Biology subjects

Manohar, P.

Publications and source records attributed to Manohar, P..

14 recordsLinked to original sources

bioRxiv2025

The Lysis Cassette of Jumbophage phiKZ

Pseudomonas jumbophage phiKZ is prominent because of the proteinaceous nuclear structure that is formed during the infection cycle, conferring resistance of the replicating phage DNA to anti-phage resistance systems. Progress has been made in deciphering the molecular basis of nucleus formation and other unique aspects of phiKZ biology, but the understanding of how it causes host lysis remains minimal. Here we present bioinformatic, physiological and molecular evidence for a "lysis cassette", the expression of which is necessary and sufficient for the temporally regulated disruption of the host envelope. This cluster contains genes for the usual components of an MGL (multigene lysis) system, including the holin, endolysin, i-spanin and o-spanin. In addition, a fifth gene in the cluster, encoding a cytoplasmic protein, was found to accelerate the timing of holin-mediated lethality when expressed in trans. Evidence is provided that suggests this lysis regulator protein interacts with the cytoplasmic domain of the phiKZ class III holin. In support of this notion, alpha-fold analysis generated a high-confidence structure of a conserved holin-lysis regulator heterodimer complex. Infections at high multiplicity resulted in slower bulk culture lysis profiles than at low multiplicity, suggesting that phiKZ might have a lysis-inhibition system.

microbiology↗

bioRxiv2023

Evaluation of Phage and Antibiotic Combinations for Staphylococcus aureus Biofilm Eradication: Insights from the Wax Worm Model

BackgroundGalleria mellonella, an advantageous model organism, to investigate the therapeutic potential of bacteriophages. Employing the Galleria mellonella (waxworm) model, this study involved infecting the larvae with pathogenic Staphylococcus aureus, followed by combination treatment with staph phages; vB_Sau_Saa90 and vB_Sau_Saa165 at the concentrations of 107 and 109 PFU/mL and oxacillin, at 100 mg/kg. The treatment modalities were executed in three distinct orders: pre-phage treatment, simultaneous treatment, and post-phage treatment. Each treatment was administered at 90 min intervals. The health state index of the waxworms was monitored throughout the study. ResultsAmong the three treatment orders, both pre-phage treatment and post-antibiotic treatment exhibited the significant outcomes. The survival percentages ranged from 40% to 50%, 30% to 50%, and 50% to 70% across strong, moderate, and weak biofilm conditions. Notably, the phages vB_Sau_Saa90 and vB_Sau_Saa165 displayed remarkable effectiveness in combination with antibiotics. This successful synergy showcased the potential of combinatorial treatment in effectively addressing biofilm-related infections. ConclusionsThis study holds considerable significance as it underscores the suitability of G. mellonella as a model organism for exploring bacteriophage-based therapeutic approaches. The studys findings shed light on the efficacy of combining bacteriophages and antibiotics to target S. aureus biofilm cells. The observed results in the waxworm model highlight a promising avenue for managing biofilm infections. However, the need for further validation through larger animal models is emphasized, as this could potentially pave the way for novel treatment strategies with broader clinical implications.

microbiology↗

bioRxiv2023

Phage-Antibiotic Combination: An Effective Method for Eradication of Staphylococcus aureus

Increasing antibiotic resistance poses a serious threat, especially in patients admitted to ICUs. The use of phages in combination with antibiotics as compassionate therapy has become a choice of treatment for pan-drug-resistant bacteria. Here, we studied the cumulative effect of phages with four antibiotics, fosfomycin, ciprofloxacin, vancomycin and oxacillin using three different treatment orders against S. aureus. The antibiotic disc synergy method showed that the plaque size of the phage increased in the subinhibitory antibiotic zone. The sub-inhibitory antibiotic amended in the agar media showed that the plaque size increased between 0.25 g/mL and 1 g/mL of antibiotics. It increases from 0.5 {+/-} 0.1 mm (phage-alone control) to 4 {+/-} 0.2 mm, 1.6 {+/-} 0.1 mm, and 1.6 {+/-} 0.4 mm with fosfomycin, ciprofloxacin, and oxacillin, respectively. Checkerboard analysis showed that phages and antibiotics were synergistic with the FIC index of less than 0.5. So, phage-antibiotic combination treatment appeared to be effective. However, the highest efficiency was observed when the antibiotics were administered after phage treatment. A maximum of 39.4-, 39.4-, and 37.0-fold reduction relative to untreated bacterial culture was observed with fosfomycin, oxacillin, and ciprofloxacin. Vancomycin antibiotic had a least 14.7-fold reduction. Finally, our findings emphasize the potential benefits of phage-antibiotic combination therapy compared with phage-alone therapy to treat S. aureus infections.

