AMR Threat Perception Assessment of Heterotrophic Bacteria From Shrimp Aquaculture Through Epidemiological Cut off Values.

BACKGROUND: Emergence and dissemination of antibiotic resistance is one of the major risks associated with the rampant usage of antibiotics in food-producing animals including aquaculture. OBJECTIVE: To determine Epidemiological Cut-OFF (ECOFF) values of heterotrophic bacterial populations from shrimp culture environments against five different antibiotics. METHODS: In this present study, bacterial samples were isolated from Penaeus vannamei culture environment in different locations of Andhra Pradesh, which is the aquaculture hub of India. The bacterial isolates were assessed for antibiotic resistance towards five antibiotics belonging to different classes (oxytetracycline, chloramphenicol, erythromycin, ciprofloxacin, and co-trimoxazole) by the disc diffusion method. Determination of Epidemiological Cut-OFF (ECOFF) values and analysis by employing normalized resistance interpretation (NRI) was carried out. RESULTS: The most dominant bacterial populations from shrimp culture were Vibrio spp. (pathogenic bacteria) followed by Bacillus spp. (probiotic bacteria). The bacterial isolates showed highest resistance towards oxytetracycline (overall 23.38%) and in location L6 (59.4%) followed by co-trimoxazole (31.1%). ECOFF values calculated by employing NRI showed that the disc diffusion data were distributed in a normalized manner. The maximum ECOFF value was obtained for ciprofloxacin (23.32 mm), while the minimum value was observed for oxytetracycline (9.05 mm). The antibiotic resistant phenotypes showed that the majority of the heterotrophic bacterial isolates (>60%) belonged to the non-wild type phenotype and primarily towards oxytetracycline (90%). CONCLUSION: The presence of non-wild antibiotic-resistant phenotypes of heterotrophic bacterial populations (which include not only pathogenic bacteria but also probiotic bacteria) indicates that shrimp culture ponds may be a reservoir for drug-resistant bacteria and there is a greater risk associated with transmission of resistant genes across bacterial flora. HIGHLIGHTS: NRI analysis of antibiotic disc diffusion data of heterotrophic bacterial populations in shrimp aquaculture environments revealed that majority of them belonged to non-wild type (90%) paticularly to oxytetracycline in comparison to other studied antibiotics (chloramphenicol, erythromycin, ciprofloxacin and co-trimoxazole).

Multidrug-resistant phenotypes among biofilm-producing staphylococcal isolates from farm-raised fish: a molecular scrutiny.

AIMS: To describe the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from farm-raised fish. METHODS AND RESULTS: Fish samples (n = 180) collected from aquaculture farms in three major districts of Kerala, yielded 45 methicillin-resistant staphylococci (MRS) (25%) isolates. All of the isolates were resistant to tested beta-lactams, and 19 (42.22%) were resistant to flouroquinolones. According to the resistance profile of each isolate, 33 of 45 (73.33%) are identified as multidrug-resistant (MDR) isolates, with an average multiple antibiotic resistance (MAR) index of 0.41 ± 0.09. Notably, virulence determinants such as classical enterotoxin (sea & seb), and SE-like toxin (seg & sei) genes co-existed with Antimicrobial Resistance Genes (ARGs) in isolates studied. Regardless of the isolates being strong biofilm producers, only 23 (51.1%) harbored icaA and icaD genes. The heterogeneity of MRSA (n = 17) clones were evident as the species belonged to three major spa types (t345, t2526, and t1309) and sequence types (STs), viz., ST772, ST88, and ST672. CONCLUSION: The current study on molecular characterization of the MRS isolates sheds light on the preventive measures that need to be implemented to limit the spread of AMR pathogens in aquaculture.

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.

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.

Multi-drug resistance, integron and transposon-mediated gene transfer in heterotrophic bacteria from Penaeus vannamei and its culture environment.

Multi-drug resistance (MDR) in bacteria is regarded as an emerging pollutant in different food production avenues including aquaculture. One hundred and sixty out of 2304 bacterial isolates from shrimp farm samples (n = 192) of Andhra Pradesh, India, were MDR. Based on biochemical identification and 16S rRNA sequencing, they were grouped into 35 bacterial species with the predominance of Vibrio parahaemolyticus (12.5%). The MDR isolates showed highest resistance toward oxytetracycline (89%) with more than 0.2 MAR (multiple antibiotic resistance), demonstrates a high-risk source. The most prevalent antibiotic-resistance gene (ARG) and mobile genetic element (MGE) detected were tetA (47.5%) and int1 (46.2%), respectively. In conjugation experiments, overall transfer frequency was found to be in the range of 1.1 × 10-9 to 1.8 × 10-3 with the transconjugants harbouring ARGs and MGEs. This study exposed the wide distribution of MDR bacteria in shrimp and its environment, which can further aggravate the already raised concerns of antibiotic residues in the absence of proper mitigation measures.

Iron-Enriched Fish Powder Improved Haemoglobin Levels in Adolescent Girls of West Jaintia Hills District of Meghalaya, India.

This study was carried out to assess the efficacy of sodium iron EDTA (NaFeEDTA) salt-enriched fish powder in addressing iron deficiency in adolescent anaemic girls. This was a 60-day randomised double-blinded, controlled intervention trial involving 123 girls age ranging from 10 to 19 years in three villages of West Jaintia Hills District of State of Meghalaya in India using soup made out of sodium iron EDTA (NaFeEDTA)-enriched fish powder (250 mg/100 g). The influence of the iron-enriched powder on blood haemoglobin levels and serum iron was determined. The research also analysed the food consumed by the study subjects during the study period and it was found that there were no significant differences between the iron-enriched and control groups. The results indicated that the girls predominantly consumed cereals with little fruits, vegetables and meat. On an average, 100 ml of soup prepared out of 10 g of fish powder per day was consumed that theoretically provided about 25 mg of iron each day. Following intervention, all the participants in the group that consumed soup made out of NaFeEDTA-enriched fish powder had significantly higher haemoglobin levels and serum iron and a lower prevalence of anaemia than the control group. The effects of NaFeEDTA salt-enriched fish powder were statistically significant and it can be inferred that NaFeEDTA-enriched fish powder was highly effective in controlling iron deficiency and reducing the prevalence of iron-deficiency anaemia among the adolescent girls.

Antibiotic resistance of culturable heterotrophic bacteria isolated from shrimp (Penaeus vannamei) aquaculture ponds.

Shrimp aquaculture is one of the fastest growing food-producing avenues, where antibiotics usage has become an issue of great concern due to the development of antimicrobial resistance in bacteria. A total of 2304 bacterial isolates from 192 samples (sediment, water, shrimp, and source water) from Andhra Pradesh, India were screened. Antibiotic resistance of bacterial isolates was highest for oxytetracycline (23.4%) followed by erythromycin (12.7%), co-trimoxazole (10%) ciprofloxacin (9.6%), and chloramphenicol (6%), of which 11.9% isolates were multi-drug resistant. Bacterial isolates from shrimp (26.7%), water (23.9%), and sediment (19.6%) samples exhibited more resistance (p ≤ 0.05) towards oxytetracycline. Higher antibacterial resistance was observed from samples of southern Andhra Pradesh (locations L6 and L7). Gram negative bacteria were more prevalent (64%) and showed significantly (p ≤ 0.01) higher resistance. This study indicated the wider distribution of antibiotic-resistant bacteria in shrimp aquaculture ponds with potential risk to humans and the environment.

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.

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.