Prasanth Manohar

Pulmonary diseases of viral origin are often followed by the manifestation of secondary infections, leading to further clinical complications and negative disease outcomes. Thus, research on secondary infections is essential. Here, we review clinical data of secondary bacterial infections developed after the onset of pulmonary viral infections. We review the most recent clinical data and current knowledge of secondary bacterial infections and their treatment in SARS-CoV-2 positive patients; case reports from SARS-CoV, MERS-CoV, SARS-CoV2 and the best-studied respiratory virus, influenza, are described. We outline treatments used or prophylactic measures employed for secondary bacterial infections. This evaluation includes recent clinical reports of pulmonary viral infections, including those by COVID-19, that reference secondary infections. Where data was provided for COVID-19 patients, a mortality rate of 15.2% due to secondary bacterial infections was observed for patients with pneumonia (41 of 268). Most clinicians treated patients with SARS-CoV-2 infections with prophylactic antibiotics (63.7%, n = 1,901), compared to 73.5% (n = 3,072) in all clinical reports of viral pneumonia included in this review. For all cases of viral pneumonia, a mortality rate of 10.9% due to secondary infections was observed (53 of 482). Most commonly, quinolones, cephalosporins and macrolides were administered, but also the glycopeptide vancomycin. Several bacterial pathogens appear to be prevalent as causative agents of secondary infections, including antibiotic-resistant strains of Staphylococcus aureus and Klebsiella pneumoniae.

Secondary bacterial infections manifest during or after a viral infection(s) and can lead to negative outcomes and sometimes fatal clinical complications. Research and development of clinical interventions is largely focused on the primary pathogen, with research on any secondary infection(s) being neglected. Here we highlight the impact of secondary bacterial infections and in particular those caused by antibiotic-resistant strains, on disease outcomes. We describe possible non-antibiotic treatment options, when small molecule drugs have no effect on the bacterial pathogen and explore the potential of phage therapy and phage-derived therapeutic proteins and strategies in treating secondary bacterial infections, including their application in combination with chemical antibiotics.

Clinical pathogens, especially Gram-negative bacteria developing resistance to third-generation cephalosporins, are making clinical outcomes more complicated and serious. This study was undertaken to evaluate the distribution of CTX-M-type extended-spectrum β-lactamases (ESBLs) in Tamil Nadu, India. For this study, clinical samples were collected from five different hospitals located in Tamil Nadu and the ESBL-producing Gram-negative isolates were characterized. MIC was performed using cefotaxime and ceftazidime. The bla ESBL-producing genes were screened using multiplex PCR for the genes, CTX-M group-1,-2,-8,-9,-26. The conjugation studies were performed using Escherichia coli AB1157 as a recipient for the isolates harbouring plasmid-borne resistance following broth-mating experiment. In total, 1500 samples were collected and 599 Gram-negative bacteria were isolated that included E. coli (n=233), Klebsiella pneumoniae (n=182), Pseudomonas aeruginosa (n=79), Citrobacter spp. (n=30), Proteus mirabilis (n=28), Salmonella spp. (n=21), Acinetobacter baumannii (n=12), Serratia spp. (n=6), Shigella spp. (n=4), Morganella morganii (n=3) and Providencia spp. (n=1). MIC results showed that 358 isolates were resistant to cefotaxime and ceftazidime. Further, ESBL gene-amplification results showed that 19 isolates had CTX-M group-1 gene including E. coli (n=16), K. pneumoniae (n=2) and P. aeruginosa (n=1) whereas one M. morganii isolate had CTX-M group-9, which was plasmid-borne. Through conjugation studies, 12/20 isolates were found to be involved in the transformation of its plasmid-borne resistance gene. Our study highlighted the importance of horizontal gene transfer in the dissemination of plasmid-borne bla CTX-M-type resistance genes among the clinical isolates.

