gyrB Gene Research Articles

A Class 4-like Chromosomal Integron Found in Aeromonas sp. Genomospecies paramedia Isolated from Human Feces.

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

In Situ Discrimination and Cultivation of Active Degraders in Soils by Genome-Directed Cultivation Assisted by SIP-Raman-Activated Cell Sorting.

The identification and in situ cultivation of functional yet uncultivable microorganisms are important to confirm inferences regarding their ecological functions. Here, we developed a new method that couples Raman-activated cell sorting (RACS), stable-isotope probing (SIP), and genome-directed cultivation (GDC)─namely, RACS-SIP-GDC─to identify, sort, and cultivate the active toluene degraders from a complex microbial community in petroleum-contaminated soil. Using SIP, we successfully identified the active toluene degrader Pigmentiphaga, the single cells of which were subsequently sorted and isolated by RACS. We further successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells, which was confirmed by gyrB gene comparison and average nucleotide identity determination. Additionally, the genotypes and phenotypes of this degrader were directly linked at the single-cell level, and its complete toluene metabolic pathways in petroleum-contaminated soil were reconstructed. Based on its unique metabolic properties uncovered by genome sequencing, we modified the traditional cultivation medium with antibiotics, amino acids, carbon sources, and growth factors (e.g., vitamins and metals), achieving the successful cultivation of RACS-sorted active degrader Pigmentiphaga sp. Our results implied that RACS-SIP-GDC is a state-of-the-art approach for the precise identification, targeted isolation, and cultivation of functional microbes from complex communities in natural habitats. RACS-SIP-GDC can be used to explore specific and targeted organic-pollution-degrading microorganisms at the single-cell level and provide new insights into their biodegradation mechanisms.

Isolation and identification of antagonistic Bacillus amyloliquefaciens HSE-12 and its effects on peanut growth and rhizosphere microbial community.

The HSE-12 strain isolated from peanut rhizosphere soil was identified as Bacillus amyloliquefaciens by observation of phenotypic characteristics, physiological and biochemical tests, 16S rDNA and gyrB gene sequencing. In vitro experiments showed that the strain possessed biocontrol activity against a variety of pathogens including Sclerotium rolfsii. The strain has the ability to produce hydrolytic enzymes, as well as volatile organic compounds with antagonistic and probiotic effects such as ethyleneglycol and 2,3-butanediol. In addition, HSE-12 showed potassium solubilizing (10.54 ± 0.19 mg/L), phosphorus solubilization (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) abilities, and was able to secrete siderophores [(Ar-A)/Ar × 100%: 56%] which promoted plant growth. After inoculating peanut with HSE-12, the available phosphorus content in rhizosphere soil increased by 27%, urease activity increased by 43%, catalase activity increased by 70% and sucrase activity increased by 50% (p < 0.05). The dry weight, fresh weight and the height of the first pair of lateral branches of peanuts increased by 24.7, 41.9, and 36.4%, respectively, compared with uninoculated peanuts. In addition, compared with the blank control, it increased the diversity and richness of peanut rhizosphere bacteria and changed the community structure of bacteria and fungi. The relative abundance of beneficial microorganisms such as Sphingomonas, Arthrobacter, RB41, and Micromonospora in rhizosphere soil was increased, while the relative abundance of pathogenic microorganisms such as Aspergillus, Neocosmospora, and Rhizoctonia was decreased.