microbiology↗

bioRxiv2022

In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. Here, we describe the Pseudomonas phage Motto which has the ability to clear P. aeruginosa infections in an animal model and also exhibited biofilm-degrading properties. The phage has substantial antibiofilm activity against strong biofilm-producing isolates (n = 10), with at least a 2-fold reduction within 24 hours. To demonstrate the safety of using phage Motto, we performed cytotoxicity studies with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in C. elegans, with a 90% survival rate when treated with the phage at an MOI of 10. ImportancePhages are often evaluated mainly on their ability to kill bacterial hosts. One important aspect, however, is often neglected: Their ability to degrade biofilms. Not all phages are able to disintegrate biofilms. While phages can kill planktonic cells, it also has often been observed that phages are not able to infect those that are embedded in biofilms. Phage Motto appears to have highly efficient enzymes that degrade biofilms, and might therefore be a highly valuable therapeutic candidate.

microbiology↗

bioRxiv2022

Phage-antibiotic combinations to control Pseudomonas aeruginosa-Candida two-species biofilms

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. This synergy has also been observed for bacteria embedded in biofilms as many phages are capable of degrading the heterogeneous material, often preventing antibiotic compounds from reaching the embedded bacteria. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by the bacterium P. aeruginosa and the fungal pathogen Candida albicans, two microorganisms commonly found together in cystic fibrosis patients. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity in disintegrating 24-hour-old pseudomonal biofilms. Also, other antibiotic combinations, such as cefotaxime, gentamicin, meropenem and tetracycline showed substantial effects on biofilms. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-hour-old dual-species biofilms but showed only negligible activity against 24-hour-old biofilms. Similarly, the combination of antibacterial compounds together with the phage showed no synergistic effects on biofilms formed by P. aeruginosa and C. albicans. This study lays the first foundation for potential therapeutic approaches to treat infections caused by bacteria and fungi using phage-antibiotic combinations.

microbiology↗

bioRxiv2022

A Lysozyme Murein Hydrolase with Broad-spectrum Antibacterial Activity from Enterobacter Phage myPSH1140

Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multi-drug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp. and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.

microbiology↗

bioRxiv2022

A multiwell-plate Caenorhabditis elegans assay for assessing the therapeutic potential of Bacteriophages against Clinical Pathogens

In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffers from batch-to-batch variations and requires manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans which feeds on bacteria, a fast growing and easy to handle organism which can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae and EHEC/ EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages. ImportanceValidating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism, C. elegans as a quick, reliable and simple alternative for testing phages. The data shows that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

microbiology↗

bioRxiv2020

Detection of NDM-1, VIM-1 and AIM-type Metallo-beta-lactamase genes in Gram-negative bacteria isolated from clinical samples in Tamil Nadu

The distribution of carbapenem-resistant Gram-negative bacteria has become an increasing public health concern in India. The aim of this study was to investigate the prevalence of carbapenem-resistant bacteria isolated from the clinical samples in Tamil Nadu, India. A total of 126 non-repetitive Gram-negative bacteria were taken for this study. The susceptibility to meropenem was determined by Minimum Inhibitory Concentration (MIC) by broth micro-dilution. The phenotypic resistance screening such as MHT (Modified Hodge test), EDTA disk synergy and CIM (carbapenem inactivation method) were performed. A multiplex PCR was used for the detection of carbapenemase-encoding genes. Among the 126 isolates studied, 82 (65.07%) meropenem-resistant isolates were identified by MIC. A total of 18 (21.9%) isolates were found to be positive for Metallo-{beta}-Lactamase production through EDTA synergy test. None of the isolates were carbapenemase producer by MHT and CIM. The isolates identified with resistance genes (8/82) were blaNDM-1 in two Klebsiella sp., two P. aeruginosa and one A. baumannii, blaVIM-1 in one P. aeruginosa and blaAIM-1 in one P. aeruginosa and one A. baumannii. The study showed the distribution and increase of carbapenem-resistant bacteria in the study region. Therefore, constant monitoring and effective elimination should be focused to reduce the spread of carbapenem-resistant isolates.

microbiology↗

bioRxiv2020

Transfer of Antibiotic Resistance Genes from Gram-positive Bacterium to Gram-negative Bacterium