Mycobacteriophages are viruses specific to mycobacteria that have gained attention as alternative therapeutic strategies for treating antibiotic-resistant infections. Mycobacteriophages are highly diverse and have been grouped into 29 clusters, 71 sub-clusters and 10 singletons based on the genome sequence. Here, we annotate the genome of PDRPxv, a lytic mycobacteriophage isolated from New Delhi; it belongs to the Siphoviridae family as determined by transmission electron microscopy. This phage survives at higher temperatures (up to 55°C) and in alkaline conditions (up to pH11). PDRPxv phage genome is 69,171 bp in length with 66.35 % GC content and encodes 107 putative open reading frames and belongs to the B1 sub-cluster. Genome annotation indicated that genes for DNA encapsidation, structural proteins, replication/transcription and lysis of the host are present in functional clusters. Structural proteins encoded by Gp10-Gp12, Gp18, Gp25 and Gp28-Gp33 were identified by mass spectrometry. Interestingly, no gene encoding a holin function was found. Single-step growth curve revealed that PDRPxv has an adsorption time of 45 min, a latency time of 135 min and an average burst size of 99 phage particles per infected cell. The short latency period and the large burst size mark the lytic nature of the PDRPxv phage, which could therefore be a promising therapeutic candidate against pathogenic Mycobacterium species.

Background: Staphylococcus aureus is one of the common opportunistic gram-positive pathogens which are often associated with nosocomial infections. Detection of methicillin-resistant S. aureus (MRSA) has become complicated due to the complex phenotypic and genomic pattern. Objective: To evaluate the sensitivity and specificity pattern of various phenotypic methods used in screening mec genes harboring MRSA. Methods: Clinical isolates of S. aureus were collected from diagnostic centers in Tamil Nadu. Phenotypic identification methods such as Minimal Inhibitory Concentration for oxacillin, oxacillin screen agar (OSA), oxacillin disk diffusion, and cefoxitin disk diffusion (CFD) tests were compared. The clinical isolates were classified into MRSA and methicillin-susceptible S. aureus (MSSA) based on the polymerase chain reaction (PCR) amplification of the mecA gene. Result: Out of 50 S. aureus, 21 were found to be MRSA based on the presence of the mecA gene. All 21 mecA-positive isolates were found to be resistant through minimum inhibitory concentration (MIC) and CFD test, having a sensitivity of 100% and specificity of 52% and 62%, respectively. OSA and oxacillin disk tests were found to have a sensitivity of 86% and specificity of 48% and 52%, respectively. Conclusion: The combination of two phenotypic methods, CFD and oxacillin MIC, can be used for the detection of MRSA in clinical laboratories.

Bacteriophages, or viruses of microbes, when used as a medical strategy, might be able to solve the current crisis mankind faces with the increasing number of pathogens being antibiotic-resistant, where chemical drugs seized to show any therapeutic effect. The so-called phage therapy may be one of the most promising alternatives to treat infections caused by antibiotic-resistant bacteria, which are killed after infection by a phage. While phages that destroy the host by lysis are chosen for therapy, many pharmacological and immunological aspects of phages as medicines have not been established so far. The immune system plays an important role in a process called phage acceptance where both, innate and adaptive immune responses of the host are involved. However, not only medical aspects but also social ones such as lacking public awareness or acceptance, and lack of structured regulatory guidelines are challenges that have to be addressed in the near future to establish phage therapy as a reliable and safe alternative for the treatment of infections. This review focuses on the unique pharmacological and immunological aspects of phages used in therapy.

phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic
resistance. Lyophilization is used for the preparation of pharmaceutical products to improve their
stability in long-term storage. the aim of this study was to improve the stability of lyophilized
bacteriophages using different excipients. Three lytic bacteriophages Escherichia phage ECP311,
Klebsiella phage KPP235 and Enterobacter phage ELP140 were subjected to lyophilization using
six different excipients: glucose, sucrose, gelatin, mannitol, polyethene glycol and sorbitol. The
lyophilized phages were stored at 4 °C and 37 °C and rehydrated using biological saline to test their
viability at 5 months interval up to 20 months. The results showed that the use of sucrose, gelatin
and their combination was beneficial in maintaining the viability of phages post-lyophilization.
When lyophilized phages were stored at 4 °C, their viability was maintained up to 20 months, but at
37 °C there was a reduction in activity after 10 months. This is one of the few studies to report the
lyophilization of phage cocktails to have viability for up to 10 months. Our study identified promising
lyophilization excipients to effectively lyophilize bacteriophages for pharmaceutical applications and
long-term storage.

