Diverse infective and lytic machineries identified in genome analysis of tailed coliphages against broad spectrum multidrug-resistant Escherichia coli / Diversas maquinarias infecciosas y líticas identificadas en el análisis del genoma de colifagos con cola contra Escherichia coli multirresistente de amplio espectro

The emergence of multidrug-resistant (MDR) E. coli with deleterious consequences to the health of humans and animals has been attributed to the inappropriate use of antibiotics. Without effective antimicrobials, the success of modern medicine in treating infections would be at an increased risk. Bacteriophages could be used as an alternative to antibiotics for controlling the dissemination of MDR bacteria. However, before their use, the bacteriophages have to be assessed for the safety aspect. In this study, three broad host range highly virulent coliphage genomes were sequenced, characterized for infective and lytic potential, and checked for the presence of virulence and resistance genes. The genome sequencing indicated that coliphages ϕEC-S-21 and ϕEC-OE-11 belonged to Myoviridae, whereas coliphage ϕEC-S-24 belonged to the Autographiviridae family derived from the Podoviridae family. The genome size of the three coliphages ranged between 24 and 145 kb, with G + C content ranging between 37 and 51%. Coding sequences (CDS) ranged between 30 and 251 amino acids. The CDS were annotated and the proteins were categorized into different modules, viz., phage structural proteins, proteins associated with DNA replication, DNA modification, bacterial cell lysis, phage packaging, and uncharacterized proteins. The presence of tRNAs was detected only in coliphage ϕEC-OE-11. All three coliphages possessed diverse infective and lytic mechanisms, viz., lytic murein transglycosylase, peptidoglycan transglycosylase, n-acetylmuramoyl-l-alanine amidase, and putative lysozyme. Furthermore, the three coliphage genomes showed neither the presence of antibiotic resistance genes nor virulence genes, which makes them desirable candidates for use in phage therapy-based applications.(AU)

Genome Characterization and Infectivity Potential of Vibriophage-ϕLV6 with Lytic Activity against Luminescent Vibrios of Penaeus vannamei Shrimp Aquaculture.

Shrimp aquaculture, especially during the hatchery phase, is prone to economic losses due to infections caused by luminescent vibrios. In the wake of antimicrobial resistance (AMR) in bacteria and the food safety requirements of farmed shrimp, aqua culturists are seeking alternatives to antibiotics for shrimp health management, and bacteriophages are fast emerging as natural and bacteria-specific antimicrobial agents. This study analyzed the whole genome of vibriophage-ϕLV6 that showed lytic activity against six luminescent vibrios isolated from the larval tanks of P. vannamei shrimp hatcheries. The Vibriophage-ϕLV6 genome was 79,862 bp long with 48% G+C content and 107 ORFs that coded for 31 predicted protein functions, 75 hypothetical proteins, and a tRNA. Pertinently, the vibriophage-ϕLV6 genome harbored neither AMR determinants nor virulence genes, indicating its suitability for phage therapy. There is a paucity of whole genome-based information on vibriophages that lyse luminescent vibrios, and this study adds pertinent data to the database of V. harveyi infecting phage genomes and, to our knowledge, is the first vibriophage genome report from India. Transmission electron microscopy (TEM) of vibriophage-ϕLV6 revealed an icosahedral head (~73 nm) and a long, flexible tail (~191 nm) suggesting siphovirus morphology. The vibriophage-ϕLV6 phage at a multiplicity of infection (MOI) of 80 inhibited the growth of luminescent V. harveyi at 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% salt gradients. In vivo experiments conducted with post-larvae of shrimp showed that vibriophage-ϕLV6 reduced luminescent vibrio counts and post-larval mortalities in the phage-treated tank compared to the bacteria-challenged tank, suggesting the potentiality of vibriophage-ϕLV6 as a promising candidate in treating luminescent vibriosis in shrimp aquaculture. The vibriophage-ϕLV6 survived for 30 days in salt (NaCl) concentrations ranging from 5 ppt to 50 ppt and was stable at 4 °C for 12 months.

Coliphage cocktails for controlling antimicrobial-resistant Escherichia coli: emphasizing polyphage and multihost interactions at different levels of multiplicity of infection.

