Novel insights into the co-selection of metal-driven antibiotic resistance in bacteria: a study of arsenic and antibiotic co-exposure.

The simultaneous development of antibiotic resistance in bacteria due to metal exposure poses a significant threat to the environment and human health. This study explored how exposure to both arsenic and antibiotics affects the ability of an arsenite oxidizer, Achromobacter xylosoxidans CAW4, to transform arsenite and its antibiotic resistance patterns. The bacterium was isolated from arsenic-contaminated groundwater in the Chandpur district of Bangladesh. We determined the minimum inhibitory concentration (MIC) of arsenite, cefotaxime, and tetracycline for A. xylosoxidans CAW4, demonstrating a multidrug resistance (MDR) trait. Following this determination, we aimed to mimic an environment where A. xylosoxidans CAW4 was exposed to both arsenite and antibiotics. We enabled the strain to grow in sub-MIC concentrations of 1 mM arsenite, 40 µg/mL cefotaxime, and 20 µg/mL tetracycline. The expression dynamics of the arsenite oxidase (aioA) gene in the presence or absence of antibiotics were analyzed. The findings indicated that simultaneous exposure to arsenite and antibiotics adversely affected the bacteria's capacity to metabolize arsenic. However, when arsenite was present in antibiotics-containing media, it promoted bacterial growth. The study observed a global downregulation of the aioA gene in arsenic-antibiotic conditions, indicating the possibility of increased susceptibility through co-resistance across the entire bacterial population of the environment. This study interprets that bacterial arsenic-metabolizing ability can rescue the bacteria from antibiotic stress, further disseminating environmental cross-resistance. Therefore, the co-selection of metal-driven antibiotic resistance in bacteria highlights the need for effective measures to address this emerging threat to human health and the environment.

Metagenomic and culture-dependent approaches unveil active microbial community and novel functional genes involved in arsenic mobilization and detoxification in groundwater.

BACKGROUND: Arsenic (As) and its species are major pollutants in ecological bodied including groundwater in Bangladesh rendering serious public health concern. Bacteria with arsenotrophic genes have been found in the aquifer, converting toxic arsenite [As (III)] to less toxic arsenate [As (V)] that is easily removed using chemical and biological trappers. In this study, genomic and metagenomic approaches parallel to culture-based assay (Graphical abstract) have made it possible to decipher phylogenetic diversity of groundwater arsenotrophic microbiomes along with elucidation of their genetic determinants. RESULTS: Seventy-two isolates were retrieved from six As-contaminated (average As concentration of 0.23 mg/L) groundwater samples from Munshiganj and Chandpur districts of Bangladesh. Twenty-three isolates harbored arsenite efflux pump (arsB) gene with high abundance, and ten isolates possessing arsenite oxidase (aioA) gene, with a wide range of minimum inhibitory concentration, MICAs (2 to 32 mM), confirming their role in arsenite metabolism. There was considerable heterogeneity in species richness and microbial community structure. Microbial taxa from Proteobacteria, Firmicutes and Acidobacteria dominated these diversities. Through these combinatorial approaches, we have identified potential candidates such as, Pseudomonas, Acinetobacter, Stenotrophomonas, Achromobacter, Paraburkholderia, Comamonas and Klebsiella and associated functional genes (arsB, acr3, arsD, arsH, arsR) that could significantly contribute to arsenite detoxification, accumulation, and immobilization. CONCLUSIONS: Culture-dependent and -independent shotgun metagenomic investigation elucidated arsenotrophic microbiomes and their functions in As biogeochemical transformation. These findings laid a foundation for further large-scale researches on the arsenotrophic microbiomes and their concurrent functions in As biogeochemical transformation in As-contaminated areas of Bangladesh and beyond.

Emergence of a novel sublineage, MYMBD21 under SA-2018 lineage of Foot-and-Mouth Disease Virus serotype O in Bangladesh.

Foot-and-Mouth Disease (FMD) hinders the growth of the livestock industry in endemic countries like Bangladesh. The management and prevention of FMD are severely impacted by the high mutation rate and subsequent frequent generation of newer genotypes of the causative agent, Foot-and-Mouth Disease Virus (FMDV). The current study was conducted in nine districts of Bangladesh during 2019-21 to characterize the circulating FMDV strains based on the VP1 sequence analysis, the major antigenic recognition site providing serotype specificity and high variability of FMDV. This study detected the first emergence of the SA-2018 lineage in Bangladesh along with the predominance of Ind-2001e (or Ind-2001BD1) sublineage of ME-SA topotype under serotype O during 2019-21. The mutational spectrum, evolutionary divergence analysis and multidimensional plotting confirmed the isolates collected from Mymensingh districts, designated as MYMBD21 as a novel sublineage under the SA-2018 lineage. Analysis of the amino acid sequence revealed several changes in the G-H loop, B-C loop and C-terminal region of VP1, revealing a 12-13% divergence from the existing vaccine strains and a 95% VP1 protein homology, with most of the mutations potentially considerable as vaccine escape mutations, evidenced by three-dimensional structural analysis. This is the first report on the emergence of the SA-2018 lineage of ME-SA topotype of FMDV serotype O in Bangladesh, as well as a possible mutational trend towards the emergence of a distinct sublineage under SA-2018 lineage, which calls for in-depth genome-wide analysis and monitoring of the FMD situation in the country to implement a strategic vaccination and effective FMD control program.