Infections due to antibiotic resistant bacteria are increasing globally and this needs
immediate attention. Bacteriophages are considered an effective alternative for
the treatment of bacterial infections. The aim of this study was to isolate and
characterize the bacteriophages that infect Escherichia coli, Klebsiella pneumoniae,
and Enterobacter species. For this, clinical bacterial isolates of the mentioned
species were obtained from diagnostic centers located in Chennai, Tamil Nadu,
India. The bacteriophages were isolated from sewage water samples collected from
Tamil Nadu, India. Phage isolation was performed using enrichment method and
agar overlay method was used to confirm the presence of bacteriophages. All the
phages were characterized for their life cycle parameters, genome analysis, and
in vitro phage cocktail activity. The three bacteriophages exhibited broad host range
activity: Escherichia virus myPSH2311 infecting E. coli belonging to six different
pathotypes, Klebsiella virus myPSH1235 infecting K. pneumoniae belonging to four
different serotypes and Enterobacter virus myPSH1140 infecting four different species
of Enterobacter. Morphological observations suggested that the bacteriophages
belonged to, Phieco32virus (Escherichia virus myPSH2311), Podoviridae (Klebsiella
virus myPSH1235), and Myoviridae (Enterobacter virus myPSH1140). The life cycles
(adsorption, latent period, and cell burst) of Escherichia virus myPSH2311, Klebsiella
virus myPSH1235 and Enterobacter virus myPSH1140 were found to be 26, 40, and
11 min, respectively. Genomic analysis revealed that Escherichia virus myPSH2311
is closely related to Escherichia phage vB_EcoP_SU10, Klebsiella virus myPSH1235
is closely related to Klebsiella phage vB_KpnP_KpV48 and Enterobacter virus
myPSH1140 is closely related to Enterobacter phage PG7 and Enterobacter phage
CC31. When phage cocktail was used against multiple bacterial mixtures, there was a
reduction in bacterial load from 106 to 103 CFU/mL within 2 h. All the three characterized
phages were found to have a broad host range activity and the prepared phage cocktails were effective against mixed bacterial population that are resistant to meropenem and
colistin, two last resort antibiotics. Infections caused by drug resistant bacteria will be a
serious threat in the future and the use of virulent bacteriophages in therapy may offer
an effective solution.

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophilization studies showed that the use of 1M sucrose, 2% gelatin and the combination of both 0.5M sucrose plus 1% gelatin could restore phage viability up to 20 months at 4˚C. For in vivo studies, it was observed that a single phage dose can reduce the E. coli infection but to achieve 100% survival rate the infected larvae should be treated with three phage doses (20 μL, 10 3 PFU/mL) at 6 hours interval. The characterized Escherichia phage myPSH1131 was found to have broad host range activity against E. coli pathogens and in vivo studies showed that multiple doses are required for effective treatment.

Use of the 16S rRNA gene in microbiota studies is limited by the lack of taxonomic and functional resolution. High resolution analyses are particularly important for understanding transmission and persistence of bacteria. The aim of our work was therefore to compare a novel reduced metagenome sequencing (RMS) approach with 16S rRNA gene sequencing to determine both the metagenome genetic diversity and the mother-to-child sharing of the microbiota in a cohort of 17 mother-child pairs. We found that although both approaches gave comparable results with respect to sample separation and taxonomy, RMS gave higher resolution and the potential for genomic-/functional assignment. Using RMS we estimated that the metagenome size increased from about 60 Mbp for 4-day-old children to about 225 Mbp for mothers. The 4-day-old children shared 7% of the metagenome sequences with the mothers, while the metagenome sequence sharing was > 30% among the mothers. We found 15 genomes shared across > 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. For the functional assignments, we identified a significant association between antibiotic usage during labor, and quantity of Fosfomycin resistance genes. In conclusion, our results show a higher functional and taxonomic resolution for RMS compared to 16S rRNA gene sequencing, where RMS enabled a detailed description of mother to child gut microbiota transmission-supporting a late recruitment of most gut bacteria and an effect of antibiotic treatment during labor on infant antibiotic resistance gene patterns.

