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.

SARS-CoV-2 infection alters the gut microbiome in diabetes patients: A cross-sectional study from Bangladesh.

Populations of different South Asian nations including Bangladesh reportedly have a high risk of developing diabetes in recent years. This study aimed to investigate the differences in the gut microbiome of COVID-19-positive participants with or without type 2 diabetes mellitus (T2DM) compared with healthy control subjects. Microbiome data of 30 participants with T2DM were compared with 22 age-, sex-, and body mass index (BMI)-matched individuals. Clinical features were recorded while fecal samples were collected aseptically from the participants. Amplicon-based (16S rRNA) metagenome analyses were employed to explore the dysbiosis of gut microbiota and its correlation with genomic and functional features in COVID-19 patients with or without T2DM. Comparing the detected bacterial genera across the sample groups, 98 unique genera were identified, of which 9 genera had unique association with COVID-19 T2DM patients. Among different bacterial groups, Shigella (25%), Bacteroides (23.45%), and Megamonas (15.90%) had higher mean relative abundances in COVID-19 patients with T2DM. An elevated gut microbiota dysbiosis in T2DM patients with COVID-19 was observed while some metabolic functional changes correlated with bidirectional microbiome dysbiosis between diabetes and non-diabetes humans gut were also found. These results further highlight the possible association of COVID-19 infection that might be linked with alteration of gut microbiome among T2DM patients.

Evolutionary dynamics of SARS-CoV-2 nucleocapsid protein and its consequences.

The emerged novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health crisis that warrants an accurate and detailed characterization of the rapidly evolving viral genome for understanding its epidemiology, pathogenesis, and containment. Here, we explored 61,485 sequences of the nucleocapsid (N) protein, a potent diagnostic and prophylactic target, for identifying the mutations to review their roles in real-time polymerase chain reaction based diagnosis and observe consequent impacts. Compared to the Wuhan reference strain, a total of 1034 unique nucleotide mutations were identified in the mutant strains (49.15%, n = 30,221) globally. Of these mutations, 367 occupy primer binding sites including the 3'-end mismatch to the primer-pair of 11 well-characterized primer sets. Noteworthily, CDC (USA) recommended the N2 primer set contained a lower mismatch than the other primer sets. Moreover, 684 amino acid (aa) substitutions were located across 317 (75.66% of total aa) unique positions including 82, 21, and 83 of those in the RNA binding N-terminal domain (NTD), SR-rich region, and C-terminal dimerization domain, respectively. Moreover, 11 in-frame deletions, mostly (n = 10) within the highly flexible linker region, were revealed, and the rest was within the NTD region. Furthermore, we predicted the possible consequence of high-frequency mutations (≥20) and deletions on the tertiary structure of the N protein. Remarkably, we observed that a high frequency (67.94% of mutated sequences) co-occuring mutations (R203K and G204R) destabilized and decreased overall structural flexibility. The N protein of SARS-CoV-2 comprises an average of 1.2 mutations per strain compared to 4.4 and 0.4 in Middle East respiratory syndrome-related coronavirus and SARS-CoV, respectively. Despite being proposed as the alternative target to spike protein for vaccine and therapeutics, the ongoing evolution of the N protein may challenge these endeavors, thus needing further immunoinformatics analyses. Therefore, continuous monitoring is required for tracing the ongoing evolution of the SARS-CoV-2 N protein in prophylactic and diagnostic interventions.

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.

Microbiome dynamics and genomic determinants of bovine mastitis.

The milk of lactating cows presents a complex ecosystem of interconnected microbial communities which can influence the pathophysiology of mastitis. We hypothesized possible dynamic shifts of microbiome composition and genomic features with different pathological conditions of mastitis (Clinical Mastitis; CM, Recurrent CM; RCM, Subclinical Mastitis; SCM). To evaluate this hypothesis, we employed whole metagenome sequencing (WMS) in 20 milk samples (CM, 5; RCM, 6; SCM, 4; H, 5) to unravel the microbiome dynamics, interrelation, and relevant metabolic functions. The WMS data mapped to 442 bacterial, 58 archaeal and 48 viral genomes with distinct variation in microbiome composition (CM > H > RCM > SCM). Furthermore, we identified a number of microbial genomic features, including 333, 304, 183 and 50 virulence factors-associated genes (VFGs) and 48, 31, 11 and 6 antibiotic resistance genes (ARGs) in CM, RCM, SCM, and H-microbiomes, respectively. We also detected different metabolic pathway and functional genes associated with mastitis pathogenesis. Therefore, profiling microbiome dynamics in different conditions of mastitis and associated microbial genomic features contributes to developing microbiome-based diagnostics and therapeutics for bovine mastitis.

Draft genome sequence of Leuconostoc falkenbergense isolated from naturally fermented buffalo milk curd.