Genomic variability correlates with biofilm phenotypes in multidrug resistant clinical isolates of Pseudomonas aeruginosa.

The multifactorial nature of Pseudomonas aeruginosa biofilm development and genomic variabilities implicates its resistance to conventional antimicrobials and virulence. Therefore, genetic determinants need to be extensively studied to block the early steps of biofilm or already formed biofilms. In this study, a total of 20 multidrug resistant (MDR) clinical P. aeruginosa isolates were evaluated for their biofilm forming abilities and related genes. Of the isolates tested, all of them showed surface attachment tendencies in nutrient limiting conditions, and classified as strong (SBF = 45%), moderate (MBF = 30%) and weak (WBF = 25%) biofilm formers. Complete genome sequencing of representative strong (DMC-27b), moderate (DMC-20c) and weak biofilm former (DMC-30b) isolates was performed. Analysis of biofilm related genes in the sequenced genomes revealed that, 80 of the 88 biofilm related genes possess 98-100% sequence identity to the reference PAO1 strain. Complete and partial sequence data of LecB proteins from tested isolates indicate that isolates containing PA14-like LecB sequences produced strong biofilms. All of the 7 pel operon protein coding genes in weak biofilm former isolate 30b showed significant nucleotide sequence variation with other tested isolates, and their corresponding proteins are 99% identical with the pel operon proteins of PA7. Bioinformatics analyses identified divergent sequence and structural features that separate PA7 like pel operon proteins from reference PAO1-like pel operon. Congo red and pellicle forming assays revealed that the sequence and structure variations may have interfered with the Pel production pathway and resulted in impaired Pel production in isolate 30b that has a PA7 like pel operon. Expression analysis also showed that both pelB and lecB genes were about 5 to 6 folds upregulated after 24 h in SBF 27b in comparison with WBF 30b. Our findings indicate significant genomic divergence in biofilm related genes of P. aeruginosa strains that affect their biofilm phenotypes.

Characterization of multidrug and heavy metal resistance of carbapenemases producing Klebsiella pneumoniae from poultry samples in Bangladesh.

BACKGROUND: Resistance to multiple drugs in Klebsiella pneumoniae (KPN) is an important issue in human and animal medicine. KPN phenotypic and genotypic aspects in poultry samples have not been comprehensively explored in Bangladesh. METHODS: This research focused on the prevalence of antibiotic resistance and the characterization of KPN in Bangladeshi poultry isolates using both phenotypic and genotypic approaches. RESULTS: A total of 32 poultry samples were randomly obtained from a commercial poultry farm in Narsingdi, Bangladesh, and 43.90% (18/41) of the isolates were confirmed to be KPN, whereas all isolates were biofilm producers. The antibiotic sensitivity test revealed the most remarkable (100%) antibiotic resistance level against Ampicillin, Doxycycline and Tetracycline while remaining susceptible to Doripenem, Meropenem, Cefoxitin and Polymyxin B. Resistance to Nalidixic acid, Nitrofurantoin, Trimethoprim, Levofloxacin, Ciprofloxacin, Cefuroxime and Chloramphenicol ranges from 18% to 70%. Minimum inhibitory concentrations for carbapenem-resistant KPN ranged from 128 to 512 mg/mL for Meropenem, Imipenem, Gentamycin and Ciprofloxacin, respectively. [Correction added on 15 June 2023, after first online publication: 512 g/mL was corrected to 512 mg/mL in the preceding sentence]. Carbapenemase-producing KPN isolates harboured a single or multiple ß-lactamase genes, blaSIM-1 , blaIMP-4 and blaOXA-48 as well as one ESBL gene (blaTEM ) and plasmid-mediated quinolone resistance gene (qnrB). Furthermore, Cr and Co outperformed Cu and Zn in antibacterial effectiveness. CONCLUSIONS: The results of this investigation showed that the high prevalence of multidrug-resistant pathogenic KPN in our chosen geographic location had displayed sensitivity to FOX/PB/Cr/Co, which might be regarded as an alternate treatment to reduce pressure of use on carbapenems.