Introduction: Infections caused by carbapenem-resistant Enterobacteriaceae are one of the serious clinical problems, considering carbapenems are the last resort of antibiotics for treating multi-drug resistant Enterobacteriaceae. Objective: In this study, we compared different phenotypic screening methods used for the identification of carbapenemase-producing Enterobacteriaceae. Methods: The clinical isolates were initially studied for their minimal inhibitory concentration and PCR based carbapenem-resistance gene analysis. The Modified Hodge Test (MHT), carbapenem inactivation method (CIM) and carbaNP test were performed. Results: The results showed that the MHT and carba NP test can produce false-negative results but carbapenem inactivation method (CIM) can produce relatively positive results for all the isolates. In the CIM method, the use of ertapenem disks produced accurate results. The carbaNP test was found to be rapid in analyzing the results but CIM was suitable for general clinical laboratories with its high sensitivity and specificity. Conclusion: This study highlighted the use of different phenotypic methods to evaluate the carbapenemase producers in the clinical laboratories.

Background: Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. The current surge in bacteriophage therapy is motivated mainly because of the emergence of antibiotic-resistant bacteria in clinics. This study evaluated the therapeutic potential of three bacteriophages isolated against Escherichia coli ec311, Klebsiella pneumoniae kp235 and Enterobacter cloacae el140 strains using Galleria mellonella. The in vitro activity of three different phages belonging to Podoviridae and Myoviridae families was studied by the double agar overlay method against multi-drug resistant strains. Larval survivability studies were performed to evaluate the potential of phages against infection using G. mellonella. Results: All the three phages were found to have potential to infect the host bacterial strains. For in vivo studies it was observed that E. coli and E. cloacae infected larvae, should be treated with three phage doses (20 μL, 10 4 PFU/mL) at 6 h interval to achieve 100% survival rate. But in the case of K. pneumoniae, a single phage dose treatment showed promising outcome. When mixed bacterial infections (all three bacterial cultures at 10 8 CFU/mL) were tested, minimum of four doses of phage cocktail (three phages) at 6 h interval was necessary to recover the larvae. All the results were confirmed by enumerating bacteria from the larvae. Conclusion: Our data shows that although in vitro studies showed high infectivity of phages, for in vivo models multiple phage doses were required for effective treatment.

Purpose. The occurrence of carbapenem-and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem-and colistin-resistance in two areas in Tamil Nadu, India. Methodology. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem-and colistin-resistance. Further, resistance genes bla NDM-1 , bla OXA-48-like , bla IMP , bla VIM , bla KPC , mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed.

The amylolytic enzyme plays a very important role in industrial applications. This study aimed to screen amylase producing Bacillus sp.
and to promote its amylolytic activity by mutagenesis. Samples were collected from coastal mud samples and starch hydrolyzing
isolates were screened. A single isolate having the highest enzyme activity was identified as Bacillus tequilensis by 16S rRNA analysis.
A starch medium was optimized and fermentation period studies revealed that the mutant strain (after 60 sec of UV exposure) had
higher activity (868 U/mL/min) than the parental strain (418 U/mL/min) after 36 hours of incubation at 37°C, pH 7.0. It was also found
that amylase from intracellular mutant strain had maximum activity; on the other side parental strain had maximum activity with an
extracellular enzyme. Optimized temperature, pH and salt concentration revealed that the intracellular amylase from mutant strain had
the maximum activity of 978 U/mL/min, 985 U/mL/min, 960 U/mL/min respectively. Varying the source of carbon in the medium had a
significant impact on enzyme activity. Metalloenzymes like amylases were reported to have strong activity towards metal ions, so
amylase activity was analysed by adding different metal ions in the medium and found that calcium ions strongly promoted amylase
activity and Fe2+, Zn2+, Cu2+, Mg2+ inhibited the activity. SDS-PAGE results showed that the molecular weight of isolated amylase to be
approximately 55.0 kDa. Our study showed the capability of mutant B. tequilensis strain to produce double the amount of intracellular
amylase than the parental strain.