This study reports the draft genome of Leuconostoc falkenbergense strain BSMRAU-M1L5, isolated from artisanal buffalo milk curd in Bangladesh. The draft genome spans 1,776,471 bp, with 50× coverage and 96 contigs.

Draft genome sequencing of Pediococcus pentosaceus strains isolated from cow milk.

We sequenced the genomes of Pediococcus pentosaceus strains MBBL4 and MBBL6, isolated from raw milk samples of healthy cows. The draft genomes of the MBBL4 and MBBL6 were 1,896,831 bp and 1,849,397 bp, respectively, and were fragmented into 58 and 42 contigs, with coverages of 118.2× and 128.7×, respectively.

Genomic insights into antibiotic resistance genes in Leuconostoc citreum strains isolated from artisanal buffalo milk curd in Bangladesh through whole-genome sequencing.

We sequenced the genome of Leuconostoc citreum strains BSMRAU-M1L6 and BSMRAU-M1L13 isolated from artisanal buffalo milk curd in Bangladesh. The draft genomes of BSMRAU-M1L6 and BSMRAU-M1L13 are 1,869,891 and 1,890,611 bp, respectively, with 50.0× coverage (both) and 65 and 75 contigs, respectively.

Draft genome sequencing of a multidrug-resistant Salmonella enterica subspecies enterica serovar Typhimurium strain isolated from chicken in Bangladesh.

Herein this study, we sequenced the genome of a multidrug-resistant Salmonella enterica serovar Typhimurium strain MBR-MFRK-23 isolated from the liver tissue of a diseased layer chicken. The 4,964,854-bp draft genome comprises 50 contigs with 50.5× coverage and 52.1% GC content and is typed as S. enterica sequence type 19.

Genome-Wide Investigation Reveals Potential Therapeutic Targets in Shigella spp.

Shigella stands as a major contributor to bacterial dysentery worldwide scale, particularly in developing countries with inadequate sanitation and hygiene. The emergence of multidrug-resistant strains exacerbates the challenge of treating Shigella infections, particularly in regions where access to healthcare and alternative antibiotics is limited. Therefore, investigations on how bacteria evade antibiotics and eventually develop resistance could open new avenues for research to develop novel therapeutics. The aim of this study was to analyze whole genome sequence (WGS) of human pathogenic Shigella spp. to elucidate the antibiotic resistance genes (ARGs) and their mechanism of resistance, gene-drug interactions, protein-protein interactions, and functional pathways to screen potential therapeutic candidate(s). We comprehensively analyzed 45 WGS of Shigella, including S. flexneri (n = 17), S. dysenteriae (n = 14), S. boydii (n = 11), and S. sonnei (n = 13), through different bioinformatics tools. Evolutionary phylogenetic analysis showed three distinct clades among the circulating strains of Shigella worldwide, with less genomic diversity. In this study, 2,146 ARGs were predicted in 45 genomes (average 47.69 ARGs/genome), of which only 91 ARGs were found to be shared across the genomes. Majority of these ARGs conferred their resistance through antibiotic efflux pump (51.0%) followed by antibiotic target alteration (23%) and antibiotic target replacement (18%). We identified 13 hub proteins, of which four proteins (e.g., tolC, acrR, mdtA, and gyrA) were detected as potential hub proteins to be associated with antibiotic efflux pump and target alteration mechanisms. These hub proteins were significantly (p < 0.05) enriched in biological process, molecular function, and cellular components. Therefore, the finding of this study suggests that human pathogenic Shigella strains harbored a wide range of ARGs that confer resistance through antibiotic efflux pumps and antibiotic target modification mechanisms, which must be taken into account to devise and formulate treatment strategy against this pathogen. Moreover, the identified hub proteins could be exploited to design and develop novel therapeutics against MDR pathogens like Shigella.

Genomic features and pathophysiological impact of a multidrug-resistant Staphylococcus warneri variant in murine mastitis.

Non-aureus staphylococci (NAS) represent a major etiological agent in dairy animal mastitis, yet their role and impact remain insufficiently studied. This study aimed to elucidate the genomic characteristics of a newly identified multidrug-resistant NAS strain, specifically Staphylococcus warneri G1M1F, isolated from murine feces in an experimental mastitis model. Surprisingly, NAS species accounted for 54.35 % of murine mastitis cases, with S. warneri being the most prevalent at 40.0 %. S. warneri G1M1F exhibited resistance to 10 major antibiotics. Whole-genome sequencing established a genetic connection between G1M1F and S. warneri strains isolated previously from various sources including mastitis milk in dairy animals, human feces and blood across diverse geographical regions. Genomic analysis of S. warneri G1M1F unveiled 34 antimicrobial resistance genes (ARGs), 30 virulence factor genes (VFGs), and 278 metabolic features. A significant portion of identified ARGs (64 %) conferred resistance through antibiotic efflux pumps, while VFGs primarily related to bacterial adherence and biofilm formation. Inoculation with G1M1F in mice resulted in pronounced inflammatory lesions in mammary and colon tissues, indicating pathogenic potential. Our findings highlight distinctive genomic traits in S. warneri G1M1F, signifying the emergence of a novel multidrug-resistant NAS variant. These insights contribute to understanding NAS-related mastitis pathophysiology and inform strategies for effective treatment in dairy animals.