Genomic diversity and molecular epidemiology of a multidrug-resistant Pseudomonas aeruginosa DMC30b isolated from a hospitalized burn patient in Bangladesh.

OBJECTIVES: Pseudomonas aeruginosa is a key opportunistic pathogen causing a wide range of community- and hospital-acquired infections in immunocompromised or catheterized patients. Here, we report the complete genome sequence of a multidrug-resistant (MDR) P. aeruginosa DMC30b to elucidate the genetic diversity, molecular epidemiology, and underlying mechanisms for antimicrobial resistance and virulence. METHODS: P. aeruginosa DMC30b was isolated from septic wound swab of a severe burn patient. Whole-genome sequencing was performed under Ion Torrent platform. The genome was assembled using the SPAdes v. 3.12.01 in an integrated Genome Analysis Platform for Ion Torrent sequence data. The genome was annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline. In-silico predictions of antimicrobial resistance genes, virulence factor genes, and metabolic functional potentials were performed using different curated bioinformatics tools. RESULTS: P. aeruginosa DMC30b was found as a MDR strain and belonged to sequence type 244 (ST244). The complete genome size is 6 994 756 bp with a coverage of 76.76x, guanine-cytosine content of 65.7% and a Benchmarking Universal Single-Copy Orthologs score of 100. The genome of P. aeruginosa DMC30b harboured two predicted plasmid replicons (e,g. IncP-6; 78 007 bp and ColRNAI; 9359 bp), 35 resistomes (antimicrobial resistance genes) conferring resistance to 18 different antibiotics (including four beta-lactam classes), and 214 virulence factor genes. It was identified as the 167th ST244 strain among ∼ 5800 whole-genome sequences of P. aeruginosa available in the NCBI database. CONCLUSION: The MDR P. aeruginosa DMC30b was identified as the 167th ST244 complete genome to be submitted to the NCBI, and the first ST244 isolate sequenced from Bangladesh. The complete genome data with high genetic diversity and underlying mechanisms for antimicrobial resistance and virulence of P. aeruginosa DMC30b will aid in understanding the evolution and phylogeny of such high-risk clones and provide a solid basis for further research on MDR or extensively drug resistant strains.

Antibiogram and virulence profiling reveals multidrug resistant Staphylococcus aureus as the predominant aetiology of subclinical mastitis in riverine buffaloes.

BACKGROUND: Staphylococcus spp. are the major causal agents of mastitis in dairy animals worldwide leading to profound economic losses and public health threats. Recently, Staphylococcus aureus has emerged as a multidrug resistant and zoonotic pathogen. This study aimed to characterize S. aureus in subclinical mastitis (SCM) milk samples of riverine buffaloes in Bangladesh through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. METHOD: We characterized S. aureus in SCM milk samples (N = 500) of riverine buffaloes through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. RESULTS: Out of 500 milk samples tested, 188 (37.6%) were found positive for SCM. From 188 SCM samples, 291 isolates were obtained with a prevalence of S. aureus in 37.4% (109/291) isolates. Phylogenetic analysis revealed the evolutionary divergence of S. aureus isolates in bubaline SCM milk samples. The antibiogram profiling showed that about 96.0% S. aureus isolates were multidrug resistant (MDR). Notably, 29 and 16 isolates harboured methicillin-resistant (mecA) and panton-valentine leucocidin (pvl) genes, respectively, and 46 plasmid-bearing isolates were MDR. Nine Staphylococcal enterotoxins (SEs/SEls) including sea (11.9%), sec (7.4%), sed (4.6%), seg (3.7%), and seh (3.7%) were detected with 72.48% toxinotypes comprising a single gene. CONCLUSION: This study therefore suggests S. aureus as the single-most aetiology (∼37.0%) of SCM in riverine buffaloes, and emergence of MDR, enterotoxin producing, and virulent S. aureus strains could impose potential threats to animal welfare and public health.

Induction of mastitis by cow-to-mouse fecal and milk microbiota transplantation causes microbiome dysbiosis and genomic functional perturbation in mice.

