Indranil Biswas's research while affiliated with University of Kansas Medical Center and other places

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrABC two-component system and overexpression of eptA gene due to upstream insertion of IS Aba1 have been shown to play a major role. Thus, the aim of our study is to characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 207 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrABC genes were characterized by whole-genome shotgun sequencing. Twenty-eight complete genomes were further characterized by hybrid assembly approach to study insertional inactivation of lpx genes and the association of IS Aba1-eptA . Several single point mutations (SNPs), like M12I in pmrA , A138T and A444V in pmrB , and E117K in lpxD, were identified. We are the first to report two novel SNPs (T7I and V383I) in the pmrC gene. Among the five colistin-resistant A. baumannii isolates where complete genome was available, the analysis showed that three of the five isolates had IS Aba1 insertion upstream of eptA . No mcr genes were identified among the isolates. We mapped the SNPs on the respective protein structures to understand the effect on the protein activity. We found that majority of the SNPs had little effect on the putative protein function; however, some SNPs might destabilize the local structure. Our study highlights the diversity of colistin resistance mechanisms occurring in A. baumannii, and IS Aba1 -driven eptA overexpression is responsible for colistin resistance among the Indian isolates. IMPORTANCE Acinetobacter baumannii is a Gram-negative, emerging and opportunistic bacterial pathogen that is often associated with a wide range of nosocomial infections. The treatment of these infections is hindered by increase in the occurrence of A. baumannii strains that are resistant to most of the existing antibiotics. The current drug of choice to treat the infection caused by A. baumannii is colistin, but unfortunately, the bacteria started to show resistance to the last-resort antibiotic. The loss of lipopolysaccharides and mutations in lipid A biosynthesis genes are the main reasons for the colistin resistance. The present study characterized 207 A. baumannii clinical isolates and constructed complete genomes of 28 isolates to recognize the mechanisms of colistin resistance. We showed the mutations in the colistin-resistant variants within genes essential for lipid A biosynthesis and that cause these isolates to lose the ability to produce lipopolysaccharides.

Protein homeostasis is an essential process that depends on concerted effort of different proteins involved in proper protein folding, deciding the fate of misfolded proteins by either refolding them into their proper conformation or degrading the misfolded proteins. In Gram-positive bacteria, AAA+ ATPases such as ClpX, ClpC, and ClpE interact with the proteolytic ClpP and degrade misfolded proteins. Clp ATPases also maintain amounts of different proteins based on the cellular need. In streptococci, these Clp ATPases play a crucial role in various physiological processes that contribute to virulence, cell growth and division, stress tolerance, competence development, and biofilm formation. Among the Clp ATPase complexes present in low G + C Gram-positive bacteria, ClpX/P is the major proteolytic system. In contrast to numerous ClpX/P substrates identified in Escherichia coli and other bacteria, very little is known about the identity of the ClpX/P substrates in streptococci. Using a proteomic approach under late logarithmic growth condition, we screened for putative substrates that might be degraded by ClpX/P; several potential substrates were identified and verified by other methods. SpxA2 is one such candidate protein, and by Western blot, we confirmed that it indeed recognizes SpxA2. We further identified Ala-Ala-Leu, located at the C-terminal of SpxA2, as a ClpX/P degradation signal. Using a green fluorescent protein reporter system, we further confirmed several ClpX/P degradation signals. Furthermore, we found that the zinc-binding domain of ClpX is needed for substrate recognition. Our in vitro data indicate that the adaptor or other accessory factors might be needed for the ClpX/P-mediated substrate recognition or degradation. Additional investigations are needed to determine the identity of these factors in the cell. IMPORTANCE Cytoplasmic Clp-related proteases play a major role in maintaining cellular proteome in bacteria. ClpX/P is one such proteolytic complex that is important for conserving protein homeostasis. In this study, we investigated the role of ClpX/P in Streptococcus mutans , an important oral pathogen. We identified several putative substrates whose cellular levels are regulated by ClpX/P in S. mutans and subsequently discovered several recognition motifs that are critical for degradation. Our study is the first comprehensive analysis of determining ClpX/P motifs in streptococci. We believe that identifying the substrates that are regulated by ClpX/P will enhance our understanding about virulence regulation in this important group of pathogens.

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5-1 μg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

Two-component signal transduction (TCS) systems are important regulatory pathways in streptococci. A typical TCS encodes a membrane-anchored sensor kinase (SK) and a cytoplasmic response regulator (RR). Approximately, 20 different types of TCSs are encoded by various streptococci. Among them, two TCSs, in particular BlpRH and ComDE, are required for bacteriocins production and competence development. The SK component of these two TCSs is highly similar and belongs to the protein kinase-10 (HPK-10) subfamily. While these two TCSs are present in streptococci, no systematic studies have been done to differentiate between these two TCSs, and the existence of these pathways in several species of the genus Streptococcus is also unknown. The lack of information about these pathways misguided researchers for decades into believing that the Streptococcus mutans BlpRH system is a ComDE system. Here, we have attempted to distinguish between the BlpRH and ComDE systems based on the location of the chromosome, genomic arrangement, and conserved residues. Using the SyntTax and NCBI databases, we investigated the presence of both TCS systems in the genome of several streptococcal species. We noticed that the NCBI database did not have proper annotations for these pathways in several species, and many of them were wrongly annotated, such as CitS or DpiB instead of BlpH. Nevertheless, our critical analyses led us to classify streptococci into two groups: class A (only the BlpRH system) and class B (both the BlpRH and ComDE systems). Most of the streptococcal groups, including bovis, pyogenic, mutans, salivarius, and suis, encode only the BlpRH system. In contrast, only in the mitis and anginosus groups were both the TCS systems present. The focus of this review is to identify and differentiate between the BlpRH and ComDE systems, and discuss these two pathways in various streptococci.