Phylogeny and symbiotic effectiveness of indigenous rhizobial microsymbionts of common bean (Phaseolus vulgaris L.) in Malkerns, Eswatini

In most legumes, the rhizobial symbionts exhibit diversity across different environments. Although common bean (Phaseolus vulgaris L.) is one of the important legumes in southern Africa, there is no available information on the genetic diversity and N2-fixing effectiveness of its symbionts in Malkerns, Eswatini. In this study, we assessed the phylogenetic positions of rhizobial microsymbionts of common bean from Malkerns in Eswatini. The isolates obtained showed differences in morpho-physiology and N2-fixing efficiency. A dendrogram constructed from the ERIC-PCR banding patterns, grouped a total of 88 tested isolates into 80 ERIC-PCR types if considered at a 70% similarity cut-off point. Multilocus sequence analysis using 16S rRNA, rpoB, dnaK, gyrB, and glnII and symbiotic (nifH and nodC) gene sequences closely aligned the test isolates to the type strains of Rhizobium muluonense, R. paranaense, R. pusense, R. phaseoli and R. etli. Subjecting the isolates in this study to further description can potentially reveal novel species. Most of the isolates tested were efficient in fixing nitrogen and elicited greater stomatal conductance and photosynthetic rates in the common bean. Relative effectiveness (RE) varied from 18 to 433%, with 75 (85%) out of the 88 tested isolates being more effective than the nitrate fed control plants.

Phenotypic and molecular characterisation of Xanthomonas axonopodis pv. punicae from pomegranate leaves

The present study has sought to provide insight into the blight-inciting phytopathogen Xanthomonas axonopodis pv. punicae, causing a devasting outbreak in pomegranate cultivation. Yellow-coloured circular with glistening featured colonies of five isolates of X. axonopodis pv. punicae have been phenotypically characterized and the identity of the isolates was confirmed by specific gyrB and 16S rRNA gene sequences. Results of BLAST analysis of PBL1 and PBL2 showed 99.64% and 99.76% identity with X. axonopodis pv. punicae strain 1(GenBank: KY354098.1) and strain 15 IARI (GenBank: JN036628.1) respectively. Further phylogenetic analysis revealed the strong association of X. axonopodis pv. punicae isolates with other Xanthomonas pathovars such as X. phaseoli pv. manihotis, X. citri pv. punicae and X. citri pv. mangiferaeindicae. An attempt was made to find the optimal growth media for bacterial proliferation and it was evident that NGA served as an excellent medium to support the multiplication of each isolate with a significant number of colonies (P < 0.05). The medium order per the best CFU/ml was NGA > YDCA > YGCA > NSA. The drug susceptibility test gained a lot of attention since most plant pathogens acquired resistance to antibiotics. Both streptomycin and amoxicillin could act as potentially effective drugs in inhibiting the growth of X. axonopodis pv. punicae (P 0.05). The H2O2 accumulation during the pathogen attack in the infected leaves was located by an endogenous peroxidase-dependent in situ histochemical staining method. Brown precipitate visible at a different colour intensity demonstrated the efficiency of PAMPs to trigger plant immunity to produce H2O2. The present study was the first report on the isolation and characterization of X. axonopodis pv. punicae isolated from blight-infected Pomegranate leaves from the mentioned regions of Tamil Nadu, India.

Identification and genetic characterization of Pseudomonas syringae pv. actinidiae from kiwifruit in Sichuan, China.

Pseudomonas syringae pv. actinidiae (Psa) causes kiwifruit bacterial canker and poses a major threat to the kiwifruit industry. This study aimed to investigate the genetic characteristics of the Psa population from kiwifruit in Sichuan, China. Sixty-seven isolates obtained from diseased plants were characterized using morphological features, multiplex-PCR and multilocus sequence analysis (MLSA). The isolates exhibited the typical colony morphology of Psa. Multiplex PCR amplification identified every isolate as Psa biovar 3. MLSA analysis of the three housekeeping genes gapA, gyrB, and pfk, revealed that the reference strains of the five described biovars were clearly distinguished by a combined phylogenetic tree, and all the tested isolates clustered with the reference strains of Psa biovar 3. Through a phylogenetic tree constructed from a single gene, it was found that pkf gene alone could distinguish biovar 3 from the other biovars. Furthermore, all Psa isolates analyzed by BOX-A1R-based repetitive extragenic palindromic (BOX)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR clustered into four groups. The clustering results of BOX- and ERIC-PCR indicated that group III had the largest number of isolates, accounting for 56.72% and 61.19% of all sixty-seven isolates, respectively, and the two characterization methods were similar and complementary. The results of this study revealed that the genomes of Psa isolates from Sichuan had rich genetic diversity, but no obvious correlation was found between clustering and geographical region. This research provides novel methodologies for rapidly detecting kiwifruit bacterial canker pathogen and a molecular differentiation at genetic level of Psa biovars diversity in China.