BACKGROUND: Mastitis pathogenesis involves a wide range of opportunistic and apparently resident microorganims including bacteria, viruses and archaea. In dairy animals, microbes reside in the host, interact with environment and evade the host immune system, providing a potential for host-tropism to favor mastitis pathogenesis. To understand the host-tropism phenomena of bovine-tropic mastitis microbiomes, we developed a cow-to-mouse mastitis model. METHODS: A cow-to-mouse mastitis model was established by fecal microbiota transplantation (FMT) and milk microbiota transplantation (MMT) to pregnant mice to assess microbiome dysbiosis and genomic functional perturbations through shotgun whole metagenome sequencing (WMS) along with histopathological changes in mice mammary gland and colon tissues. RESULTS: The cow-to-mouse FMT and MMT from clinical mastitis (CM) cows induced mastitis syndromes in mice as evidenced by histopathological changes in mammary gland and colon tissues. The WMS of 24 samples including six milk (CM = 3, healthy; H = 3), six fecal (CM = 4, H = 2) samples from cows, and six fecal (CM = 4, H = 2) and six mammary tissue (CM = 3, H = 3) samples from mice generating 517.14 million reads (average: 21.55 million reads/sample) mapped to 2191 bacterial, 94 viral and 54 archaeal genomes. The Kruskal-Wallis test revealed significant differences (p = 0.009) in diversity, composition, and relative abundances in microbiomes between CM- and H-metagenomes. These differences in microbiome composition were mostly represented by Pseudomonas aeruginosa, Lactobacillus crispatus, Klebsiella oxytoca, Enterococcus faecalis, Pantoea dispersa in CM-cows (feces and milk), and Muribaculum spp., Duncaniella spp., Muribaculum intestinale, Bifidobacterium animalis, Escherichia coli, Staphylococcus aureus, Massilia oculi, Ralstonia pickettii in CM-mice (feces and mammary tissues). Different species of Clostridia, Bacteroida, Actinobacteria, Flavobacteriia and Betaproteobacteria had a strong co-occurrence and positive correlation as the indicator species of murine mastitis. However, both CM cows and mice shared few mastitis-associated microbial taxa (1.14%) and functional pathways regardless of conservation of mastitis syndromes, indicating the higher discrepancy in mastitis-associated microbiomes among lactating mammals. CONCLUSIONS: We successfully induced mastitis by FMT and MMT that resulted in microbiome dysbiosis and genomic functional perturbations in mice. This study induced mastitis in a mouse model through FMT and MMT, which might be useful for further studies- focused on pathogen(s) involved in mastitis, their cross-talk among themselves and the host.

Circulating Phylotypes of White Spot Syndrome Virus in Bangladesh and Their Virulence.

White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the south-east (Cox's Bazar) and south-west (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal-Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox's Bazar and Satkhira) and over the study periods (2017-2019). The mean Log load of WSSV varied from 8.40 (Cox's Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71 ± 0.76 ppt, 3.7 ± 0.1 ppm, 34.11 ± 0.38 °C and 8.23 ± 0.38, respectively, in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at the 167th position in the isolates from Cox's Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 × 109 copies of WSSV per mL resulted in a median LT50 value of 73 h and 75 h, respectively. The in vivo trial showed higher mean Log WSSV copies (6.47 ± 2.07 per mg tissue) in BD1-challenged shrimp PL compared to BD2 (4.75 ± 0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 h and 62 h of challenge, respectively, with mean Log WSSV copies of 12.06 ± 0.48 and 9.95 ± 0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs), penaeidin and lysozyme expression were lower in the BD1-challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.

First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera.

BACKGROUND AND AIM: Fowl cholera (FC) caused by Pasteurella multocida is a highly contagious bacterial disease of global importance for poultry production. The severity and incidence of FC caused by P. multocida may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment, and the bacterial strain. This study aimed to investigate the genetic diversity of multidrug-resistant P. multocida strains isolated from FC outbreaks in laying hens from commercial farms of Bangladesh. MATERIALS AND METHODS: We collected 57 samples of suspected FC, including 36 live and 21 dead laying hens. P. multocida isolates were characterized by biochemical and molecular-biological methods. RESULTS: Twenty-two strains of P. multocida were isolated from these samples through phenotypic and genotypic characterization. The strains were grouped into two distinct random amplification of polymorphic DNA (RAPD) biotypes harboring a range of pathogenic genes; exbB, ompH, ptfA, nanB, sodC, and hgbA. In this study, 90.90% and 81.82% P. multocida strains were multidrug-resistant and biofilm formers, respectively. Whole-genome sequencing of the two representative RAPD phylotypes confirmed as P. multocida type B: L2:ST122, harboring a number of virulence factors-associated genes (VFGs), and antimicrobial resistance (AMR) genes (ARGs). In addition, pan-genome analysis revealed 90 unique genes in the genomes of P. multocida predicted to be associated with versatile metabolic functions, pathogenicity, virulence, and AMR. CONCLUSION: This is first-ever report on the association of P. multocida genotype B: L2:ST122 and related VFGs and ARGs in the pathogenesis of FC in laying hens. This study also provides a genetic context for future researches on the evolutionary diversity of P. multocida strains and their host adaptation.