Unveiling the gut bacteriome diversity and distribution in the national fish hilsa (Tenualosa ilisha) of Bangladesh.

The field of fish microbiome research has rapidly been advancing, primarily focusing on farmed or laboratory fish species rather than natural or marine fish populations. This study sought to reveal the distinctive gut bacteriome composition and diversity within the anadromous fish species Tenualosa ilisha (hilsa), which holds the status of being the national fish of Bangladesh. We conducted an analysis on 15 gut samples obtained from 15 individual hilsa fishes collected from three primary habitats (e.g., freshwater = 5, brackish water = 5 and marine water = 5) in Bangladesh. The analysis utilized metagenomics based on 16S rRNA gene sequencing targeting the V3-V4 regions. Our comprehensive identification revealed a total of 258 operational taxonomic units (OTUs). The observed OTUs were represented by six phyla, nine classes, 19 orders, 26 families and 40 genera of bacteria. Our analysis unveiled considerable taxonomic differences among the habitats (freshwater, brackish water, and marine water) of hilsa fishes, as denoted by a higher level of shared microbiota (p = 0.007, Kruskal-Wallis test). Among the identified genera in the gut of hilsa fishes, including Vagococcus, Morganella, Enterobacter, Plesiomonas, Shigella, Clostridium, Klebsiella, Serratia, Aeromonas, Macrococcus, Staphylococcus, Proteus, and Hafnia, several are recognized as fish probiotics. Importantly, some bacterial genera such as Sinobaca, Synechococcus, Gemmata, Serinicoccus, Saccharopolyspora, and Paulinella identified in the gut of hilsa identified in this study have not been reported in any aquatic or marine fish species. Significantly, we observed that 67.50% (27/40) of bacterial genera were found to be common among hilsa fishes across all three habitats. Our findings offer compelling evidence for the presence of both exclusive and communal bacteriomes within the gut of hilsa fishes, exhibiting potential probiotic properties. These observations could be crucial for guiding future microbiome investigations in this economically significant fish species.

Characterization of ß-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh.

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to assess the prevalence of P. aeruginosa in clinical, environmental, and poultry sources in Bangladesh, along with their antibiotic susceptibility and the profiling of ß-lactamase and virulence genes using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n = 15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n = 22), Poultry Market (PM; n = 30) and Mymensingh Medical College Hospital (MCCH; n = 23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Hence, this study provides valuable and novel insights on the resistance and virulence of circulating P. aeruginosa within the clinical, environmental, and poultry environments of Bangladesh. These findings are crucial for understanding the emergence of ß-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both human and animal populations.

Unveiling distinct genetic features in multidrug-resistant Escherichia coli isolated from mammary tissue and gut of mastitis induced mice.

Escherichia coli is one of the major pathogens causing mastitis in lactating mammals. We hypothesized that E. coli from the gut and mammary glands may have similar genomic characteristics in the causation of mastitis. To test this hypothesis, we used whole genome sequencing to analyze two multidrug resistant E. coli strains isolated from mammary tissue (G2M6U) and fecal sample (G6M1F) of experimentally induced mastitis mice. Both strains showed resistance to multiple (>7) antibiotics such as oxacillin, aztreonam, nalidixic acid, streptomycin, gentamicin, cefoxitin, ampicillin, tetracycline, azithromycin and nitrofurantoin. The genome of E. coli G2M6U had 59 antimicrobial resistance genes (ARGs) and 159 virulence factor genes (VFGs), while the E. coli G6M1F genome possessed 77 ARGs and 178 VFGs. Both strains were found to be genetically related to many E. coli strains causing mastitis and enteric diseases originating from different hosts and regions. The G6M1F had several unique ARGs (e.g., QnrS1, sul2, tetA, tetR, emrK, blaTEM-1/105, and aph(6)-Id, aph(3″)-Ib) conferring resistance to certain antibiotics, whereas G2M6U had a unique heat-stable enterotoxin gene (astA) and 7192 single nucleotide polymorphisms. Furthermore, there were 43 and 111 unique genes identified in G2M6U and G6M1F genomes, respectively. These results indicate distinct differences in the genomic characteristics of E. coli strain G2M6U and G6M1F that might have important implications in the pathophysiology of mammalian mastitis, and treatment strategies for mastitis in dairy animals.

Draft genome sequencing of Enterococcus avium strains isolated from bovine mastitis.

