The CRISPR/Cas system as an antimicrobial resistance strategy in aquatic ecosystems.

With the growing population, demand for food has dramatically increased, and fisheries, including aquaculture, are expected to play an essential role in sustaining demand with adequate quantities of protein and essential vitamin supplements, employment generation, and GDP growth. Unfortunately, the incidence of emerging/re-emerging AMR pathogens annually occurs because of anthropogenic activities and the frequent use of antibiotics in aquaculture. These AMR pathogens include the WHO's top 6 prioritized ESKAPE pathogens (nosocomial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), extended-spectrum beta lactases (ESBLs) and carbapenemase-producing E. coli, which pose major challenges to the biomagnification of both nonnative and native antibiotic-resistant bacteria in capture and cultured fishes. Although implementing the rational use of antibiotics represents a promising mitigation measure, this approach is practically impossible due to the lack of awareness among farmers about the interplay between antimicrobial use and the emergence of antimicrobial resistance (AMR). Nevertheless, to eradicate these 'superbugs,' CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR associate protein) has turned out to be a novel approach owing to its ability to perform precise site-directed targeting/knockdown/reversal of specific antimicrobial resistance genes in vitro and to distinguish AMR-resistant bacteria from a plethora of commensal aquatic bacteria. Along with highlighting the importance of virulent multidrug resistance genes in bacteria, this article aims to provide a holistic picture of CRISPR/Cas9-mediated genome editing for combating antimicrobial-resistant bacteria isolated from various aquaculture and marine systems, as well as insights into different types of CRISPR/Cas systems, delivery methods, and challenges associated with developing CRISPR/Cas9 antimicrobial agents.

Genotyping of Acinetobacter baumannii isolates from a tertiary care hospital in Cochin, South India.

Acinetobacter baumannii poses a significant challenge in healthcare settings across the globe, with isolates exhibiting carbapenem resistance at unprecedented rates. Here, we characterized a collection of A. baumannii isolates (n=64) recovered during the period September 2020 - November 2021 at a teaching hospital in Cochin, South India. The species identity of the isolates was confirmed with bla OXA-51-like PCR. The major carbapenemase determinants identified were bla OXA-23-like (45, 70.3 %) and bla NDM-1 (31, 48.4 %); co-occurrence of these genes was also observed in 27 (42.2 %) isolates. Other resistance genes identified included bla PER (34, 53.1 %), aac(6')-Ib-cr (42, 65.6 %), qnrS (25, 39.1 %), sul1 (32, 50 %), sul2 (33, 51.6 %), strA/strB (36, 56.3 %), aphA1-Iab (35, 54.7 %) and tetB (32, 50 %). Mapping PCR revealed the insertion element, ISAbaI upstream of bla OXA-23-like in all isolates possessing this gene. Concerning disinfectant resistance, all isolates carried the quaternary ammonium compound (QAC) resistance gene, qacEΔ1. Minimal inhibitory concentration (MIC) of benzalkonium chloride was high among the isolates and ranged from 8 to 128 µg ml-1. However, low MICs were observed for chlorhexidine and triclosan, with the majority (54, 80.6 %) of isolates showing an MIC of 2 µg ml-1 for chlorhexidine and all isolates exhibiting MICs of ≤0.125 µg ml-1 for triclosan. Further, all isolates were strong biofilm-producers, as assessed by the crystal violet-based microtitre plate assay. The ApaI-pulsed-field gel electrophoresis (PFGE) revealed the multi-clonal nature of the isolates, with 16 clusters and 16 unique pulsotypes identified at a cut-off of 80 %. In short, this study provides useful data on the molecular features of A. baumannii from this region, which could be helpful to assess the local epidemiology of this pathogen and also to devise infection control strategies.

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.

Resistance profiles and genotyping of extended-spectrum beta-lactamase (ESBL) -producing and non-ESBL-producing E. coli and Klebsiella from retail market fishes.