Draft genome sequence of a carbapenem-resistant clinical Acinetobacter baumannii revealing co-existence of four classes of ß-lactamases.

OBJECTIVES: Nosocomial carbapenem-resistant Acinetobacter baumannii is a challenge in the treatment of intensive care unit (ICU) patients. The presence of antimicrobial resistance genes (ARGs) and mobile genetic elements can further complicate effects to combat antibiotic resistance in this post-antibiotic era. The aim of this study was to analyse the molecular basis of carbapenem resistance in A. baumannii strain DMC-32a. METHODS: Strain DMC-32a, isolated from a wound swab of an ICU patient, was screened phenotypically and genotypically for carbapenem resistance. The isolate was subjected whole-genome sequencing (WGS) to investigate the resistance mechanisms. RESULTS: Strain DMC-32a was resistant to all tested antibiotics belonging to seven classes, except for the polymyxins. MICs determined against imipenem and meropenem were 512 mg/L and >512 mg/L, respectively. Gene-specific PCR confirmed the presence of blaNDM-1 and intI1. WGS confirmed the isolate as sequence type ST1053, the presence of four classes of ß-lactamases [A (blaPER-7, blaTEM-116), B (blaNDM-1), C (blaADC-26) and D (blaOXA-23, blaOXA-51)], and four genes encoding aminoglycoside-modifying enzymes [aph(6)-Id, aph(3'')-Ib, aph(3')-VI and ant(3'')-IIc] conferring resistance to streptomycin, amikacin, kanamycin and spectinomycin. The analysis also confirmed the presence of a class 1 integron gene cassette harbouring ARGs to rifamycin (arr-2) and phenicols (cmlA5). No plasmid replicon was found from the sequence data. CONCLUSION: The co-existence of four ß-lactamase classes in A. baumannii DMC-32a has not been reported previously from Bangladesh and not in this ST elsewhere. The emergence of such a nosocomial pathogen creates the need for effective therapeutics for critically-ill patients and for controlling hospital-acquired infections.

Microbiome signature and diversity regulates the level of energy production under anaerobic condition.

The microbiome of the anaerobic digester (AD) regulates the level of energy production. To assess the microbiome diversity and composition in different stages of anaerobic digestion, we collected 16 samples from the AD of cow dung (CD) origin. The samples were categorized into four groups (Group-I, Group-II, Group-III and Group-IV) based on the level of energy production (CH4%), and sequenced through whole metagenome sequencing (WMS). Group-I (n = 2) belonged to initial time of energy production whereas Group-II (n = 5), Group-III (n = 5), and Group-IV (n = 4) had 21-34%, 47-58% and 71-74% of CH4, respectively. The physicochemical analysis revealed that level of energy production (CH4%) had significant positive correlation with digester pH (r = 0.92, p < 0.001), O2 level (%) (r = 0.54, p < 0.05), and environmental temperature (°C) (r = 0.57, p < 0.05). The WMS data mapped to 2800 distinct bacterial, archaeal and viral genomes through PathoScope (PS) and MG-RAST (MR) analyses. We detected 768, 1421, 1819 and 1774 bacterial strains in Group-I, Group-II, Group-III and Group-IV, respectively through PS analysis which were represented by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes and Fibrobacteres phyla (> 93.0% of the total abundances). Simultaneously, 343 archaeal strains were detected, of which 95.90% strains shared across four metagenomes. We identified 43 dominant species including 31 bacterial and 12 archaeal species in AD microbiomes, of which only archaea showed positive correlation with digester pH, CH4 concentration, pressure and temperature (Spearman correlation; r > 0.6, p < 0.01). The indicator species analysis showed that the species Methanosarcina vacuolate, Dehalococcoides mccartyi, Methanosarcina sp. Kolksee and Methanosarcina barkeri were highly specific for energy production. The correlation network analysis showed that different strains of Euryarcheota and Firmicutes phyla exhibited significant correlation (p = 0.021, Kruskal-Wallis test; with a cutoff of 1.0) with the highest level (74.1%) of energy production (Group-IV). In addition, top CH4 producing microbiomes showed increased genomic functional activities related to one carbon and biotin metabolism, oxidative stress, proteolytic pathways, membrane-type-1-matrix-metalloproteinase (MT1-MMP) pericellular network, acetyl-CoA production, motility and chemotaxis. Importantly, the physicochemical properties of the AD including pH, CH4 concentration (%), pressure, temperature and environmental temperature were found to be positively correlated with these genomic functional potentials and distribution of ARGs and metal resistance pathways (Spearman correlation; r > 0.5, p < 0.01). This study reveals distinct changes in composition and diversity of the AD microbiomes including different indicator species, and their genomic features that are highly specific for energy production.