A Novel RAA Combined Test Strip Method Based on Dual Gene Targets for Pathogenic Vibrio vulnificus in Aquatic Products.

Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water.

Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

A peptide nucleic acid probe-based multiplex qPCR assay for rapid and accurate detection and quantification of fish-pathogenic Edwardsiella species

Edwardsiellosis causes severe economic losses in the aquaculture industry worldwide. Accurate identification of the fish pathogenic Edwardsiella species is crucial for preventing disease spread and providing effective treatment strategies in the aquaculture industry. These species have high phenotypic and genetic similarities (> 99% in 16S rRNA sequences), which makes their differentiation challenging. In this study, we developed a multiplex real-time PCR diagnostic assay based on the single-copy DNA gyrase subunit B (gyrB) gene using a peptide nucleic acid (PNA) probe to discriminate among four fish pathogenic Edwardsiella species, E. tarda, E. piscicida, E. anguillarum, and E. ictaluri. Through phylogenetic analysis based on gyrB sequences (1462 bp), 57 Korean Edwardsiella isolates were affiliated to E. piscicida, E. anguillarum, and E. tarda, accounting for host and geographical origin specificity. The assay targeted a conserved region in gyrB, and designed four probes, each capable of distinguishing among the four Edwardsiella species, even in cases of 1–3 nucleotide mismatch, based on differences in melting temperature. The assay exhibited high specificity and sensitivity, with reaction efficiency in the range 85–100% and a reliable quantifiable limit of 102 copies per reaction for all fluorescence signals. In addition, it could detect and quantify the four species, even in mixed infections, allowing accurate identification within 2 h in a single reaction. This PNA probe-based multiplex qPCR assay could be a practical and reliable diagnostic tool in the field and provide correct taxonomic assignment for further research on the widened host range and niche, pathogenicity, and susceptibility of each Edwardsiella species, to help establish appropriate treatment and vaccine strategies.

Siderophore-producing Pantoea ferrattrahens sp. nov. isolated from a clinical specimen and Pantoea ferramans sp. nov. isolated from soil at the bottom of a pond.

Two Gram-negative facultative anaerobes were isolated from a sepsis patient with pancreatic cancer (strain PAGU 2156T ) and soil at the bottom of a pond (strain PAGU 2198T ), respectively. These two strains formed haloes around the colonies on chrome azurol S agar plates, indicating the production of siderophores. Two isolates assigned to the genus Pantoea based on the 16S rRNA gene were differentiated from established species by using polymorphic taxonomies. Phylogenetic analysis using four housekeeping genes (gyrB, rpoB, atpD, and infB) showed that strain PAGU 2156T is closely related to Pantoea cypripedii LMG 2657T (89.9%) or Pantoea septica LMG 5345T (95.7%). Meanwhile, strain PAGU 2198T formed a single clade with Pantoea rodasii DSM 26611T (93.6%) and Pantoea rwandensis DSM 105076T (93.3%). The average nucleotide identity values obtained from the draft genome assembly showed ≤90.2% between strain PAGU 2156T and closely related species and ≤81.5% between strain PAGU 2198T and closely related species. Based on various phenotypes, biochemical properties, and whole-cell fatty acid composition compared with related species, it was concluded that each strain should be classified as a new species of the genus Pantoea. In this manuscript, Pantoea ferrattrahens sp. nov. and Pantoea ferramans sp. nov. with strain PAGU 2156T (=NBRC 115930T = CCUG 76757T ) and strain PAGU 2198T (=NBRC 114265T = CCUG 75151T ) are proposed as each type strain.