We performed whole-genome sequencing of four multidrug-resistant Enterococcus avium strains isolated from milk (4M1), feces (4F1 and 4F2), and farm soil (4S1) of mastitic dairy cows. The draft genomes of E. avium strains 4M1, 4F1, 4F2, and 4S1 were approximately 4.2 Mbp, with 39.1% GC content and 66.5× coverage.

Gut and flesh microbiome sequencing of the Bangladesh national fish hilsa (Tenualosa ilisha).

The gut and flesh microbiome of the national fish of Bangladesh, Tenualosa ilisha, were analyzed using 16S rRNA gene sequencing. Our findings revealed a significant microbial disparity between sample categories and the habitat of hilsa fish, which will serve as a valuable foundation for further comprehensive studies on the hilsa microbiome.

Whole-Genome Sequence of the Multidrug-Resistant Staphylococcus warneri Strain G1M1F, Isolated from Mice with Mastitis.

We sequenced the genome of Staphylococcus warneri G1M1F, a multidrug-resistant strain isolated from fecal samples of mice with induced mastitis. The complete genome of G1M1F consists of one chromosome of 2,504,515 bp and two plasmid replicons of 28,679 and 8,615 bp; it comprises 32.7% GC content, with 60× genome coverage.

Whole-Genome Sequence of Multidrug-Resistant Klebsiella pneumoniae MNH_G2C5, Isolated from Bovine Clinical Mastitis Milk.

Klebsiella pneumoniae is one of the most common and important mastitis-causing bacteria, and strain MNH_G2C5 was isolated from the milk of a cow suffering from clinical mastitis in a dairy farm of the Gazipur district of Bangladesh. The MNH_G2C5 genome was estimated to be 4,589,728 bp, with 65.5% genome coverage.

Draft genome sequence of a multidrug-resistant Klebsiella pneumoniae fecal isolate from a cow with clinical mastitis.

Klebsiella pneumoniae is one of the most important mastitis-causing pathogens. The multidrug-resistant K. pneumoniae strain MNH_G2C5F was isolated from the feces of a cow with clinical mastitis. The MNH_G2C5F strain had a genome size of 5,381,832 bp (85.0× coverage) and typed as sequence type 273 (ST273).

Improvement of growth, yield and associated bacteriome of rice by the application of probiotic Paraburkholderia and Delftia.

Plant probiotic bacteria enhance growth and yield of crop plants when applied at the appropriate time and dose. Two rice probiotic bacteria, Paraburkholderia fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 promote growth and yield of plants. However, no information is available on application of these two bacteria on growth, yield, and diversity and population of bacteriome in roots and rhizosphere soils of the treated rice plants. This study aimed to assess the effect of BRRh-4 and BTL-M2 application on growth, yield and bacteriome in roots and rhizosphere soil of rice under varying doses of N, P and K fertilizers. Application of BRRh-4 and BTL-M2 strains significantly (p < 0.05) increased seed germination, growth and yield of rice compared to an untreated control. Interestingly, the grain yield of rice by these bacteria with 50% less of the recommended doses of N, P, and K fertilizers were statistically similar to or better than the rice plants treated with 100% doses of these fertilizers. Targeted amplicon (16S rRNA) sequence-based analysis revealed significant differences (PERMANOVA, p = 0.00035) in alpha-diversity between the root (R) and rhizosphere soil (S) samples, showing higher diversity in the microbial ecosystem of root samples. Additionally, the bacteriome diversity in the root of rice plants that received both probiotic bacteria and chemical fertilizers were significantly higher (PERMANOVA, p = 0.0312) compared to the rice plants treated with fertilizers only. Out of 185 bacterial genera detected, Prevotella, an anaerobic and Gram-negative bacterium, was found to be the predominant genus in both rhizosphere soil and root metagenomes. However, the relative abundance of Prevotella remained two-fold higher in the rhizosphere soil metagenome (52.02%) than in the root metagenome (25.04%). The other predominant bacterial genera detected in the rice root metagenome were Bacillus (11.07%), Planctomyces (4.06%), Faecalibacterium (3.91%), Deinococcus (2.97%), Bacteroides (2.61%), and Chryseobacterium (2.30%). On the other hand, rhizosphere soil metagenome had Bacteroides (12.38%), Faecalibacterium (9.50%), Vibrio (5.94%), Roseomonas (3.40%), and Delftia (3.02%). Interestingly, we found the presence and/or abundance of specific genera of bacteria in rice associated with the application of a specific probiotic bacterium. Taken together, our results indicate that improvement of growth and yield of rice by P. fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 is likely linked with modulation of diversity, structures, and signature of bacteriome in roots and rhizosphere soils. This study for the first time demonstrated that application of plant growth promoting bacteria significantly improve growth, yield and increase the diversity of bacterial community in rice.