The emergence of antibiotic resistance due to uncontrolled use of antibiotics in non-humans, poses a major threat for treating bacterial infections in humans. Added to this is the possibility of transfer of resistance from Gram-positive bacteria to Gram-negative bacteria. Therefore, the possibility of resistance gene transfer from a non-human originated pathogenic bacterium to a pathogenic bacterium infecting humans needs evaluation. In this study, poultry litter samples collected from Tamil Nadu, India were screened for the presence of meropenem- and cefotaxime-resistant Staphylococcus sciuri. Standard microbiological techniques and 16S rRNA analysis were used to confirm S. sciuri. In the resistant isolates, resistance genes such as blaNDM-1, blaOXA-48-like, blaKPC, blaVIM, blaIMP and blaCTX-M were screened. Transconjugation studies were performed using donor, S. sciuri and recipient, E. coli AB1157 (Strr). A total of 26 meropenem-resistant and 24 cefotaxime resistant S. sciuri were isolated from poultry litter samples. The presence of blaNDM-1 (n=2), blaIMP (n=8), blaCTX-M-9 (n=5) and blaCTX-M-2 (n=1) was detected. Transconjugation results confirmed that S. sciuri carrying plasmid-borne resistance gene blaNDM-1 conjugated to E. coli AB1157. The transferability of resistance genes from S. sciuri to E. coli could be another possible reason for spread of antibiotic-resistant bacteria in humans.

microbiology↗

bioRxiv2020

A Biological Inventory of Prophages in A. baumannii Genomes Reveal Distinct Distributions in Classes, Length and Genomic Positions

Acinetobacter baumannii is of major clinical importance as the bacterial pathogen often causes hospital acquired infections, further complicated by the high prevalence of antibiotic resistant strains. Aside from natural tolerance to certain antibiotic classes, resistance is often acquired by the exchange of genetic information via conjugation but also by the high natural competence exhibited by A. baumannii. In addition, bacteriophages are able to introduce resistance genes but also toxins and virulence factors via phage mediated transduction. In this work, we analysed the complete genomes of 177 A. baumannii strains for the occurrence of prophages, and analysed their taxonomy, size and positions of insertion. Among all the prophages that were detected, Siphoviridae and Myoviridae were the two most commonly found families, while the average genome size was determined as 3.98 Mbp. Our data shows the wide variation in the number of prophages in A. baumannii genomes and the prevalence of certain prophages within strains that are most "successful" or potentially beneficial to the host. Our study also revealed that only two specific sites of insertion within the genome of the host bacterium are being used, with few exceptions only. Lastly, we analysed the existence of genes that are encoded in the prophages, which confer antimicrobial resistance (AMR). Several phages carry AMR genes, including OXA-23 and NDM-1, illustrating the importance of lysogenic phages in the acquisition of resistance genes.

bioinformatics↗

bioRxiv2020

Dissemination of carbapenem-resistance and plasmids encoding carbapenemases in Gram-negative bacteria isolated from India

Carbapenem resistance in Gram-negative bacteria is an ongoing public-health problem of global dimensions leaving very few treatment options for severely infected patients. This study focuses on the dissemination of plasmid-borne carbapenemase genes in Gram-negative bacteria in Tamil Nadu, India. A total of 151 non-repetitive isolates belonging to 11 genera were collected from a diagnostic center in Tamil Nadu. E. coli (n=57) isolates were classified as, Enteropathogenic (n=12), Enteroaggregative (n=9), Enterohemorrhagic (n=8), Enterotoxigenic (n=3), Enteroinvasive (n=1) and unclassified E. coli (n=24). Of the 45 Klebsiella species, 14 were K1 whereas 11 were K2 serotype and in 20 Klebsiella serotype could not be determined. Other isolates (n=49) consisted of P. aeruginosa, S. typhi, E. cloacae, A. baumannii, S. marcescens, A. xylosoxidans, P. mirabilis and E. meningoseptica. Of the 151 isolates, 71% (n=107) and 68% (n=103) were found to be resistant to meropenem and imipenem respectively. The most prevalent beta-lactamase gene was blaNDM-1 (21%, 12/57) followed by blaOXA-181 (16%, 9/57), blaGES-9 (n=8), blaOXA-23 (n=7), blaIMP-1 (n=3), blaGES-1 (n=11) and blaOXA-51 (n=9). The unusual presence of blaOXA-23 was seen in E. coli (n=4), and blaOXA-23 and blaOXA-51 (IncA/C) in K. pneumoniae (n=3). Plasmid incompatibility (inc/rep) typing results showed that the plasmids carrying resistance genes (n=11) belonged to IncX, IncA/C, IncFIA-FIB and IncFIIA groups. E. coli and K. pneumoniae were able to transfer plasmid-borne carbapenemase via conjugation. This study highlights the prevalence of carbapenem resistance and the acquisition of plasmid-borne carbapenemase genes in Gram-negative bacteria highlighting the role of plasmid transfer in disseminating resistance.

