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)

Detection of lytic phage infecting flavour-producing strain of Lacticaseibacillus paracasei in the dairy effluents of Kerala.

The performance of the starter culture is a critical factor that decides the quality of fermented milk. Dahi is a fermented milk product popular in India made using a mixed starter culture of lactic acid bacteria comprising acid and flavour producers. The prevalence of bacteriophages in the dairy environment can critically affect the activity of these starter cultures resulting in starter failure. As there is little information available on the occurrence of bacteriophages in the dairy environment of Kerala, this research communication examines the presence of lytic bacteriophages acting against three potential flavour-producing strains of Lacticaseibacillus paracasei (Lc. paracasei). Dairy effluent samples were screened for the presence of phages against the strains of Lc. paracasei by the multiple host enrichment method. Plates showing clearance zone in spot assay were confirmed for the presence of phages by double-layer agar assay. The plaques obtained in the double-layer agar assay were purified for further identification by next-generation sequencing. A bacteriophage infecting one of the three strains of Lc. paracasei was detected by the plaque assay and the blast annotation of the bacteriophage sequence found 86.05% similarity of the phage to Siphoviridae family. The study endorses the need for monitoring phages in the dairy environment to control phage-related starter failure in the state of Kerala.

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.

Tetracycline resistance potential of heterotrophic bacteria isolated from freshwater fin-fish aquaculture system.

AIMS: This study investigated the tetracycline resistance potential of heterotrophic bacteria isolated from twenty-four freshwater fin-fish culture ponds in Andhra Pradesh, India. METHODS AND RESULTS: A total of 261 tetracycline resistant bacteria (tetR) were recovered from pond water, pond sediment, fish gills, fish intestine, and fish feed. Bacteria with high tetracycline resistance (tetHR) (n = 30) that were resistant to tetracycline concentrations above 128  µg mL-1 were predominantly Lactococcus garvieae followed by Enterobacter spp., Lactococcus lactis, Enterobacter hormaechei, Staphylococcus arlettae, Streptococcus lutetiensis, Staphylococcus spp., Brevundimonas faecalis, Exiguobacterium profundum, Lysinibacillus spp., Stutzerimonas stutzeri, Enterobacter cloacae, and Lactococcus taiwanensis. Resistance to 1024 µg mL-1 of tetracycline was observed in L. garvieae, S. arlettae, Enterobacter spp., B. faecalis. Tet(A) (67%) was the predominant resistance gene in tetHR followed by tet(L), tet(S), tet(K), and tet(M). At similar concentrations of exposure, tetracycline procured at the farm level (69.5% potency) exhibited lower inhibition against tetHR bacteria compared to pure tetracycline (99% potency). The tetHR bacteria showed higher cross-resistance to furazolidone (100%) followed by co-trimoxazole (47.5%) and enrofloxacin (11%). CONCLUSIONS: The maximum threshold of tetracycline resistance at 1024 µg mL-1 was observed in S. arlettae, Enterobacter spp., B. faecalis, and L. garvieae and tet(A) was the major determinant found in this study.

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.

A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage.

A 2-step microtiter plate assay was developed to simultaneously check wide values of MOIs of bacteriophages, ranging between MOI-0.0001 and MOI-10000 in the first step and optimize the most suitable MOI (lowest quantity of phage) for inhibiting the growth of the target bacteria in the second step. The results of the first step revealed that the effective MOI of coliphage-ɸ5 for controlling the growth of antimicrobial resistant (AMR) E. coli was between 4.36 and 43.6 for E.coli-EC-3; between 38.2 and 382 for E.coli-EC-7 and between 81.5 and 815 for E.coli-EC-11. The optimum MOI of coliphage-ɸ5 determined in the second step was 17.44, 191 and 326 for controlling the growth of E.coli-EC-3; E.coli-EC-7 and E.coli-EC-11, respectively. The effective MOI of vibriophage-ɸLV6 for controlling luminescent Vibrio harveyi in the first step was found to be between 18.3 and 183 and the optimum MOI as determined in the second step was 79. The sequential 2-step microtiter plate method yielded faster optimization of MOI and was economical compared to the conventional flask method. The measurement of OD values at 550 nm and 600 nm showed similar trend and replicate data from 5-wells and 3-wells yielded identical pattern indicating that the measuring absorbance data in 3-replicate wells at either OD550 or OD600 is sufficient to generate quantifiable phage lysis data. The 2-step microtiter plate assay finds application in phage therapy in human health care, agriculture and animal agriculture for determining the optimum MOIs for selected bacteriophages.

