Genomic landscape of NDM-1 producing multidrug-resistant Providencia stuartii causing burn wound infections in Bangladesh.

The increasing antimicrobial resistance in Providencia stuartii (P. stuartii) worldwide, particularly concerning for immunocompromised and burn patients, has raised concern in Bangladesh, where the significance of this infectious opportunistic pathogen had been previously overlooked, prompting a need for investigation. The two strains of P. stuartii (P. stuartii SHNIBPS63 and P. stuartii SHNIBPS71) isolated from wound swab of two critically injured burn patients were found to be multidrug-resistant and P. stuartii SHNIBPS63 showed resistance to all the 22 antibiotics tested as well as revealed the co-existence of blaVEB-6 (Class A), blaNDM-1 (Class B), blaOXA-10 (Class D) beta lactamase genes. Complete resistance to carbapenems through the production of NDM-1, is indicative of an alarming situation as carbapenems are considered to be the last line antibiotic to combat this pathogen. Both isolates displayed strong biofilm-forming abilities and exhibited resistance to copper, zinc, and iron, in addition to carrying multiple genes associated with metal resistance and the formation of biofilms. The study also encompassed a pangenome analysis utilizing a dataset of eighty-six publicly available P. stuartii genomes (n = 86), revealing evidence of an open or expanding pangenome for P. stuartii. Also, an extensive genome-wide analysis of all the P. stuartii genomes revealed a concerning global prevalence of diverse antimicrobial resistance genes, with a particular alarm raised over the abundance of carbapenem resistance gene blaNDM-1. Additionally, this study highlighted the notable genetic diversity within P. stuartii, significant informations about phylogenomic relationships and ancestry, as well as potential for cross-species transmission, raising important implications for public health and microbial adaptation across different environments.

Unveiling a high-risk epidemic clone (ST 357) of 'Difficult to Treat Extensively Drug-Resistant' (DT-XDR) Pseudomonas aeruginosa from a burn patient in Bangladesh: A resilient beast revealing coexistence of four classes of beta lactamases.

OBJECTIVES: Pseudomonas aeruginosa (P. aeruginosa) stands out as a key culprit in the colonization of burn wounds, instigating grave infections of heightened severity. In this study, we have performed comparative whole genome analysis of a difficult to treat extensively drug resistant P. aeruginosa isolated from a burn patient in order to elucidate genomic diversity, molecular patterns, mechanisms and genes responsible for conferring antimicrobial resistance and virulence. METHOD: P. aeruginosa SHNIBPS206 was isolated from an infected burn wound of a critically injured burn patient. Whole genome sequencing was carried out and annotated with Prokka. Sequence type, serotype, antimicrobial resistance genes and mechanisms, virulence genes, metal resistance genes and CRISPR/Cas systems were investigated. Later, pangenome analysis was carried out to find out genomic diversity. RESULT: P. aeruginosa SHNIBPS206 (MLST 357, Serotype O11) was resistant to 14 antibiotics including carbapenems and harboured all four classes of beta lactamase producing genes: Class A (blaPME-1, blaVEB-9), Class B (blaNDM-1), Class C (blaPDC-11) and Class D (blaOXA-846). Mutational analysis of Porin D gave valuable insights. Several efflux pump, virulence and metal resistance genes were also detected. Pangenome analysis revealed high genomic diversity among different strains of P. aeruginosa. CONCLUSION: To our knowledge, this is the first report of an extensively drug resistant ST 357 P. aeruginosa from Bangladesh, which is an epidemic high-risk P. aeruginosa clone. Further research and in-depth comprehensive studies are required to investigate the prevalence of such high-risk clone of P. aeruginosa in Bangladesh.

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.

Genomic characterization of the dominating Beta, V2 variant carrying vaccinated (Oxford-AstraZeneca) and nonvaccinated COVID-19 patient samples in Bangladesh: A metagenomics and whole-genome approach.