Bioaccumulation and detoxification of trivalent arsenic by Achromobacter xylosoxidans BHW-15 and electrochemical detection of its transformation efficiency.

Arsenotrophic bacteria play an essential role in lowering arsenic contamination by converting toxic arsenite [As (III)] to less toxic and less bio-accumulative arsenate [As (V)]. The current study focused on the qualitative and electrocatalytic detection of the arsenite oxidation potential of an arsenite-oxidizing bacteria A. xylosoxidans BHW-15 (retrieved from As-contaminated tube well water), which could significantly contribute to arsenic detoxification, accumulation, and immobilization while also providing a scientific foundation for future electrochemical sensor development. The minimum inhibitory concentration (MIC) value for the bacteria was 15 mM As (III). Scanning Electron Microscopy (SEM) investigation validated its intracellular As uptake capacity and demonstrated a substantial association with the MIC value. During the stationary phase, the strain's As (III) transformation efficiency was 0.0224 mM/h. Molecular analysis by real-time qPCR showed arsenite oxidase (aioA) gene expression increased 1.6-fold in the presence of As (III) compared to the untreated cells. The immobilized whole-cell also showed As (III) conversion up to 18 days. To analyze the electrochemical oxidation in water, we developed a modified GCE/P-Arg/ErGO-AuNPs electrode, which successfully sensed and quantified conversion of As (III) into As (V) by accepting electrons; implying a functional As oxidase enzyme activity in the cells. To the best of our knowledge, this is the first report on the electrochemical observation of the As-transformation mechanism with Achromobacter sp. Furthermore, the current work highlighted that our isolate might be employed as a promising candidate for arsenic bioremediation, and information acquired from this study may be helpful to open a new window for the development of a cost-effective, eco-friendly biosensor for arsenic species detection in the future.

Multilocus sequence typing of multidrug-resistant Salmonella strains circulating in poultry farms of Bangladesh.

Salmonella is one of the most important foodborne zoonotic pathogens, and becoming multidrug-resistant (MDR), which represents a serious public health concern worldwide. This study aimed to identify the circulating MDR strains of Salmonella through cutting edge molecular techniques including gene specific PCR, RAPD-PCR, ribosomal gene sequencing, and multilocus sequence types (MLST) in the poultry industry of Bangladesh. Two hundred Salmonella isolates were retrieved from 154 samples comprising droppings (n = 60), cloacal swabs (n = 60), feeds (n = 14), feeding water (n = 14), and handler's swab (n = 6) from 14 commercial layer farms of Bangladesh. The isolates were confirmed as Salmonella through invA gene specific PCR, and further genotyping was done by RAPD-PCR, and 16S rRNA sequencing. The isolates were distributed into 18 different genotypes according to RAPD typing. The phylogenetic analysis identified three diverging phylogroups such as S. enterica Litchfield, S. enterica Enteritidis and S. enterica Kentucky with 11, 8, and 6 strains, respectively. The in vitro antibiogram profiling the Salmonella isolates through disc diffusion method using 13 commercially available antibiotics revealed highest resistance against doxycycline (91.5%) followed by tetracycline and ampicillin (86.0%, in each), and 72.0% isolates as MDR, being resistant to ≥ 5 antibiotics. The MLST typing was carried out based on the PCR amplification of seven housekeeping genes (aroC, hisD, hemD, purE, secA, thrA, and dnaN). MLST typing also revealed three sequence types (STs) such as ST11, ST198, and ST214 in these isolates, and eBURST analysis showed ST11 as the founder genotype. The three STs were highly resistant to tetracyclines and quinolone group of antibiotics, and all of the isolates harboring S. enterica Litchfield showed the highest resistance. Circulating common MLSTs with MDR properties in different farms confirmed the possibility of a common route of intra-farm transmission. We report for the first time of the association serovar Litchfield (ST11) in avian salmonellosis with MDR properties which is an urgent public health concern in Bangladesh.

Microbial co-infections in COVID-19: Associated microbiota and underlying mechanisms of pathogenesis.