Prevalence of Vibrio mimicus in Fish, Fishery Products, and Environment of South West Coast of Kerala, India.

BACKGROUND: Vibrio mimicus is a seafood-borne bacterium involved in incidences of human infections following consumption of raw or undercooked seafood. Regular monitoring of seafood for V.mimicus is necessary for risk assessment and to establish mitigation measures. METHOD: During the period 2017-2020, a total of 250 samples comprising finfish, shellfish, water, ice, and sediment samples were collected from fish markets, fish landing centers, and fish farms in the Ernakulum district on the Southwest coast of Kerala, India. V. mimicus was isolated using enrichment in alkaline peptone water for 18 h followed by plating on thiosulfate citrate bile salts sucrose agar and then incubated at 37°C for 18-24 h. The presumptive V. mimicus isolates were confirmed by biochemical characterization and molecularly with vmh gene-specific for V. mimicus. RESULTS: The study revealed that the prevalence of V. mimicus is 5.6% in the total of samples screened. The highest occurrence was observed in brackish water fish (19%) followed by freshwater fish (18%) and marine fish (2%) samples. The study points out the risk of brackish water fishes as potential carriers of this pathogen. This requires preventive measures to mitigate health hazards associated with V. mimicus entering into the seafood production chain.

Tropical shrimp aquaculture farms harbour pathogenic Vibrio parahaemolyticus with high genetic diversity and Carbapenam resistance.

In characterization of food borne pathogens from the environment, assessment of virulence, genetic diversity and AMR are essential preludes to formulate preventive strategies and to combat the spread. This study aimed to identify and characterize pathogenic Vibrio parahaemolyticus in the coastal aquaculture farms of Kerala, India. Twenty-seven ß-haemolytic V. parahaemolyticus were isolated from 7 out of 40 farms studied. Among the 27 isolates, 15 possessed the tdh gene and 4 had trh. ERIC PCR and PFGE illustrated the presence of pathogenic isolates that shared genetic similarity with clinical strains. One pathogenic isolate was identified to be multidrug resistant (MDR) and 59% exhibited a MAR index of 0.2 or above. Seventy four percent of the pathogenic isolates were ESBL producers and 3.7% of them were carbapenemase producers phenotypically. This asks for adoption of control measures during farming to prevent the transmission of pathogenic V. parahaemolyticus to the environment and food chain.

Methicillin-Resistant Staphylococcus aureus in Seafood: Prevalence, Laboratory Detection, Clonal Nature, and Control in Seafood Chain.

Methicillin-resistant Staphylococcus aureus (MRSA), a versatile pathogen bearing multiple virulence determinants, is increasingly being detected in various food-producing animals, including fish. In addition, it is a potential food poisoning agent. MRSA is not an inherent microbiota of fish; its presence is attributed to pre- or postharvest contamination through fish handlers, water, ice, and processing equipment. Several reviews have been written on MRSA in clinical as well as the food animal-producing sector, but information specific to MRSA in seafood is scant. This review puts forth insights on MRSA detection in seafood, antibiotic resistance, diversity of clones in seafood, and possible control measures in seafood production chain. Emphasis has been given on assessing the variations in the protocols employed for isolation and identification in different food matrices and lay the foundation for researchers to develop optimized procedure.