Molecular characterization and differential effects of levofloxacin and ciprofloxacin on the potential for developing quinolone resistance among clinical Pseudomonas aeruginosa isolates.

Fluoroquinolones are some of the most used antimicrobial agents for the treatment of Pseudomonas aeruginosa. This study aimed at exploring the differential activity of ciprofloxacin and levofloxacin on the selection of resistance among P. aeruginosa isolates at our medical center. 233 P. aeruginosa clinical isolates were included in this study. Antimicrobial susceptibility testing (AST) was done using disk diffusion and broth microdilution assays. Random Amplification of Polymorphic DNA (RAPD) was done to determine the genetic relatedness between the isolates. Induction of resistance against ciprofloxacin and levofloxacin was done on 19 isolates. Fitness cost assay was done on the 38 induced mutants and their parental isolates. Finally, whole genome sequencing was done on 16 induced mutants and their 8 parental isolates. AST results showed that aztreonam had the highest non-susceptibility. RAPD results identified 18 clusters. The 19 P. aeruginosa isolates that were induced against ciprofloxacin and levofloxacin yielded MICs ranging between 16 and 256 μg/mL. Levofloxacin required fewer passages in 10 isolates and the same number of passages in 9 isolates as compared to ciprofloxacin to reach their breakpoints. Fitness cost results showed that 12 and 10 induced mutants against ciprofloxacin and levofloxacin, respectively, had higher fitness cost when compared to their parental isolates. Whole genome sequencing results showed that resistance to ciprofloxacin and levofloxacin in sequenced mutants were mainly associated with alterations in gyrA, gyrB and parC genes. Understanding resistance patterns and risk factors associated with infections is crucial to decrease the emerging threat of antimicrobial resistance.

The outstanding diversity of rhizobia microsymbionts of common bean (Phaseolus vulgaris L.) in Mato Grosso do Sul, central-western Brazil, revealing new Rhizobium species.

Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).

First report of bacterial panicle blight (BPB) in Rice caused by Burkholderia gladioli in Bangladesh.

Bacterial panicle blight (BPB) has become one of the most destructive diseases of rice worldwide and Burkholderia gladioli and B. glumae are two major pathogens causing BPB (1). This disease causes several types of damage, most importantly grain spotting, rot, and panicle blight, which can result in yield losses of 75% or more (1,3). In recent years, symptoms including sheath rot, grain spotting, grain rot, and panicle blight have been observed in both inbred and hybrid rice varieties. These symptoms resemble those of BPB and cause cultivar-dependent yield losses. (3) also reported the same symptoms for BPB. To confirm the cause of the disease, 21 rice panicles (Haridhan, a local variety) with typical BPB symptoms were collected from a farmer's field in the region of Mymensingh, Bangladesh during the rainy season in mid-October, 2021. Due to the severity of the outbreak, the panicles became dark brown and produced chaffy grains; nearly 100% of the rice panicles in that field were severely infected. To identify the causal pathogen(s), 1g of rice grains from 20 plants with typical BPB symptoms were surface-sterilized by immersing them in 70% ethanol for a few seconds followed by sodium hypochlorite solution (3%) for 1min. The grains were then rinsed with sterilized distilled water three times. Surface-sterilized grains were then ground with a mortar and pestle; 5mL of sterile distilled water was added during grinding. The extracted suspension (20µL) was then either streaked or spread onto the selective medium (S-PG) (2). Bacterial colonies showing purple color on the S-PG medium were selected and purified as candidate pathogens. For molecular characterization, species specific primers targeting gyrB gene were used to perform PCR and resulted in 479bp as reported by (4). To verify further, the PCR products of 16SF & 16SR were amplified and sequenced partially producing around 1400bp (1) and five 16SF partial sequences were deposited into NCBI GenBank (OP108276 to OP108280). 16S rDNA and gyrB revealed almost 99% homology with Burkholderia gladioli (KU851248.1, MZ425424.1) and B. gladioli (AB220893, CP033430) respectively using BLAST analysis. These purified bacterial isolates produced a diffusible light-yellow pigment on King's B medium indicating toxoflavin production (3). The candidate five bacterial isolates were then confirmed by inoculating 10ml suspension 108CFU/mL into the panicles and sheaths of BRRIdhan28 in net house condition as described previously (1). All of the bacterial isolates obtained from the spotted rice grains produced light brown lesions on the inoculated leaf sheath as well as spotting on the grain. To fulfill Koch's postulates, the bacteria were re-isolated from the symptomatic panicles and were confirmed as B. gladioli by analyzing the sequences of gyrB and 16s rDNA genes. Taken together, these results confirmed that B. gladioli is responsible for causing BPB in the rice grain samples that we collected. To our knowledge, this is the first report of BPB caused by B. gladioli in Bangladesh and further research is necessary to develop an effective disease management technique, or else the production of rice will be severely hampered.