With the excessive genome plasticity, Acinetobacter baumannii can acquire and disseminate antimicrobial resistance (AMR) genes often associated with mobile genetic elements (MGEs). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution, and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii (CRAb) to understand the role of diverse MGEs and their genetic context responsible for disseminating carbapenem resistance genes. For this, 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between 2018 and 2019 were analyzed. AMR determinants, the genetic context of resistance genes, and molecular epidemiology were studied using whole-genome sequencing. This study observed an increased prevalence of blaOXA–23 followed by dual carbapenemases, blaOXA–23, and blaNDM. This study identified three novel Oxford MLST sequence types. The majority of the isolates belonged to the dominant clone, IC2, followed by less prevalent clones such as IC7 and IC8. This study identified variations of AbaR4 and AbGRI belonging to the IC2 lineage. To the best of our knowledge, this is the first study that provides comprehensive profiling of resistance islands, their related MGEs, acquired AMR genes, and the distribution of clonal lineages of CRAb from India.

In naturally competent bacteria, DNA transformation through horizontal gene transfer is an evolutionary mechanism to receive extracellular DNA. Bacteria need to maintain a state of competence to accept foreign DNA, and this is an energy‐driven phenomenon that is tightly controlled. In Streptococcus, competence development is a complex process that is not fully understood. In this study, we used Streptococcus mutans, an oral bacterium, to determine how cell density affects competence development. We found that in S. mutans the transformation efficiency is maximum when the transforming DNA was added at low cell density and incubated for 2.5 h before selecting for transformants. We also found that S. mutans cells remain competent until the mid‐logarithmic phase, after which the competence decreases drastically. Surprisingly, we observed that individual components of Clp proteolytic complexes differentially regulate competence. If the transformation is carried out at the early growth phase, both ClpP protease and ClpX ATPase are needed for competence. In contrast, we found that both ClpC and ClpE negatively affect competence. We also found that if the transformation is carried out at the mid‐logarithmic growth phase ClpX is still required for competence, but ClpP negatively affects competence. While the exact reason for this differential effect of ClpP and ClpX on transformation is currently unknown, we found that both ClpC and ClpE have a negative effect on transformation, which was not reported before.

With the excessive genome plasticity, Acinetobacter baumannii has the capability to acquire and disseminate antimicrobial resistance genes that are often associated with mobile genetic elements (MGE). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii to understand the role of diverse MGE and their genetic context that are responsible for the dissemination of carbapenem resistance genes. For this, a total of 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between the years 2018 and 2019 were analysed. Antimicrobial resistance determinants, genetic context of resistance genes and molecular epidemiology were studied using whole genome sequencing. A high prevalence of bla OXA-23 was observed followed by the presence of dual carbapenemase, bla OXA-23 and bla NDM. Three novel Oxford sequence types were identified. Majority of the isolates belonged to dominant clone, IC2 followed by less prevalent clones such as IC7 and IC8. Complex diverse AbaR4 like and AbGRI-like islands belonging to IC2 lineage were identified. To the best of our knowledge, this is the first study that provides a comprehensive profiling of resistance islands along with the MGE, acquired antimicrobial resistance genes and the distribution of clonal lineages of carbapenem resistant A. baumannii from India.

When bacteria encounter environmental stresses, the expression of various proteins collectively known as heat shock proteins is induced. These heat shock proteins are necessary for cell survival specifically under conditions that induce protein denaturation.

Streptococcus mutans , a dental pathogen, encodes the ComDE two-component system comprised of a histidine kinase (ComD) and a response regulator (ComE). This system is necessary for production of bacteriocins and development of genetic competence. ComE interacts with its cognate promoters to activate the transcription of bacteriocin and competence related genes. Previous transcriptomic studies indicated that expressions of bacteriocin genes were upregulated in the presence of oxygen. To understand the relationship between the aerobic condition and bacteriocin expression, we analyzed the S. mutans ComE sequence and its close homologs. Surprisingly, we noticed the presence of cysteine (Cys) residues located at positions 200 and 229, which are highly conserved among the ComE homologs. Here we investigated the role of Cys residues of S. mutans ComE in the activation of bacteriocin transcription using the P nlmA promoter that expresses bacteriocin NlmA. We constructed both single mutants and double mutants by replacing the Cys residues with serine and performed complementation assays. We observed that the presence of Cys residues is essential for P nlmA activation. With purified ComE mutant proteins we found that ComE double mutants displayed a nearly two-fold lower association rate than wild-type ComE. Furthermore, ANS fluorescence studies indicated that the double mutants displayed wider conformation changes than wild-type ComE. Finally, we demonstrated that close streptococcal ComE homologs successfully activate the P nlmA expression in vivo . This is the first report suggesting that S. mutans ComE and its homologs can sense the oxidation status of the cell, a phenomenon similar to the AgrA system of Staphylococcus aureus but with different outcome. IMPORTANCE Streptococci are an important species that prefer to grow under anaerobic or microaerophilic environments. Studies have shown that streptococci growth in an aerobic environment generates oxidative stress responses by activating various defense systems including production of antimicrobial peptides called bacteriocins. This study highlights the importance of a two-component response regulator (ComE) that senses the aerobic environment and induces bacteriocin production in Streptococcus mutans , a dental pathogen. We believe increased bacteriocin secretion under aerobic conditions is necessary for survival and colonization of S. mutans in the oral cavity by inhibiting other competing organisms. Redox sensing by response regulator might be a wide-spread phenomenon, since two other ComE homologs from pathogenic streptococci that inhabit diverse environmental niches also perform a similar function.

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role. To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-synonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A. baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified. Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.