Long-term adaptation of ParA, RelE/ParE partition system, replication protein and phage proteins encoding low-cost plasmids of Escherichia species isolated from diarrheic children of North East India.

AIMS: The prevalent distribution of plasmid-mediated ß-lactam resistance is the most pressing global problem in enteric diseases. The current work aims to characterize plasmid-carrying ß-lactam resistant Enterobacteriaceae isolates from North East India for horizontal gene transfer (HGT) and plasmid adaptation study. METHODS AND RESULTS: In vitro transconjugation and transformation showed overall high conjugation frequency (4.11 × 10-1-9.2 × 10-1) and moderate transformation efficiency/µg DNA (1.02 × 102 -1 × 103), and the highest conjugation frequency (9.2 × 10-1) and transformation efficiency (1 × 103) for Escherichia species S-10. Intra/intergenus plasmid transformation efficiency was highest for the transformation of Klebsiella pneumoniae S-2 to Shigellaflexneri S-42 (1.3 × 103) and lowest for Escherichia species S-10 to Escherichia fergusonii S-30 (2 × 102). In the plasmid stability test, S-10 was detected with the highest plasmid carrying frequency (83.44%) and insignificant segregational loss rate (0.0004) until the 60th day with low plasmid cost on the host. The above findings were also validated by whole-plasmid sequencing of Escherichia species S-10. The genome was identified with two plasmids constituting multiple phage proteins, relaxosomal protein NikA, replication protein RepA, and the plasmid maintenance proteins (ParA, RelE/ParE), thus assisting stable plasmid maintenance. CONCLUSIONS: The results thus indicate that the high conjugation ability and low plasmid fitness cost might lead to horizontal gene transfer of the plasmid to the environment due to their prolonged adaptation in nonselective conditions, intensifying the infection's severity.

Identification of Arg32Ser, His92Tyr and Leu147Phe novel mutations in chromosomally mediated ß-lactamase SHV and in silico characterization to understand their substrate activity imparting resistance.

The emergence of ß-lactam resistance is yearning for clinical significance in Enterobacteriaceae, which are categorized under global priority pathogen lists by the World Health Organization. Likewise, the prevalence of numerous ß-lactamase enzymes, mutational propensity in such bacteria, and their role in accelerating resistance is still a major concern. Thus, the present work intends to characterize the ß-lactamase producing bacteria isolated from acute diarrheal patients to understand their chromosomally acquired resistance pattern through molecular characterization and in silico approaches. The current study highlights the first identified Escherichia fergusonii and Escherichia marmotae species and their ß-lactamase encoding genes, blaOKP-A, blaNDM and blaOXA from the unexplored Enterobacteriaceae family from North East India. First-ever reported point mutations such as Arg32Ser, His92Tyr, and Leu147Phe were observed in BlaSHV protein of two Klebsiella pneumoniae isolates S-35 and S-46. In molecular docking, non-catalytic site H-bond interactions of Arg 218, Ala 223, Asn 128, Ser 126, Gln 95, Asp 100, Tyr 101, Ser 102, Ala 274 with a low binding affinity towards BlaSHV was found. This correlates with the high imipenem, ceftazidime, cefuroxime, ceftriaxone, and cefpodoxime resistance in Klebsiella pneumoniae S-35 with the complementary effect of mutations Arg32Ser and Leu147Phe. Besides, the role of His92Tyr mutation in controlling the resistance in Klebsiella pneumoniae S-46 is also illustrated. Thus, our study highlights the novel mutations of ß-lactamase and its clinical importance with altered resistance profiles. This could be useful to design better therapeutics and to readjust antibiotic treatment regimes against them and control to grow more resistance under selective pressure.Communicated by Ramaswamy H. Sarma.