Background: Colistin is one of the oldest antibiotics in the polymyxin group, and is used mostly against gram-negative bacteria. Because of developing resistance among clinical isolates colistin has become an alternative drug for multidrug resistant bacteria. Objectives: To determine colistin resistance among isolates from Tamil Nadu, India. Methods: We included 94 gram-negative isolates from two centers in Tamil Nadu in the present study. Isolates were identified by 16S rRNA sequencing. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Results: The isolates identified at species level included 48 Escherichia coli, 9 Klebsiella pneumoniae, 10 Pseudomonas aeruginosa, 5 Proteus mirabilis, 4 Salmonella enterica, 3 Enterobacter hormaechei, 3 Enterobacter cloacae, 2 Achromobacter xylosoxidans, 2 Acinetobacter baumannii, 1 Providencia vermicola, 1 Acinetobacter towneri, 1 Enterobacter gergoviae, 2 Providencia rettgeri, 1 Enterobacter asburiae, 1 Pseudomonas stutzeri, and 1 Salmonella typhi. The MIC of colistin ranged from 0.12 μg/ml to 128 μg/ml. The MIC 50 was 1 μg/mL and MIC 90 was >128 μg/ml. The MIC ≥ 8 μg/mL was resistant breakpoint for all the species. A total of 27 isolates were resistant to colistin. Colistin resistant isolates included E. coli (9/48), K. pneumoniae (6/9), P. aeruginosa (3/10), A. baumannii (1/2), P. mirabilis (4/5), E. cloacae (1/3), P. rettgeri (2/2), and S. enterica (1/4). Carbapenem susceptibility of colistin resistant isolates was tested and 14 were found to be resistant to meropenem. Conclusions: Our study indicates the emergence of colistin resistant isolates from clinical samples among different groups of gram-negative organisms. Resistance to both carbapenem and colistin occurs. Developing new antibiotics and programs to reduce nosocomial infections is necessary especially for multidrug resistant isolates.

This study presents different fuels (Glycine and Urea) that can be used to synthesize nanocrystalline forsterite by the sol-gel combustion method. The weight change of precursor during thermal treatment was studied by ther-mo-gravimetric analysis (TGA). Pure forsterite was characterized by heating microscopy, Fourier transform infra-red spectroscopy, X-ray Diffraction, Brunauer-Emmett-Teller, Scanning Electron Microscopy, and Energy dispersive X-ray spectroscopy. The HAP (hydroxyapatite) deposition ability, degradation and dissolution behaviour of forsterite was examined in simulated body fluid (SBF). The combusted forsterite precursor showed distinct thermal behaviour for each fuel when analyzed by heating microscopy. BET analysis showed that the particle size of forsterite synthesized using glycine was 28 nm, specific surface area 65.11 m 2 /g and average pore diameter 16.4 nm while using urea 1.951 μm, 0.939 m 2 /g, and 30.5 nm are the respective parameters. The dissolution of forsterite pointed to the consumption of Ca and P ions from SBF, the negligible release of Si ion into the SBF and these ionic interactions with SBF can be altered as per the material properties. The forsterite showed good antibacterial activity against S. aureus but lower activity against E. coli. The bactericidal activity of forsterite indicated that it can be used to inhibit biofilm formation in dental, bone implants and bacterial infection during surgical operations.

Hydrolytic amylases produced from microorganisms are of high demand due to its larger industrial applications. In this study, α-amylase producing bacteria were isolated from the marine environment and produced enzyme was partially purified. Samples were collected from marine environment and bacterial isolates were screened and identified by 16S rRNA analysis as Bacillus drentensis, Bacillus tequilensis and Bacillus subtilis. Under optimized conditions such as pH, temperature and salt concentrations, higher enzyme activity was observed than normal conditions. In Bacillus drentensis, the enzyme activity of 16.73 U/mL was obtained in pH 5.0 at 40 °C with 1% salt concentration. Interestingly, this acidic amylase was stable even at larger salt concentrations. In fermentation period studies, Bacillus drentensis had maximum activity after 48 h incubation, with the cell mass of 2.5 (OD), protein content of 3.1 (mg/mL) and the enzyme activity was 26.33 U/mL. Partial purification by (NH4)2SO4 precipitation at 40% saturation yielded 5.79% enzymes with the specific activity of 46.1 U/mL and the dialyzed extract yielded 14.01% enzyme with 293.1 U/mL of specific activity. SDS-PAGE results revealed that the molecular mass of isolated amylase was approximately 55kDa. Many Bacillus sp. were used to produce amylases but to the best of our knowledge it is the first report on production of α-amylase from Bacillus drentensis with the high specific activity of 293.1 U/mL.