AIM: This study elucidates the in-vitro bactericidal effectiveness of polyphage cocktail combinations of 2, 4, 6, 8, and 10 individual coliphages against a cocktail of 20 AMR Escherichia coli. METHODS AND RESULTS: Different polyphage cocktails viz., 45 two-phage combinations, 28 four-phage combinations, 15 six-phage combinations, 6 eight-phage combinations, and 1 ten-phage combination were formulated using a pool of ten coliphages that were isolated from two different geographical locations (East and West coasts of India). The different polyphage cocktails were tested at four different levels of Multiplicity of Infection (MOI) viz., MOI-1, MOI-10, MOI-100, and MOI-1000. All the 2, 4, 6, 8, and 10-phage cocktails were found to be effective in controlling the growth of a cocktail of 20 AMR bacteria when tested at MOI-1000 and MOI-100 but variations in antibacterial activity were observed at lower MOIs of 10 and 1. The ten coliphage cocktail showed lytic activity against 100% of AMR E. coli from farmed brackish water shrimp, 96% of laboratory collection of AMR E. coli, 92% of AMR E. coli from farmed freshwater fish, and 85% of AMR E. coli from market shrimp. CONCLUSION: Polyphage cocktails of 2, 4, 6, 8, and 10 coliphages applied at an MOI of 1000 effectively suppressed the growth of antimicrobial-resistant E. coli. The results indicated phage-phage synergy in the lytic activity of several coliphage combinations at higher MOIs of 1000 and 100 while phage-phage antagonism was evidenced at lower MOIs of 10 and 1.

Diverse infective and lytic machineries identified in genome analysis of tailed coliphages against broad spectrum multidrug-resistant Escherichia coli.

The emergence of multidrug-resistant (MDR) E. coli with deleterious consequences to the health of humans and animals has been attributed to the inappropriate use of antibiotics. Without effective antimicrobials, the success of modern medicine in treating infections would be at an increased risk. Bacteriophages could be used as an alternative to antibiotics for controlling the dissemination of MDR bacteria. However, before their use, the bacteriophages have to be assessed for the safety aspect. In this study, three broad host range highly virulent coliphage genomes were sequenced, characterized for infective and lytic potential, and checked for the presence of virulence and resistance genes. The genome sequencing indicated that coliphages ϕEC-S-21 and ϕEC-OE-11 belonged to Myoviridae, whereas coliphage ϕEC-S-24 belonged to the Autographiviridae family derived from the Podoviridae family. The genome size of the three coliphages ranged between 24 and 145 kb, with G + C content ranging between 37 and 51%. Coding sequences (CDS) ranged between 30 and 251 amino acids. The CDS were annotated and the proteins were categorized into different modules, viz., phage structural proteins, proteins associated with DNA replication, DNA modification, bacterial cell lysis, phage packaging, and uncharacterized proteins. The presence of tRNAs was detected only in coliphage ϕEC-OE-11. All three coliphages possessed diverse infective and lytic mechanisms, viz., lytic murein transglycosylase, peptidoglycan transglycosylase, n-acetylmuramoyl-l-alanine amidase, and putative lysozyme. Furthermore, the three coliphage genomes showed neither the presence of antibiotic resistance genes nor virulence genes, which makes them desirable candidates for use in phage therapy-based applications.

Gene sequencing analysis of tailed phages identified diverse (Kayfunavirus and Berlinvirus) coliphages in aquatic niche against AMR Escherichia coli.

Escherichia coli has been recognized as a pathogen of concern in the antimicrobial resistance (AMR) perspective. Globally initiatives were taken to control AMR. Bacteriophages are recognized as promising alternative to antibiotics. Harnessing broad-spectrum bacteriophages for augmenting phage repositories is being prioritized across continents for future health care needs. In this context, a study was conducted to isolate coliphages against a collection of AMR E. coli isolated from diverse aquatic niche. Thirty pooled water samples (5 each from rivers, aquaculture ponds, lake, sewage treatment plant, domestic waste and canals) were analysed, and fifty-four lytic coliphages were isolated against the wide range of E. coli host strains. Broad host-spectrum phages were isolated predominantly from sewage water samples. Enriched phages were quantified, and the concentrations ranged from 106 to 107 PFU/mL. Ten phages, viz. ФEC-S-18, ФEC-S-21, ФEC-S-22, ФEC-S-23, ФEC-S-24, ФEC-S-25, ФEC-S-28, ФEC-S-30, ФEC-S-39 and ФEC-S-49, exhibited lytic activity against more than ten AMR strains of E. coli. PCR analysis of the 54 phages using the major capsid protein (MCP) specific primers coupled with gene sequence analysis identified two phages related to Berlinvirus and 35 phages to Kayfunavirus of Autographiviridae. However, the remaining 17 phages did not show amplification using the MCP primers. The study has demonstrated that aquatic environment harboured phages with broad host spectrum that can potentially be used as agents for biological control of E. coli for infection control and food safety.

Predominance of genetically diverse ESBL Escherichia coli identified in resistance mapping of Vembanad Lake, the largest fresh-cum-brackishwater lake of India.