Studies on antimicrobial resistance (AMR) profiles and epidemiological affirmation for AMR transmission are limited in fisheries and aquaculture settings. Since 2015, based on Global Action Plan on AMR by World Health Organization (WHO) and World Organization for Animal Health (OIE), several initiatives have been under taken to enhance the knowledge, skills and capacity to establish AMR trends through surveillance and strengthening of epidemiological evidence. The focus of this study was to determine the prevalence of antimicrobial resistance (AMR), its resistance profiles and molecular characterization with respect to phylogroups, antimicrobial resistance genes (ARGs), virulence genes (VGs), quaternary ammonium compounds resistance (QAC) genes and plasmid typing in retail market fishes. Pulse field gel electrophoresis (PFGE) to understand the genetic lineage of the two most important Enterobacteriaceae members, E. coli and Klebsiella sp. was performed. 94 fish samples were collected from three different sites viz., Silagrant (S1), Garchuk (S2) and North Guwahati Town Committee (NGTC) Region (S3) in Guwahati, Assam. Out of the 113 microbial isolates from the fish samples, 45 (39.82%) were E. coli; 23 (20.35%) belonged to Klebsiella genus. Among E. coli, 48.88% (n = 22) of the isolates were alerted by the BD Phoenix M50 instrument as ESBL, 15.55% (n = 7) as PCP and 35.55% (n = 16) as non-ESBL. E. coli (39.82%) was the most prevalent pathogen among the Enterobacteriaceae members screened and showed resistance to ampicillin (69%) followed by cefazoline (64%), cefotaxime (49%) and piperacillin (49%). In the present study, 66.66% of E. coli and 30.43% of Klebsiella sp. were categorized as multi drug resistance (MDR) bacteria. CTX-M-gp-1, with CTX-M-15 variant (47%), was the most widely circulating beta-lactamase gene, while other ESBL genes blaTEM (7%), blaSHV (2%) and blaOXA-1-like (2%) were also identified in E. coli. Out of the 23 isolates of Klebsiella, 14(60.86%) were ampicillin (AM)-resistant (11(47.82%) K. oxytoca, 3(13.04%) K. aerogenes), whereas 8(34.78%) isolates of K. oxytoca showed intermediate resistance to AM. All Klebsiella isolates were susceptible to AN, SCP, MEM and TZP, although two K. aerogenes were resistant to imipenem. DHA and LAT genes were detected, respectively, in 7(16%) and 1(2%) of the E. coli strains while a single K. oxytoca (4.34%) isolate carried MOX, DHA and blaCMY-2 genes. The fluoroquinolone resistance genes identified in E. coli included qnrB (71%), qnrS (84%), oqxB (73%) and aac(6)-Ib-cr (27%); however, in Klebsiella, these genes, respectively, had a prevalence of 87%, 26%, 74% and 9%. The E. coli isolates belonged to phylogroup A(47%), B1(33%) and D(14%). All of the 22(100%) ESBL E. coli had chromosome-mediated disinfectant resistance genes viz., ydgE, ydgF, sugE(c), mdfA while 82% of ESBL E. coli had emrE. Among the non-ESBL E. coli isolates, 87% of them showed the presence of ydgE, ydgF and sugE(c) genes, while 78% of the isolates had mdfA and 39% had emrE genes respectively. 59% of the ESBL and 26% of the non-ESBL E. coli had showed the presence of qacEΔ1. The sugE(p) was present in 27% of the ESBL-producing E. coli and in 9% of non-ESBL isolates. Out of the 3 ESBL-producing Klebsiella isolates, 2(66.66%) K. oxytoca isolates were found harboring plasmid-mediated qacEΔ1 gene while one (33.33%) K. oxytoca isolate had sugE(p) gene. IncFI was the most prevalent plasmid type detected in the isolates studied, with A/C (18%), P (14%), X, Y (9% each) and I1-Iγ (14%, 4%). 50% (n = 11) of the ESBL and 17% (n = 4) of the non-ESBL E. coli isolates harboured IncFIB and 45% (n = 10) ESBL and one (4.34%) non-ESBL E. coli isolates harboured IncFIA. Dominance of E. coli over other Enterobacterales and diverse phylogenetic profiles of E. coli and Klebsiella sp. suggests the possibility of contamination and this may be due to compromised hygienic practices along the supply chain and contamination of aquatic ecosystem. Continuous surveillance in domestic markets must be a priority in addressing antimicrobial resistance in fishery settings and to identify any unwarranted epidemic clones of E. coli and Klebsiella that can challenge public health sector.