First Isolation and Identification of Aeromonas veronii in a Captive Giant Panda (Ailuropoda melanoleuca).

The objective of this study was to understand biological characteristics of one bacteria strain named as VPG which was isolated from multiple organs of a dead captive giant panda cub. Here, we use biochemical tests, 16S rRNA and gyrB genes for bacterial identification, the disk diffusion method for antibiotic resistance phenotype, smart chip real-time PCR for the antibiotic resistance genotype, multiplex PCR for determination of virulence genes, and the acute toxicity test in mice for testing the pathogenicity of isolates. The isolate was identified as A. veronii strain based on the biochemical properties and genetic analysis. We found that the strain carried 31 antibiotic resistance genes, revealed antimicrobial resistance phenotypically to several antibiotics including penicillin, ampicillin, oxacillin, amoxicillin, imipenem, and vancomycin, and carried virulence genes including aer, act, lip, exu, ser, luxs, and tapA. The main pathological changes in giant panda were congestion, necrotic lesions and a large number of bacteria in multiple organs. In addition, the LD50 in Kunming mice infected with strain VGP was 5.14 × 107 CFU/mL by intraperitoneal injection. Infection with strain VGP led to considerable histological lesions such as hemorrhage of internal organs, necrosis of lymphocytes and neurons in Kunming mice. Taken together, these results suggest that infection with strain VGP would be an important causes of death in this giant panda cub.

Molecular identification of Pseudomonas aeruginosa and antibiotic resistance testing

Pseudomonas aeruginosa is a Gram-negative bacillus considered an opportunistic pathogen, usually associated with nosocomial infections in immunocompromised patients. Among the infections caused, the most frequent are urinary tract infections, wound infections, pneumonia, and bacteremia. It has intrinsic and acquired resistance to antibiotics, due to the low permeability of its outer membrane, the overexpression of efflux pumps, and the production of enzymes that degrade the antibiotic. In this study, 61 samples of P. aeruginosa collected from the General Hospital “Dr. Aquiles Calles Ramírez" from the city of Tepic, which were phenotypically identified using the Vitek2 automated system. Subsequently, they were molecularly identified with the polymerase chain reaction technique, targeting the gyrB gene that codes for the B subunit of DNA gyrase. In 60 of the samples, amplification of the gyrB gene was obtained and only one, no amplification was observed. The results showed that infections caused by P. aeruginosa are more frequent in male patients, in age range of 41-60 years. Urine culture being the origin of the sample that most predominated. The sensitivity-resistance analysis to antibiotics showed that most of the strains are resistant to β-lactams, except for meropenem, which presented a high sensitivity in most of the strains, as well as in the case of ciprofloxacin, amikacin, and gentamicin.

Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa.