The novel coronavirus infectious disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has traumatized the whole world with the ongoing devastating pandemic. A plethora of microbial domains including viruses (other than SARS-CoV-2), bacteria, archaea and fungi have evolved together, and interact in complex molecular pathogenesis along with SARS-CoV-2. However, the involvement of other microbial co-pathogens and underlying molecular mechanisms leading to extortionate ailment in critically ill COVID-19 patients has yet not been extensively reviewed. Although, the incidence of co-infections could be up to 94.2% in laboratory-confirmed COVID-19 cases, the fate of co-infections among SARS-CoV-2 infected hosts often depends on the balance between the host's protective immunity and immunopathology. Predominantly identified co-pathogens of SARS-CoV-2 are bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Acinetobacter baumannii, Legionella pneumophila and Clamydia pneumoniae followed by viruses including influenza, coronavirus, rhinovirus/enterovirus, parainfluenza, metapneumovirus, influenza B virus, and human immunodeficiency virus. The cross-talk between co-pathogens (especially lung microbiomes), SARS-CoV-2 and host is an important factor that ultimately increases the difficulty of diagnosis, treatment, and prognosis of COVID-19. Simultaneously, co-infecting microbiotas may use new strategies to escape host defense mechanisms by altering both innate and adaptive immune responses to further aggravate SARS-CoV-2 pathogenesis. Better understanding of co-infections in COVID-19 is critical for the effective patient management, treatment and containment of SARS-CoV-2. This review therefore necessitates the comprehensive investigation of commonly reported microbial co-pathogens amid COVID-19, their transmission pattern along with the possible mechanism of co-infections and outcomes. Thus, identifying the possible co-pathogens and their underlying molecular mechanisms during SARS-CoV-2 pathogenesis may shed light in developing diagnostics, appropriate curative and preventive interventions for suspected SARS-CoV-2 respiratory infections in the current pandemic.

Genome-wide genetic marker analysis and genotyping of Escherichia fergusonii strain OTSVEF-60.

Poultry originated Escherichia fergusonii (POEF), an emerging bacterial pathogen, causes a wide range of intestinal and extra-intestinal infections in the poultry industry which incurred significant economic losses worldwide. Chromosomal co-existence of antibiotics and metal resistance genes has recently been the focal point of POEF isolates besides its pathogenic potentials. This study reports the complete genome analysis of POEF strain OTSVEF-60 from the poultry originated samples of Bangladesh. The assembled draft genome of the strain was 4.2 Mbp containing 4503 coding sequences, 120 RNA (rRNA = 34, tRNA = 79, ncRNA = 7), and three intact phage signature regions. Forty-one broad range antibiotic resistance genes (ARGs) including dfrA12, qnrS1, blaTEM-1, aadA2, tet(A), and sul-2 along with multiple efflux pump genes were detected, which translated to phenotypic resistant patterns of the pathogen to trimethoprim, fluoroquinolones, ß-lactams, aminoglycoside, tetracycline, and sulfonamides. Moreover, 22 metal resistance genes were found co-existing within the genome of the POEF strain, and numerous virulence genes (VGs) coding for cit (AB), feo (AB), fep (ABCG), csg (ABCDEFG), fliC, ompA, gadA, ecpD, etc. were also identified throughout the genome. In addition, we detected a class I integron gene cassette harboring dfrA12, ant (3″)-I, and qacEΔ-sul2 genes; 42 copies of insertion sequence (IS) elements; and two CRISPR arrays. The genomic functional analysis predicted several metabolic pathways related to motility, flagellar assembly, epithelial cell invasion, quorum sensing, biofilm formation, and biosynthesis of vitamin, co-factors, and secondary metabolites. We herein for the first time detected multiple ARGs, VGs, mobile genetic elements, and some metabolic functional genes in the complete genome of POEF strain OTSVEF-60, which might be associated with the pathogenesis, spreading of ARGs and VGs, and subsequent treatment failure against this emerging avian pathogen with currently available antimicrobials.

Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline.

Infecting millions of people, the SARS-CoV-2 is evolving at an unprecedented rate, demanding advanced and specified analytic pipeline to capture the mutational spectra. In order to explore mutations and deletions in the spike (S) protein - the most-discussed protein of SARS-CoV-2 - we comprehensively analyzed 35,750 complete S protein-coding sequences through a custom Python-based pipeline. This GISAID-collected dataset of until 24 June 2020 covered six continents and five major climate zones. We identified 27,801 (77.77% sequences) mutated strains compared to reference Wuhan-Hu-1 wherein 84.40% of these strains mutated by only a single amino acid (aa). An outlier strain (EPI_ISL_463893) from Bosnia and Herzegovina possessed six aa substitutions. We also identified 11 residues with high aa mutation frequency, and each contains four types of aa variations. The infamous D614G variant has spread worldwide with ever-rising dominance and across regions with different climatic conditions alongside L5F and D936Y mutants, which have been documented throughout all regions and climate zones, respectively. We also found 988 unique aa substitutions spanned across 660 residues, which differed significantly among different continents (p = .003) and climatic zones (p = .021) as inferred with the Kruskal-Wallis test. Besides, 17 in-frame deletions at four sites adjacent to receptor-binding-domain were determined that may have a possible impact on attenuation. This study provides a fast and accurate pipeline for identifying mutations and deletions from the large dataset for coding and also non-coding sequences as evidenced by the representative analysis on existing S protein data. By using separate multi-sequence alignment, removing ambiguous sequences and in-frame stop codons, and utilizing pairwise alignment, this method can derive both synonymous and non-synonymous mutations (strain_ID reference aa:mutation position:strain aa). We suggest that the pipeline will aid in the evolutionary surveillance of any SARS-CoV-2 encoded proteins and will prove to be crucial in tracking the ever-increasing variation of many other divergent RNA viruses in the future. The code is available at https://github.com/SShaminur/Mutation-Analysis.

