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.

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.

Gamma radiation-induced in vitro hormetic apogamy in the fern Pityrogramma calomelanos (L.) link.

Pityrogramma calomelanos (L.) Link, popularly known as "Silver fern" has significant importance as a medicinal plant used traditionally for its astringent, analgesic, anti-haemorrhagic, anti-hypertensive, anti-pyretic and anthelminthic properties. This fern demonstrates an increased morphogenetic potential towards sporophyte formation, upon exposure to low doses of gamma radiation. Young sporophytic leaf crosier cultures were established in vitro on agar based Knop's media with and without 20 g/l sucrose. The cultures were subjected to 60Co radiations in the range of 2.5-100 Gy. Apospory (production of gametophytes on sporophytic tissue without spores) was observed on leaf tissue cultured on Knops media with and without sucrose in P. calomelanos, at the end of 60 days. 5 Gy treated explants showed high number of aposporous gametophytes and was comparable to the control. Other tested doses reduced the aposporous gametophyte production significantly. In the second phase of the experimentation, the cultures were retained on the gametophyte induction media for a period of 4 weeks. Aposporous gametophytes were observed to proliferate with occasional development of antheridia. At the end of 4 weeks, morphogenetic development on the gametophytic tissue resulted in a significantly higher number of apogamous sporophytes (production of sporophytes without fusion of gametes) were obtained on 5 Gy treated tissue as compared to control and all the other treated explants. Apogamous sporophytes thus produced were successfully grown in the greenhouse and transferred to the field. Thus the use of gamma radiation in vitro not only reduced the need for sucrose for induction of apospory in P.calomelanos, it also exhibited hormesis at 5 Gy for improved sporophyte production.