The current study determines the density of Vibrio spp. and isolates V. cholerae and Vibrio mimicus from fish-anatomical-sites, prawn, crab and mussel samples recovered from fish markets, freshwater and brackish water. Virulence and antibiotic resistance profiling of isolates were carried out using standard molecular and microbiology techniques. Vibrio spp. was detected in more than 90% of samples [134/144] and its density was significantly more in fish than in other samples. Vibrio. cholerae and V. mimicus were isolated in at least one sample of each sample type with higher isolation frequency in fish samples. All the V. cholerae isolates belong to non-O1/non-O139 serogroup. One or more V. cholerae isolates exhibited intermediate or resistance against each of the eighteen panels of antibiotics used but 100% of the V. mimicus were susceptible to amikacin, gentamycin and chloramphenicol. Vibrio cholerae exhibited relatively high resistance against polymyxin, ampicillin and amoxicillin/clavulanate while V. mimicus isolates exhibited relatively high resistance against nitrofurantoin, ampicillin and polymixin. The multiple-antibiotic-resistance-index [MARI] for isolates ranges between 0 and 0.67 and 48% of the isolates have MARI that is >0.2 while 55% of the isolates exhibit MultiDrug Resistance Phenotypes. The percentage detection of acc, ant, drf18, sul1, mcr-1, blasvh, blaoxa, blatem, blaoxa48, gyrA, gyrB and parC resistance-associated genes were 2%, 9%, 14%, 7%, 2%, 25%, 7%, 2%, 2%, 32%, 25% and 27% respectively while that for virulence-associated genes in increasing other was ace [2%], tcp [11%], vpi [16%], ompU [34%], toxR [43%], rtxC [70%], rtxA [73%] and hyla [77%]. The study confirmed the potential of environmental non-O1/non-O139 V. cholerae and V. mimicus to cause cholera-like infection and other vibriosis which could be difficult to manage with commonly recommended antibiotics. Thus, regular monitoring of the environment to create necessary awareness for this kind of pathogens is important in the interest of public health.

Acinetobacter entericus sp. nov., isolated from the gut of plastic-eating insect larvae Zophobas atratus.

A Gram-negative, non-motile and rod-shaped strain, BIT-DXN8T, was isolated from the gut of plastic-eating insect larvae Zophobas atratus. The taxonomic position of this new isolate was examined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1411 bp) indicated that the most similar strain to BIT-DXN8T was Acinetobacter bouvetii DSM 14964T (98.5%), followed by Acinetobacter haemolyticus CIP 64.3T (98.2%) and Acinetobacter pullicarnis S23T (98.2%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (fusA, gyrB, recA, rplB and rpoB) and genome sequences, placed strain BIT-DXN8T in a separate lineage among the genus Acinetobacter of the family Moraxellaceae. The average nucleotide identity and digital DNA-DNA hybridization values of the strain when compared to all other species within the genus Acinetobacter were below 96 and 70 %, respectively. The physiological and biochemical tests confirm the affiliation of strain BIT-DXN8T to the present species within the genus Acinetobacter, but with some specific phenotypic differences. Therefore, strain BIT-DXN8T is considered to represent a novel species, for which the name Acinetobacter entericus sp. nov. is proposed. The type strain is BIT-DXN8T (=CCTCC AB 2022117T=KCTC 92696T).

The Mass Spectrometry Identification, Antimicrobial Genes Detection, and Proteomics Analysis of Stutzerimonas stutzeri Strain Was Isolated from Industrial Wastewater