INTRODUCTION Protease is an enzyme that chiefly involved in cleaving the long peptide chains into short fragments and is produced by various sources such as plants, animals, and microorganisms. Among these, microorganisms were noteworthy,since it can grow in a shorter period, produce a specialized enzyme, cheaper and reliable 1. In culture medium, large amount of extra cellular proteases is produced by the bacteria which belong to genus Bacillus, they are largely present in the soil and mud regions 2, 3. Marine microorganism producing protease has upraised magnetism by the reason of habituation to extreme conditions 4. Protease enzyme finds application in various fields like meat tenderization, detergents, silver recovery, laundry, stone washing jeans, pulp and paper manufacture, leather de-hairing and tanning, de-sizing of textiles and de-inking of paper 5, 6. Hence the yield of protease can be enhanced by exposing the strain to ultraviolet radiation and is universally practiced for inducing strain improvement 7. Our present study mainly focused on to isolate protease producing strain and tooptimize the cultural condition such as temperature, pH, and fermentation period along with exposure of strains to ultraviolet radiation to investigate the increased protease activity.

– Virulence factors of E. faecalis have been found to be involved in the pathogenesis of dental diseases. In this study, prevalence of gelatinase and the effect of factors such as glucose concentration and pH have been investigated. 62% of the isolates (n=26) were positive for gelE in 42 E.faecalis isolates which were amplified in PCR. Among the gelE positive isolates, 65% (n=17) of the isolates were from Dental Caries. The results showed increasing activity of gelatinase as the concentration of glucose increased upto 1%. We also observed that the gelatinase retained its activity between pH 6 and 8.

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

Background 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. Minimal inhibitory concentration of imipenem and meropenem were determined using micro-broth dilution method. E. coli pathotyping, Klebsiella serotyping, screening for beta-lactamases and plasmid incompatibility grouping was performed.Results 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 iso...

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 bacteri...

Introduction: Infections caused by carbapenem-resistant Enterobacteriaceae are one of the serious clinical problems, considering carbapenems are the last resort of antibiotics for treating multi-drug resistant Enterobacteriaceae. Objective: In this study, we compared different phenotypic screening methods used for the identification of carbapenemase-producing Enterobacteriaceae. Methods: The clinical isolates were initially studied for their minimal inhibitory concentration and PCR based carbapenem-resistance gene analysis. The Modified Hodge Test (MHT), carbapenem inactivation method (CIM) and carbaNP test were performed. Results: The results showed that the MHT and carba NP test can produce false-negative results but carbapenem inactivation method (CIM) can produce relatively positive results for all the isolates. In the CIM method, the use of ertapenem disks produced accurate results. The carbaNP test was found to be rapid in analyzing the results but CIM was suitable for general...

Infections caused by extended-spectrum ß-lactamase (ESBL) and carbapenemase producing Gram negative bacteria are an emerging global health problem. For the present study, the clinical isolates (n=58) were collected from two diagnostic centers in Tamil Nadu, India. The bacterial isolates were screened for cefotaxime and meropenem resistance. Further, the presence of ESBL resistant gene CTX-M and carbapenem-resistant genes blaNDM-1, blaOXA-48, blaIMP, blaVIM, blaKPC and integrons were studied. Transconjugation studies and DNA fingerprinting was performed. A total of 58 clinical isolates that included Escherichia coli (n=33), Klebsiella pneumoniae (n=8), Pseudomonas aeruginosa (n=6), Enterobacter cloacae (n=6) and Proteus mirabilis (n=5) were analyzed. MIC results showed that 77.6% (45/58) and 36.2% (21/58) of the clinical isolates were resistant to cefotaxime and meropenem, respectively. The blaCTX-M was present in 34 isolates, the presence of blaNDM-1 in 6 isolates and the blaOXA-48 ...

Background Colistin is one of the oldest antibiotics in the polymyxin group, and is used mostly against gramnegative bacteria. Because of developing resistance among clinical isolates colistin has become an alternative drug for multidrug resistant bacteria. Objectives To determine colistin resistance among isolates from Tamil Nadu, India. Methods We included 94 gram-negative isolates from two centers in Tamil Nadu in the present study. Isolates were identified by 16S rRNA sequencing. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Results The isolates identified at species level included 48 Escherichia coli, 9 Klebsiella pneumoniae, 10 Pseudomonas aeruginosa, 5 Proteus mirabilis, 4 Salmonella enterica, 3 Enterobacter hormaechei, 3 Enterobacter cloacae, 2 Achromobacter xylosoxidans, 2 Acinetobacter baumannii, 1 Providencia vermicola, 1 Acinetobacter towneri, 1 Enterobacter gergoviae, 2 Providencia rettgeri, 1 Enterobacter asburiae, 1 Pseudomonas stutzeri, a...