Prevalence of S. aureus and/or MRSA from seafood products from Indian seafood products.

Compared to the clinical sector, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the food sector is relatively low. However, their presence in seafood is a significant public health concern. In India, fish and fishery products are maximally manually handled compared to other food products. In this study, 498 fish samples were collected under various conditions (fresh, chilled or dressed) and representatives from their surroundings. These samples were screened for the prevalence of Staphylococcus aureus, determining its antimicrobial resistance, MRSA and genetic profile. It is observed that 15.0% and 3.0% of the total samples were screened positive for S. aureus and MRSA, respectively. The S. aureus strain MRSARF-10 showed higher resistance to linezolid, co-trimoxazole, cefoxitin, ofloxacin, gentamicin, rifampicin, ampicillin/sulbactam and Piperacillin-tazobactam. This MRSA, spa type t021 and SCCmec type V strain isolated from dried ribbon fish (Family Trachipteridae) carried virulence factors for exoenzymes such as aureolysin, serine, toxin genes and a novel MLST ST 243, as revealed from its draft-genome sequence. This highly pathogenic, multidrug-resistant and virulent S. aureus novel strain is circulating in the environment with chances of spreading among the seafood workers and the environment. It is further suggested that Good Hygienic Practices recommended by World Health Organization need to be followed during the different stages of seafood processing to provide pathogen-free fish and fishery products to the consumers.

Genomic insights of beta-lactamase producing Klebsiella quasipneumoniae subsp. similipneumoniae belonging to sequence type 1699 from retail market fish, India.

Klebsiella quasipneumoniae is a recently described species and often misidentified as Klebsiella pneumoniae. Here, we report the genomic characterization of Klebsiella quasipneumoniae subsp. similipneumoniae (India238 strain) isolated from fish. The annotated genome acknowledged the presence of blaCTX-M-15, blaOKP-B-1, fosA5, oqxAB and virulence genes. The strain with ST1699 and serotypes KL52 and OL103 also harboured insertion sequences (ISs): ISKpn26 and ISEc9. Three complete phage genomes were identified in contigs 1 and 6 of the bacterial genome, enhancing the prospects of genome manipulation. The study highlights the pitfall of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae. This is the first Indian study documenting the incidence of extended-spectrum beta-lactamase (ESBL)-producing K. quasipneumoniae subsp. similipneumoniae from a non-clinical environment, equipped with virulomes and associated mobile genetic elements. Given that fish can act as a potential vector for transmission of antimicrobial resistance genes, our findings have paramount importance on human health.

Genotypes and phenotypes of methicillin-resistant staphylococci isolated from shrimp aquaculture farms.

The population of methicillin-resistant (MR) staphylococci in aquatic environment is rarely investigated. Here, we characterized a collection of MR staphylococci recovered from shrimp aquaculture farms (n = 37) in Kerala, India. A total of 261 samples yielded 47 MR isolates (16 S. aureus, 13 S. haemolyticus, 11 S. epidermidis, 3 S. saprophytics and 2 each of S.intermedius and S. kloosii). Multi-drug resistance was evident in 72.3% of the isolates, with resistance mainly towards erythromycin (78.7%), norfloxacin and trimethoprim-sulfamethoxazole (53.2%), and gentamicin (34%). Major resistance genes identified included mecA (100%), ermC (38.3%), aacA-aphD (21.3%), tetK (14.9%) and tetM (21.3%). Almost 60% of the isolates carried type V SCCmec (Staphylococcal Cassette Chromosome mec), and the remaining harboured untypeable SCCmec elements. Comprehensive genotyping of the methicillin-resistant Staphylococcus aureus isolates revealed high prevalence of ST772-t345-V (sequence type-spa type-SCCmec type) (75%), followed by minor representations of ST6657-t345-V and ST3190-t12353. The isolates of S. haemolyticus and S. epidermidis were genotypically diverse as shown by their pulsed-field gel electrophoresis (PFGE) profiles. Genes encoding staphylococcal enterotoxins were observed in 53.2% of the isolates. Various genes involved in adhesion and biofilm formation were also identified. In conclusion, our findings provide evidence that shrimp aquaculture settings can act as reservoirs of methicillin-resistant staphylococci.