A large amount of organic matter, heavy metals, and even antibiotics are present in industrial wastewater, aquaculture waters, and various types of sewage, along with abundant microorganisms. To date, only a few studies involving the resistance and proteomics of Stutzerimonas stutzeri in high-salt wastewater are available. Herein, a comprehensive assessment of a newly isolated Stutzerimonas stutzeri strain, which is present in high-salt wastewater, was performed using mass spectrometry, genetic identification, and biochemical analysis to characterize the genetic and biochemical properties. Growth experiments revealed that the Stutzerimonas stutzeri strain had a moderate growth rate in nutrient broth, and the bacterial count was not high. Further analysis highlighted an apparent susceptibility of this strain to most antibiotics but some resistance to chloramphenicol and minocycline. A resistance gene assay results showed that the gene gyrB was associated with antibiotic resistance in this Stutzerimonas stutzeri strain. Proteomic analysis revealed for the first time the co-existence of two drug-resistance-related proteins (Multidrug/solvent RND membrane fusion protein and MexE) in Stutzerimonas stutzeri. Moreover, Stutzerimonas stutzeri isolated from high-salt wastewater was subjected to drug resistance gene detection, and the total protein of Stutzerimonas stutzeri was detected by protein mass spectrometry analysis. The subcellular classification shows that the 50 proteins with the highest abundance are divided into cell inner membrane, cell outer membrane, cytoplasm, cytoplasmic side, membrane, multi-pass membrane protein, and peripheral membrane protein, among which the proportion of cytoplasmic components is the highest. Overall, this study’s findings provide a new perspective for further research on the characteristics of Stutzerimonas stutzeri in high-salt wastewater.

Multidrug-Resistant Salmonella Species and Their Mobile Genetic Elements from Poultry Farm Environments in Malaysia.

The prevalence and persistent outbreaks of multidrug-resistant (MDR) Salmonella in low-income countries have received growing attention among the public and scientific community. Notably, the excessive use of antibiotics in chicken feed for the purpose of treatment or as prophylaxis in the poultry industry have led to a rising rate of antimicrobial resistance. Therefore, this study aimed to determine the presence of antimicrobial-resistant Salmonella species and its mobile genetic elements from soil and effluent samples of 33 randomly selected poultry farms in Selangor, Malaysia. Salmonella species were isolated on selective media (CHROMagar™ Salmonella). VITEK® 2 system was used to identify the isolates and their antimicrobial susceptibility. Subsequently, eight isolates were subjected to the whole genome sequencing (WGS). Based on the results, Salmonella spp. was detected in 38.1% (24/63) of samples, with the highest resistance to ampicillin (62.5%), followed by ampicillin/sulbactam (50.0%) and ciprofloxacin (45.8%). Meanwhile, the identified serovars were Salmonella enterica subspecies enterica serovar Weltevreden (S. Weltevreden), S. Jedburgh, and S. Brancaster. The most prevalent resistance genes detected include qnrS1, blaTEM-176, dfrA14, and tet(A). The IncX1 plasmid, with encoded resistance genes, was also detected in four isolates. Furthermore, mutations in the quinolone resistant-determining regions (QRDR) were discovered, specifically in the gyrA, gyrB, and parC genes. In short, surveillance such as continuous monitoring of antimicrobial resistance and emerging trends in resistance patterns through farm environmental samples could provide information to formulate public health interventions for effective infection prevention and disease control.

Bradyrhizobium xenonodulans sp. nov. isolated from nodules of Australian Acacia species invasive to South Africa

A genealogical concordance approach was used to delineate strains isolated from Acacia dealbata and Acacia mearnsii root nodules in South Africa. These isolates form part of Bradyrhizobium based on 16S rRNA sequence similarity. Phylogenetic analysis of six housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) confirmed that these isolates represent a novel species, while pairwise average nucleotide identity (ANIb) calculations with the closest type strains (B. cosmicum 58S1T, B. betae PL7HG1T, B. ganzhouense CCBAU 51670 T, B. cytisi CTAW11T and B. rifense CTAW71T) resulted in values well below 95–96%. We further performed phenotypic tests which revealed that there are high levels of intraspecies variation, while an additional analysis of the nodA and nifD loci indicated that the symbiotic loci of the strains are closely related to those of Bradyrhizobium isolates with an Australian origin. Strain 14ABT (=LMG 31415 T = SARCC-753 T) is designated as the type strain of the novel species for which we propose the name Bradyrhizobium xenonodulans sp. nov.