Bangladesh is experiencing a second wave of COVID-19 since March 2021, despite the nationwide vaccination drive with ChAdOx1 (Oxford-AstraZeneca) vaccine from early February 2021. Here, we characterized 19 nasopharyngeal swab (NPS) samples from COVID-19 suspect patients using genomic and metagenomic approaches. Screening for SARS-CoV-2 by reverse transcriptase polymerase chain reaction and metagenomic sequencing revealed 17 samples of COVID-19 positive (vaccinated = 10, nonvaccinated = 7) and 2 samples of COVID-19 negative. We did not find any significant correlation between associated factors including vaccination status, age or sex of the patients, diversity or abundance of the coinfected organisms/pathogens, and the abundance of SARS-CoV-2. Though the first wave of the pandemic was dominated by clade 20B, Beta, V2 (South African variant) dominated the second wave (January 2021 to May 2021), while the third wave (May 2021 to September 2021) was responsible for Delta variants of the epidemic in Bangladesh including both vaccinated and unvaccinated infections. Noteworthily, the receptor binding domain (RBD) region of S protein of all the isolates harbored similar substitutions including K417N, E484K, and N501Y that signify the Beta, while D614G, D215G, D80A, A67V, L18F, and A701V substitutions were commonly found in the non-RBD region of Spike proteins. ORF7b and ORF3a genes underwent a positive selection (dN/dS ratio 1.77 and 1.24, respectively), while the overall S protein of the Bangladeshi SARS-CoV-2 isolates underwent negative selection pressure (dN/dS = 0.621). Furthermore, we found different bacterial coinfections like Streptococcus agalactiae, Neisseria meningitidis, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Pseudomonas plecoglossicida, expressing a number of antibiotic resistance genes such as tetA and tetM. Overall, this approach provides valuable insights on the SARS-CoV-2 genomes and microbiome composition from both vaccinated and nonvaccinated patients in Bangladesh.

Temporal landscape of mutational frequencies in SARS-CoV-2 genomes of Bangladesh: possible implications from the ongoing outbreak in Bangladesh.

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh-originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA X, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in nonspecific proteins (NSP2-3, NSP-12(RdRp), NSP-13(Helicase)), S-Spike, ORF3a, and N-Nucleocapsid protein were common in the circulating strains with varying degrees and the most unique mutations (UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, Envelope protein (E) and accessory factors (NSP7a, ORF 6, ORF7b) suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed a significant association (p = 0.003) with sex and age of the COVID-19-positive cases. So, identification of these mutational accumulation patterns may greatly facilitate vaccine development deciphering the age and the sex-dependent differential susceptibility to COVID-19.

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.

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.

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.

Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin.

INTRODUCTION: Effective sanitation strategies for poultry farms require an appropriate selection of the disinfectant based on the contaminants present and their sensitivity to the disinfectants. AIM: The current study investigated the prevalence of streptococci/micrococci in poultry farms of Bangladesh and the efficacy of commercial disinfectants (Savlon, Lysol, Quatovet, Virkon S, and Virocid) along with alcohol against these pathogens to adopt appropriate strategies. MATERIALS AND METHODS: Conventional approaches and the 16S rRNA gene sequencing were performed to confirm the isolates at the species level along with microtiter biofilm assay to determine their biofilm-forming ability. Efficacy of the disinfectants was tested against those isolates using agar well diffusion and minimum inhibitory concentration (MIC) test by broth dilution method using different dilutions of the disinfectants. RESULTS: Staphylococcus lentus (n = 32), Micrococcus luteus (n = 7), and Micrococcus aloeverae (n = 4) were confirmed among 102 presumptively screened streptococci/micrococci isolates from 43 samples. No single disinfectant showed equally high efficacy against all three bacterial species in agar well diffusion test, although Virocid showed the lowest MIC against all three of them. Lysol was least effective among the commercial disinfectants by both MIC and diffusion method, although each commercial disinfectant was more effective than alcohol. Considering both the average diameter of the inhibition zones and the MIC values, efficacy can be interpreted as Virocid > Quatovet > Savlon > Virkon S > Lysol. Although the efficacy decreased with decreasing concentration, the disinfectants retained a satisfactory level of efficacy at 50% concentration. Among test pathogens, M. aloeverae was the most sensitive to the disinfectants and the weakest biofilm producers, whereas 4/14 S. lentus and 1/5 M. luteus were strong biofilm producers, which may cause more reduction in the efficacy in environmental conditions. CONCLUSION: As no ideal disinfectant was found in the study, the efficacy of the disinfectants should be routinely evaluated and validated to ensure the sanitation standards in the poultry sector.