microbiology↗

bioRxiv2019

Identification of blaGIM-1 in Acinetobacter variabilis isolated from the hospital environment in Tamil Nadu, India

BackgroundEmergence of carbapenem resistance mechanisms among Gram-negative bacteria is a worrisome health problem. Here, we focused on to identify the presence of carbapenem-resistant bacteria among the samples collected from hospital environments in Tamil Nadu.\n\nMethodsA total of 30 hospital environmental samples were collected between August 2017 and January 2018 from hospitals located in Chennai and Vellore such as lift switches, stair rails, switchboards, nursing desks, used nursing gloves, door handles, wheelchairs, touch screens, chairs and from pillars inside the hospitals.\n\nResults and discussionA total of 22 carbapenem-resistant Gram-negative bacteria were isolated that included Escherichia coli, Klebsiella sp., Enterobacter sp., Salmonella sp., Pseudomonas aeruginosa and Acinetobacter sp. Interestingly, blaGIM-1 was detected in Acinetobacter variabilis strain isolated in samples collected from hospitals. Unlike other studies, the identified GIM-1 was not plasmid encoded, and this is the first report for the presence of GIM-1 (German imipenemase) in India.\n\nConclusionExtensive surveillance programs are necessary to trace the uncontrolled spread of carbapenem-resistance genes in order to reduce the rapid spread of resistance.

microbiology↗

bioRxiv2018

Role of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033 and PA2070 in Pseudomonas aeruginosa

To evaluate the presence of biofilm-specific antibiotic-resistant genes, PA0756-0757, PA5033 and PA2070 in Pseudomonas aeruginosa isolated from clinical samples in Tamil Nadu. For this cross-sectional study, 24 clinical isolates (included pus, urine, wound, and blood) were collected from two diagnostic centers in Chennai from May 2015 to February 2016. Biofilm formation was assessed using microtiter dish biofilm formation assay and minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) were determined for planktonic and biofilm cells (MBC assay). Further, PCR amplification of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033 and PA2070 were performed. Biofilm formation was found to be moderate/strong in 16 strains. MBC for planktonic cells showed that 4, 7, 10 and 14 strains were susceptible to gentamicin, ciprofloxacin, meropenem and colistin respectively. In MBC assay for biofilm cells (MBC-B), all the 16 biofilm producing strains were resistant to ciprofloxacin and gentamicin whereas nine and four were resistant to meropenem, and colistin respectively. The biofilm-specific antibiotic-resistant genes PA0756-0757 was found in 10 strains, 6 strains with PA5033 and 9 strains with PA2070 that were found to be resistant phenotypically. This study highlighted the importance of biofilm-specific antibiotic resistance genes PA0756-0757, PA5033, and PA2070 in biofilm-forming P. aeruginosa.

microbiology↗

bioRxiv2017

Genetic diversity and mother-child overlap of the gut associated microbiota determined by reduced genome sequencing

The genetic diversity and sharing of the mother-child associated microbiota remain largely unexplored. This severely limits our functional understanding of gut microbiota transmission patterns. The aim of our work was therefore to use a novel reduced metagenome sequencing in combination with shotgun and 16S rRNA gene sequencing to determine both the metagenome genetic diversity and the mother-to-child sharing of the microbiota. For a cohort of 17 mother-child pairs we found an increase of the collective metagenome size from about 100 Mbp for 4-day-old children to about 500 Mbp for mothers. The 4-day-old children shared 7% of the metagenome sequences with the mothers, while the metagenome sequence sharing was more than 30% among the mothers. We found 15 genomes shared across more than 50% of the mothers, of which 10 belonged to Clostridia. Only Bacteroides showed a direct mother-child association, with B. vulgatus being abundant in both 4-day-old children and mothers. In conclusion, our results support a common pool of gut bacteria that are transmitted from adults to infants, with most of the bacteria being transmitted at a stage after delivery.

microbiology↗