Antibiotic Resistance Profiles and Molecular Characteristics of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli and Klebsiella pneumoniae Isolated From Shrimp Aquaculture Farms in Kerala, India.

This study was undertaken to evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae in selected shrimp aquaculture farms (n = 37) in Kerala, South India and to characterize the isolates using molecular tools. Overall, a low prevalence of ESBL-producers was found in the farms, most likely due to the reduced antibiotic usage in the shrimp farming sector. Out of the 261 samples (77 shrimp and 92 each of water and sediment), 14 (5.4%) tested positive for ESBL-E. coli or ESBL-K. pneumoniae. A total of 32 ESBL-E. coli and 15 ESBL- K. pneumoniae were recovered from these samples. All ESBL isolates were cefotaxime-resistant with minimal inhibitory concentration (MIC) ≥32 µg/ml. Of all isolates, 9 (28.1%) E. coli and 13 (86.7%) K. pneumoniae showed simultaneous resistance to tetracycline, ciprofloxacin, and trimethoprim-sulfamethoxazole. PCR analysis identified CTX-M group 1 (bla CTX-M-15 ) as the predominant ESBL genotype in both E. coli (23, 71.9%) and K. pneumoniae (15, 100%). Other beta-lactamase genes detected were as follows: bla TEM and bla SHV (11 K. pneumoniae), bla CTX-M group 9 (9 E. coli), and bla CMY-2 (2 E. coli). Further screening for AMR genes identified tetA and tetB (13, 40.6%), sul1 (11, 34.4%), sul2 (9, 28.1%), catA and cmlA (11, 34.4%), qepA and aac(6')-Ib-cr (9, 28.1%) and strAB and aadA1 (2, 6.3%) in E. coli, and qnrB (13, 86.7%), qnrS (3, 20%), oqxB (13, 86.7%), tetA (13, 86.7%), and sul2 (13, 86.7%) in K. pneumoniae isolates. Phylogenetic groups identified among E. coli isolates included B1 (4, 12.5%), B2 (6, 18.8%), C (10, 31.3%), D (3, 9.4%), and E (9, 28.1%). PCR-based replicon typing (PBRT) showed the predominance of IncFIA and IncFIB plasmids in E. coli; however, in K. pneumoniae, the major replicon type detected was IncHI1. Invariably, all isolates of K. pneumoniae harbored virulence-associated genes viz., iutA, entB, and mrkD. Epidemiological typing by pulsed-field gel electrophoresis (PFGE) revealed that E. coli isolates recovered from different farms were genetically unrelated, whereas isolates of K. pneumoniae showed considerable genetic relatedness. In conclusion, our findings provide evidence that shrimp aquaculture environments can act as reservoirs of multi-drug resistant E. coli and K. pneumoniae.

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.

Edible Mushrooms as Functional Ingredients for Development of Healthier and More Sustainable Muscle Foods: A Flexitarian Approach.

Consumers are increasingly interested in nutritious, safe and healthy muscle food products with reduced salt and fat that benefit their well-being. Hence, food processors are constantly in search of natural bioactive ingredients that offer health benefits beyond their nutritive values without affecting the quality of the products. Mushrooms are considered as next-generation healthy food components. Owing to their low content of fat, high-quality proteins, dietary fibre and the presence of nutraceuticals, they are ideally preferred in formulation of low-caloric functional foods. There is a growing trend to fortify muscle food with edible mushrooms to harness their goodness in terms of nutritive, bioactive and therapeutic values. The incorporation of mushrooms in muscle foods assumes significance, as it is favourably accepted by consumers because of its fibrous structure that mimics the texture with meat analogues offering unique taste and umami flavour. This review outlines the current knowledge in the literature about the nutritional richness, functional bioactive compounds and medicinal values of mushrooms offering various health benefits. Furthermore, the effects of functional ingredients of mushrooms in improving the quality and sensory attributes of nutritionally superior and next-generation healthier muscle food products are also highlighted in this paper.