Antibiotic resistance among Gram negative bacteria has become one of the major and serious clinical and health problems globally. Increasing number of data regarding the identification of new species getting resistance to multiple drugs (Multi - drug resista nce) is reported worldwide. Metallo beta - lactamases (NDM - 1) which helps bacterium to resist beta antibiotics including Carbapenems become the most concern problem. Aim of this study was to identify the antibiotic susceptibility pattern by disk diffusion method and to design a primer to detect the prevalence of bla NDM - 1 by polymerase chain reaction in clinical isolates. Out of 39 isolates tested for antibiotic susceptibility pattern majority of the isolates were found to be resistant to Mero penem (98%) and Imipenem (95%). PCR amplification results showed the presence of bla NDM - 1 in 5 isolates (2 strains of Klebsiella pneumoniae Salmonella typhi and 1 Pseudomonas aeruginosa ). 50% prevalence of species in our study area...

Abstract Aquaculture being a prime source of food for the majority of the global population, the industry is facing enormous loss due to infectious diseases. The use of antibiotics in combating infections is a breakthrough for aquaculture in the past, but the rising antibiotic resistance is posing an immense threat to their livelihood due to strict regulatory guidelines. Alternative therapeutic measures are required to continue the aquaculture production to meet the global need. There is anticipation in using bacteriophages, especially as a biocontrol agent. In this review, we have focused on collecting pieces of evidence to support the application of bacteriophages or phage-derived endolysins in aquaculture to fight bacterial infections in a post-antibiotic era. We have also summarised the prospects of phage applications in aquaculture and highlighted the importance of government initiatives in regulating and escorting the reliable method as a biocontrol strategy in a comprehensive manner in the aquaculture industry. In the future, studies have to be accomplished in aquaculture to prove the effectiveness of phages or phage-derived endolysins to intensify their use on a large scale as a prime source of biocontrol agents.

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 respect...

Mycobacteriophages are viruses specific to mycobacteria that have gained attention as alternative therapeutic strategies for treating antibiotic-resistant infections. Mycobacteriophages are highly diverse and have been grouped into 29 clusters, 63 sub-clusters and 10 singletons based on the genome sequence. Here, we annotate the genome of PDRPxv, a lytic mycobacteriophage isolated from New Delhi; it belongs to the Siphoviridae family as determined by transmission electron microscopy. This phage survives at higher temperatures (up to 55 °C) and in alkaline conditions (up to pH11). The phage genome is 69,171 bp in length with 66.35% GC content and encodes 107 putative open reading frames. The PDRPxv phage belongs to the B1 sub-cluster. Genome annotation indicated that genes for DNA encapsidation, structural proteins, replication/transcription and lysis of the host are present in functional clusters. Structural proteins encoded by Gp10-Gp12, Gp18, Gp25 and Gp28-Gp33 were identified by mass spectrometry. Interestingly, no gene encoding a holin function was found. Single-step growth curve revealed that PDRPxv has an adsorption time of 45 minutes, a latency time of 135 minutes and an average burst size of 99 phage particles per infected cell. The short latency period and the large burst size mark the lytic nature of the PDRPxv phage, which could therefore be a promising therapeutic candidate against pathogenic Mycobacterium species.

Phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic resistance. Lyophilization is used for the preparation of pharmaceutical products to improve their stability in long-term storage. The aim of this study was to improve the stability of lyophilized bacteriophages using different excipients. Three lytic bacteriophagesEscherichiaphage ECP311,Klebsiellaphage KPP235 andEnterobacterphage ELP140 were subjected to lyophilization using six different excipients: glucose, sucrose, gelatin, mannitol, polyethylene glycol and sorbitol. The lyophilized phages were stored at 4 °C and 37 °C and rehydrated using biological saline to test their viability at 5 months interval up to 20 months. The results showed that the use of sucrose, gelatin and their combination was beneficial in maintaining the viability of phages post-lyophilization. When lyophilized phages were stored at 4 °C, their viability was maintained up to 20 months, but at 37 °C there was a r...