Antimicrobial resistance (AMR) burden in Escherichia coli along the 90 km stretch of Vembanad Lake, Kerala, India, was assessed. Seventy-seven percent of water samples drawn from 35 different stations of the lake harbored E. coli. Antibiotic susceptibility test performed on 116 E. coli isolates revealed resistance to ≥ one antibiotic with 39 AMR profiles in 81%, multidrug resistance in 30%, and extended spectrum ß-lactamase (ESBL) producers in 32%. Of all the 15 antibiotics tested, the probability of isolating cefotaxime-resistant E. coli was the highest (P ≤ 0.05) in the lake. Genetically diverse ESBL types, namely blaTEM-116, blaCTX-M -152, blaCTX-M -27, blaCTX-M -55, blaCTX-M-205, and blaSHV-27, were identified in the lake. This is probably the first report in India for the presence of blaCTX-M-205 (blaCTX-M-group 2) in the Vembanad Lake. ST11439 and single and double loci variants of ST443 and ST4533 were identified in multilocus sequence typing (MLST). Inc plasmids (B/O, F, W, I1, FIIA, HI1, P-1α, K/B, and N) identified in the lake evidences the resistance transmission potential of the E. coli isolated from the lake. Molecular typing (ERIC-PCR, MLST, and PBRT) delineated diverse E. coli, both between and within the sampling stations. Low multiple antibiotic resistance index (average MAR< 0.2) indicates a lower risk of the lake to the human population, but the occurrence of genetically diverse ESBL E. coli in the Vembanad Lake signals health hazards and necessitates pragmatic control measures.

Whole genome sequence analysis of multi drug resistant community associated methicillin resistant Staphylococcus aureus from food fish: detection of clonal lineage ST 28 and its antimicrobial resistance and virulence genes.

Methicillin-resistant staphylococcus aureus (MRSA) sequence type 28 (ST 28) and spa type t021 is a CC30, prototype of ST-30, Community Associated-MRSA (CA-MRSA) (lukS-lukF +). It is a multi-drug resistant strain harbouring staphylococcal endotoxins, haemolysins, ureolysin, serine protease, and antimicrobial resistance genes. In this study, we report the draft genome sequence of this MRSA isolated from the most commonly used food fish, ribbon fish (Trichiurus lepturus). The total number of assembled paired-end high-quality reads was 7,731,542 with a total length of  2.8Mb of 2797 predicted genes. The unique ST28/ t021 CA- MRSA in fish is the first report from India, and in addition to antibiotic resistance is known to co-harbour virulence genes, haemolysins, aureolysins and endotoxins. Comprehensive comparative genomic analysis of CA-MRSA strain7 can help further understand their diversity, genetic structure, diversity and a high degree of virulence to aid in fisheries management.

Effect of electron beam irradiation on the biochemical, microbiological and sensory quality of Litopenaeus vannamei during chilled storage.

The effects of electron beam irradiation (0, 2.5, 5.0, 7.5 and 10 kGy) on the quality characteristics of vacuum packed headless vannamei stored under chilled condition (2 °C) was assessed. The total volatile base nitrogen, non protein nitrogen content, peroxide value and thiobarbituric acid reactive substances showed an increasing trend during storage. Texture analysis indicated that shrimp irradiated at above 2.5 kGy dose had a lower hardness than control. L* value showed a decreased trend during storage (51.87-13.89). Moreover, b* and a* value showed a gradual increase during storage. The total viable count was lower in the irradiated sample than control. A similar trend also observed for psychrophilic, pseudomonas and H2S former count. There is a significant (p < 0.05) reduction in Brochothrix thermosphacta and lactobacillus count in the irradiated sample. Based on the microbial and sensory analysis control had a shelf life up to 12th day. However, electron beam irradiated sample had extended shelf life of 15-23 days with respect to dose level.

Protein hydrolysate from yellowfin tuna red meat as fortifying and stabilizing agent in mayonnaise.

Present study addresses the fortification of mayonnaise with fish protein hydrolysate by partial replacement of egg yolk in the product. Red meat of yellowfin tuna (Thunnus albacares), a by-product from tuna canning industry was used as the source of protein hydrolysate (TPH). Substitution of egg yolk with hydrolysate imparted noticeable fish flavor to the product only at higher levels of replacement (50% and above). Emulsion stability of mayonnaise samples was not significantly affected at the given range of substitution. The preliminary product acceptability parameters indicated higher desirability with a sensory score of 7.6 ± 0.7 for a replacement ratio of 1:2::TPH:egg yolk. Hence, the same combination was further subjected to morphological, rheological characterization, and opted for storage stability studies. Fortified mayonnaise exhibited lower particle size, indicative of higher emulsion quality which was also evident in the rheological properties of the sample. Results indicated better oxidative and physicochemical stability for fortified samples compared to control under chilled conditions, suggesting the applicability of fish protein hydrolysate as fortifying and stabilizing agent in mayonnaise preparations.