Evidence of combined effect of amino acid substitutions within G-H and B-C loops of VP1 conferring serological heterogeneity in foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) serotype A exhibits a higher degree of genetic and antigenic diversity resulting in frequent vaccine failure due to serological mismatch between the vaccine and heterologous strains. Currently, knowledge on the molecular basis of antigenic relationships among the FMDVs is limited; nevertheless, intratype antigenic variation due to mutation(s) is widely considered as the main hurdle to appropriate FMD vaccine development. Here, we studied genetic and antigenic variations of four FMDV serotype A isolates, BAN/GA/Sa-197/2013 (BAN-197), BAN/CH/Sa-304/2016 (BAN-304), BAN/DH/Sa-307/2016 (BAN-307) and BAN/DH/Sa-310/2017 (BAN-310) circulating in Bangladesh during 2013-2017. Initially, antigenic relationships (r1 -values) of the field isolates were evaluated by the two-dimensional microneutralization test (2D-MNT) using the hyperimmune antisera raised in cattle against the vaccine strain, BAN-304. Interesingly, the results showed protective serological cross-reactivity (r1 -values > 0.4) between the vaccine strain and the field isolates, BAN-307 and BAN-310, except BAN-197 that substantially mismatched (r1  = 0.129 ± 0.043) with the BAN-304. Although VP1-based phylogeny grouped all the isolates within the same sublineage C (a subgroup of VP3Δ59 variant) under the lineage A/ASIA/G-VII, strikingly, computational analyses of the viral capsid proteins demonstrated significant deviation at the VP1 G-H loop of BAN-197 from the vaccine strain, while VP(2-4) of both isolates were structurally conserved. To bridge the gap of how the distortion of the G-H loop and consequent antigenic hetergeneity occurred in BAN-197, we performed in silico combinatorial substitutions of the VP1 mutant amino acids (aa) of BAN-197 with the respective residues in BAN-304. Remarkably, our analyses revealed that two substitutions of distantly located aa at B-C (T48I:threonine â†’ isoleucine) and G-H (A143V:alanine â†’ valine) loops, in combination, distorted the VP1 G-H loop. Overall, this work contributes to understanding the molecular basis of antigenic relationships operating in serotype A FMDVs and the selection of suitable vaccine strain(s) for effective prophylaxis of FMD based on VP1-based analyses.

Mutational insights into the envelope protein of SARS-CoV-2.

The ongoing mutations in the structural proteins of SARS-CoV-2 are the major impediment for prevention and control of the COVID-19 disease. Presently we focused on evolution of the envelope (E) protein, one of the most enigmatic and less studied protein among the four structural proteins (S, E, M and N) associated with multitude of immunopathological functions of SARS-CoV-2. In the present study, we comprehensively analyzed 81,818 high quality E protein sequences of SARS-CoV-2 globally available in the GISAID database as of 20 August 2020. Compared to Wuhan reference strain, our mutational analysis explored only 1.2 % (982/81818) mutant strains undergoing a total of 115 unique amino acid (aa) substitutions in the E protein, highlighting the fact that most (98.8 %) of the E protein of SARS-CoV-2 strains are highly conserved. Moreover, we found 58.77 % (134 of 228) nucleotides (nt) positions of SARS-CoV-2 E gene encountering a total of 176 unique nt-level mutations globally, which may affect the efficacy of real time RT-PCR-based molecular detection of COVID-19. Importantly, higher aa variations observed in the C-terminal domain (CTD) of the E protein, particularly at Ser55-Phe56, Arg69 and the C-terminal end (DLLV: 72-75) may alter the binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 and thus could play a key role in COVID-19 pathogenesis. Furthermore, this study revealed the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. Our analysis also observed a triple cysteine motif harboring mutation (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of the structural proteins including the envelope protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.