Clinical pathogens especially Gram-negative bacteria developing resistance to third-generation cephalosporins are making the clinical outcome more complicated and serious. This study was undertaken to evaluate the distribution of extended-spectrum beta-lactamases in Tamil Nadu regions in India. For this study, clinical samples were collected from five different hospitals located in Tamil Nadu and ESBL producing Gram-negative isolates were characterized. Minimal inhibitory concentration (MIC) was performed using cefotaxime and ceftazidime. The blaESBL producing genes were screened using multiplex PCR for the genes, CTX-M group-1,-2,-8,-9,-26. Conjugation studies were performed using E. coli AB1157 as a recipient for the isolates harbouring plasmid-borne resistance following broth-mating experiment. In total, 1500 samples were collected and 599 Gram-negative bacteria were isolated that included Escherichia coli (n=233), Klebsiella pneumoniae (n=182), Pseudomonas aeruginosa (n=79), Cit...

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 ...

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.MethodsA 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.Results 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 ...

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophi...

Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. The current surge in bacteriophage therapy is motivated mainly because of the emergence of antibiotic-resistant bacteria in clinics. This study evaluated the therapeutic potential of three bacteriophages isolated against Escherichia coli ec311, Klebsiella pneumoniae kp235 and Enterobacter cloacae el140 strains using Galleria mellonella. The in vitro activity of three different phages belonging to Podoviridae and Myoviridae families was studied by the double agar overlay method against multi-drug resistant strains. Larval survivability studies were performed to evaluate the potential of phages against infection using G. mellonella. All the three phages were found to have potential to infect the host bacterial strains. For in vivo studies it was observed that E. coli and E. cloacae infected larvae, should be treated with three phage doses (20 μL, 10 PFU/mL) at 6 h interval to ac...

The amylolytic enzyme plays a very important role in industrial applications. This study aimed to screen amylase producing Bacillus sp. and to promote its amylolytic activity by mutagenesis. Samples were collected from coastal mud samples and starch hydrolyzing isolates were screened. A single isolate having the highest enzyme activity was identified as Bacillus tequilensis by 16S rRNA analysis. A starch medium was optimized and fermentation period studies revealed that the mutant strain (after 60 sec of UV exposure) had higher activity (868 U/mL/min) than the parental strain (418 U/mL/min) after 36 hours of incubation at 37°C, pH 7.0. It was also found that amylase from intracellular mutant strain had maximum activity; on the other side parental strain had maximum activity with an extracellular enzyme. Optimized temperature, pH and salt concentration revealed that the intracellular amylase from mutant strain had the maximum activity of 978 U/mL/min, 985 U/mL/min, 960 U/mL/min respe...

The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem- and colistin-resistance in two areas in Tamil Nadu, India. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem- and colistin-resistance. Further, resistance genes blaNDM-1, blaOXA-48-like, blaIMP, blaVIM, blaKPC, mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed. A total of 89 bacterial isolates were studied which included Escherichia coli (n=43), Klebsiella pneumoniae (n=18), Pseudomonas aeruginosa (n=10), Enterobacter cloacae (n=6), Acinetobacter baumannii (n=5), Klebsiella oxytoca (n=4), Proteus mirabilis (n=2) and Salmonella paratyphi (n=1). MIC testing showed that 58/89 (65 %) and 29/89 (32 %) isolates were resistant to ...

Bioprecipitation is a process of precipitating water by precipitation causing microorganisms by its ice nucleating properties. The concept of rain-making bacteria is known since 1980’s but lack of research data makes it unrevised. Pseudomonas syringae is a Gram-negative bacterium mostly known to have ice-nucleating properties causing plant diseases. Their huge numbers of pathovars were identified in different hosts each having different modes of action. As always known for its pathogenesis in plant species with its ice-nucleating gene (ina), a concept of ice minus bacteria was created in 1970’s which is against wild type P.syringae. So the bacterium lacking ice nucleating gene (ina) competed with wild type strain and succeeded. But findings say that a bacterium (wild type Pseudomonas syringae) was found on rain drops of different parts of the world and that bacterium is literally raining. More studies in this bacterium as a rainmaking element may give as a better chance to know more about its role in life cycle.