthesis genes were detectable. From this study, the selected antagonistic bacteria can be used to control the bacterial leaf blight disease of purple rice in rice paddy fields.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India

IntroductionThe importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. MethodA total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. ResultsOf these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. ConclusionThe in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

Comparative evaluation of critical concentrationsfor detecting borderline rifampin resistance in Mycobacterium tuberculosis

Background: Rifampin plays an important role in tuberculosis treatment. In recent years, the introduction of molecular testing techniques has enabled the rapid detection of rifampin resistance, leading to discrepancies with conventional methods. The World Health Organization (WHO) has analyzed mutations in the rpoB gene that induce rifampin resistance and identified certain mutations causing borderline resistance, which are often undetected using conventional tests. Consequently, the WHO has lowered the rifampin resistance criterion concentration from 1.0 to 0.5 μg/mL in 7H10 and MGIT 960. The present study aimed to evaluate the impact of this change in critical concentration on the detection of borderline rifampin resistance in Mycobacterium tuberculosis. Methods: Tuberculosis strains submitted for antituberculosis drug susceptibility testing from May 2021–2022 were analyzed. Three institutions participated; the Seoul Clinical Laboratories used the agar proportion method, whereas the Samsung Medical Center and Seoul National University Bundang Hospital utilized the MGIT 960 system to test both the original and revised concentrations. Mutations were confirmed through rpoB gene sequencing for strains showing discrepancies. Results: A total of 1,596 valid susceptibility tests were conducted during the study period. Rifampin resistance was detected in 35 cases (2.19%) at 1.0 μg/mL and in 38 cases (2.38%) at 0.5 μg/mL, whereas isoniazid resistance was observed in 158 cases (9.90%). Among the three rifampin discrepancy strains, one harbored an H445L mutation, whereas the remaining two exhibited an L452P mutation. These mutations were classified as borderline resistant. Conclusion: Applying the new rifampin critical concentration resulted in a 0.19% increase in resistance rate and an 8.57% increase in detection cases. Additionally, despite testing with large number of rifampin-susceptible strains, no false resistance results were obtained. Therefore, the application of the new critical concentration is considered beneficial for the management of rifampin-resistant tuberculosis.

Prevalence of Beijing Central Asian/Russian Cluster 94-32 among Multidrug-Resistant M. tuberculosis in Kazakhstan.

The Beijing genotype is the most distributed M. tuberculosis family in Kazakhstan. In this study, we identified dominant Beijing clusters in Kazakhstan and assessed their drug susceptibility profiles and association with the most widely spread mutation Ser531Leu of the rpoB gene and the mutation Ser315Thr of the katG gene associated with resistance to rifampicin and isoniazid, respectively. M. tuberculosis isolates (n = 540) from new TB cases were included in the study. MIRU-VNTR genotyping was performed for 540 clinical isolates to determine M. tuberculosis families using 24 loci. RD analysis was additionally performed for the Beijing isolates. The identification of mutations in the drug-resistance genes of M. tuberculosis was performed with allele-specific real-time PCR and Sanger sequencing. The Beijing genotype was identified in 60% (324/540) of the clinical isolates. Central Asian/Russian cluster 94-32 was the most distributed cluster among the Beijing isolates (50.3%; 163/324). Three other dominant Beijing clusters were identified as 94-33 (3.4%; 11/324), 100-32 (3.1%; 10/324) and 99-32 (3.1%; 10/324). The Beijing genotype was associated with drug-resistant TB (p < 0.0001), including multidrug-resistant TB (p < 0.0001), in our study. An association of the mutation Ser531Leu of the rpoB gene with the Beijing genotype was found (p < 0.0001; OR = 16.0000; 95%CI: 4.9161-52.0740). Among the Beijing isolates, cluster 94-32 showed an association with MDR-TB (p = 0.021). This is why the evaluation of the Beijing genotype and its clusters is needed to control MDR-TB in Kazakhstan.

Pseudomonas aphyarum sp. nov., Pseudomonas fontis sp. nov., Pseudomonas idahonensis sp. nov. and Pseudomonas rubra sp. nov., isolated from in, and around, a rainbow trout farm.

During a large-scale bacterial culturing effort of biofilms in the vicinity of a rainbow trout aquaculture facility in Idaho, USA, 10 isolates were identified as having pathogen-inhibiting activity and were characterized further. These isolates were shown to be Gram-negative, rod-shaped bacteria belonging to the genus Pseudomonas. Whole-genome comparisons and multi-locus sequence analysis using four housekeeping genes (16S rRNA, gyrA, rpoB and rpoD) showed that these 10 isolates clustered into four distinct species groups. These comparisons also indicated that these isolates were below the established species cutoffs for the genus Pseudomonas. Further phenotypic characterization using API 20NE, API ZYM and Biolog GENIII assays and chemotaxonomic analysis of cellular fatty acids were carried out. Based on the genomic, physiological and chemotaxonomic properties of these isolates, we concluded that these strains composed four novel species of the genus Pseudomonas. The proposed names are as follows: Pseudomonas aphyarum sp. nov. consisting of strains ID233, ID386T and ID387 with ID386T (=DSM 114641T=ATCC TSD-305T) as the type strain; Pseudomonas rubra sp. nov. consisting of strains ID291T, ID609 and ID1025 with ID291T (=DSM 114640T=ATCC TSD-303T) as the type strain; Pseudomonas idahonensis sp. nov. consisting of strains ID357T and ID1048 with ID357T (=DSM 114609T=ATCC TSD-304T) as the type strain; and Pseudomonas fontis sp. nov. consisting of strains ID656T and ID681 with ID656T (=DSM 114610T=ATCC TSD-306T) as the type strain.

Evaluation of Metabarcoding Methods for Plant Disease Surveillance

Metabarcoding holds great potential for general plant pathogen surveillance by providing an untargeted profile of the host microbiome. However, the standard marker utilized for microbiome analysis of prokaryotes, the 16 rRNA gene, offers limited diagnostic utility as it provides phylogenetic resolution primarily at the genus level, and universal primers often co-amplify plant DNA. Here, we evaluated two recently published universal primer sets targeting the DNA gyrase and RNA polymerase ß subunit genes ( gyrB and rpoB, respectively), relative to a plant discriminating 16S primer set (799F/1115R), as disease surveillance tools. Comparative analysis of a mock bacterial community, as well as naturally infected citrus variegated chlorosis samples, indicated that the gyrB method displayed optimal performance in the amplification of DNA across a broad taxonomic spectrum of plant-pathogenic bacteria, providing resolution at the species and, in some cases, subspecies levels. It also generated high-quality datasets with minimal to no co-amplification of plant DNA, outperforming the rpoB and 16S assays in these regards. Further evaluation revealed that the gyrB method displayed an overall linear trend in the detection of several diverse bacterial pathogens; however, it was at least an order of magnitude less sensitive than a standard real-time PCR assay. Finally, we demonstrate the potential of this method to disentangle a mixed population of Pantoea spp. associated with a rice-bacterial blight outbreak and identify putative novel pathogens that would otherwise be overlooked with conventional PCR-based tests. This work represents steps toward establishing a robust, untargeted metabarcoding method for general plant disease surveillance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .