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Background Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. Results Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. Conclusion The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.

Colistin resistance has increased due to the increasing and inappropriate use of this antibiotic. The mechanism involves modification of lipid A with phosphoethanolamine (PEtN) and/or 4-amino-4deoxy- l -arabinose (L-Ara4N). EptA and eptB catalyze the transfer of phosphoethanolamine to lipid A. In this study, gene network was constructed to find the associated genes related to colistin resistance, and further in vitro validation by transcriptional analysis was performed. In silico studies showed that eptB gene is a highly interconnected node in colistin resistance gene network. To ascertain these findings twelve colistin-resistant clinical isolates of Escherichia coli were selected in which five were harboring the plasmid-mediated mcr -1. Screening for colistin resistance was performed by broth microdilution (BMD) method and Rapid polymyxin NP test. PCR confirmed the presence of the eptA and eptB genes in all isolates and five isolates were harboring mcr -1. Transcriptional expression in five isolates harboring mcr -1, showed an enhanced expression of eptB when exposed under sub-inhibitory colistin stress. The present study for the first time highlighted genetic interplay between mcr -1 and eptA and eptB under colistin exposure.

Dissemination of class D OXA-type carbapenemases is one of the significant causes of beta-lactam resistance in Gram-negative bacteria. The amino acid residues present near the active site are involved in hydrolytic mechanism of class D carbapenemases, though it is not identified in OXA-23. Here, with the help of site-directed mutagenesis, we aimed to explicate the importance of the residues W165, L166 and V167 of the possible omega loop and residue D222 in the short β5-β6 loop on the activity of OXA-23. All the residues were substituted with alanine. The resultant proteins were assayed for the changes in activity in E. coli cells and purified for in vitro activity, and stability assessment. E. coli cells harboring OXA-23_W165A and OXA-23_L166A, individually, exhibited a significant decrease in resistance towards beta-lactam antibiotics as compared to OXA-23. Further, purified OXA-23_W165A and OXA-23_L166A imparted about >4-fold decrease in catalytic efficiency and displayed reduced thermal stability as compared to OXA-23. Bocillin-FL binding assay revealed that W165A substitution results in improper N-carboxylation of K82, leading to deacylation deficient OXA-23. Therefore, we infer that the residue W165 maintains the integrity of N-carboxylated lysine (K82) of OXA-23 and the residue L166 might be responsible for properly orientating the antibiotic molecules.

The recent emergence of multidrug-resistant (MDR) Klebsiella pneumoniae with hypervirulent traits causing severe infections and considerable mortality is a global cause for concern. The challenges posed by these hypermucoviscous strains of K. pneumoniae with regard to their optimal treatment, management, and control policies are yet to be answered. We studied a series of extensively drug-resistant (XDR) and hypervirulent K. pneumoniae ST5235 isolates with resistance to carbapenems and polymyxins causing neonatal sepsis in a tertiary care hospital in India. A total of 9 K. pneumoniae isolates from 9 cases of neonatal sepsis were studied with respect to their clinical relevance, antimicrobial susceptibility profile, presence of extended spectrum β lactamase (ESBL) production, and responsible genes, carbapenemases (classes A, B, and D), and aminoglycoside-resistant genes. Hypervirulence genes encoding hypermucoid nature, iron uptake, and siderophores were detected by multiplex PCR. The plasmid profile was studied by replicon typing. Isolates were typed by multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC) PCR to study the sequence types (STs) and clonal relation, respectively. The neonates in the studied cases had history of pre-maturity or low birth weight with maternal complications. All the cases were empirically treated with piperacillin–tazobactam and amikacin followed by imipenem/meropenem and vancomycin and polymyxin B as a last resort. However, all the neonates finally succumbed to the condition (100%). The studied isolates were XDR including resistance to polymyxins harboring multiple ESBL genes and carbapenemase genes ( bla NDM and bla OXA −48 ). Hypervirulence genes were present in various combinations with rmpA/A2 genes present in all the isolates. IncFI plasmids were detected in these isolates. All belonged to ST5235. In ERIC PCR, 6 different clusters were seen. The study highlighted the emergence and burden of XDR hypervirulent isolates of K. pneumoniae causing neonatal sepsis in a tertiary care hospital.

Abstract Background Carbapenem-Resistant Enterobacterales (CRE) has been categorized as pathogens of critical priority by World Health organization (WHO) as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion bla OXA−78 and bla OXA−58 of in CRE of clinical origin. Methods Bacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of bla OXA−58 and bla OXA−78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of bla OXA−58 and bla OXA−78 genes and further characterization of the CRE isolates. Results The carbapenem-resistant Escherichia coli (BJD_(E)C456) and Serratia marcescens (BJD_(S)M81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor bla OXA−58 and bla OXA−78, a variant of bla OXA−51. Horizontal transfer of bla OXA−58 and bla OXA−78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to bla OXA−78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of bla OXA−58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_(E)C180 belonged to ST2437 while BJD_(S)M81 was of an unknown sequence type. The nucleotide sequences of bla OXA−78 (OQ533021) and bla OXA−58 (OQ533022) have been deposited in GenBank. Conclusions The study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated bla OXA−78 and bla OXA−58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Objective The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. Results 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

Objective This study was designed to discover the dissemination of virulence genes in Methicillin-resistant Staphylococcus aureus from clinical, community and environmental settings . Results This study includes 1165 isolates collected from hospital, community and environmental settings . Among them sixty three were confirmed as MRSA with varied SCC mec types viz; type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII. The virulence gene such as sea (n = 54), seb (n  = 21), eta (n  = 27), etb (n  = 2), cna (n  = 24), ica (n = 2) and tst (n = 30) was also revealed from this study. The study underscores coexistence of resistance cassette and virulence genes among clinical and environment isolates which is first of its kind from this part of the world.

Objective This study was designed to investigate the transcriptional response of OmpF and OmpC along with an antisense RNA, MicF under concentration gradient carbapenem exposure. Result An elevation in the expression of OmpF gene under concentration gradient imipenem stress from a particular concentration was observed. For OmpC gene a significant decrease in the expression was noticed under concentration gradient imipenem and meropenem stress. The study showed reduction in the expression of OmpC gene against imipenem and meropenem possibly preventing the entry of carbapenem antibiotic inside the cell indicating a possible role in carbapenem resistance.

Global spread of KPC poses to be a serious threat complicating treatment options in hospital settings. The present study investigates the genetic environment of blaKPC-2 among clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of India. The study isolates were collected from different wards and clinics of Silchar Medical College and Hospital, India, from 2012-2013. The presence of blaKPC was confirmed by genotypic characterization followed by sequencing. Cloning of the blaKPC-2 gene was performed and the genetic environment of this gene was characterized as well. Transferability of the resistance gene was determined by transformation assay and Southern hybridization. Additionally, restriction mapping was also carried out. Two isolates of P. aeruginosa were found to harbor blaKPC-2, were resistant towards aminoglycosides, quinolone and β-lactam-β-lactamase inhibitor combination. In both the isolates, the resistance determinant was associated with class 1 integron and horizontally transferable. Both the isolates were co-harboring blaNDM-1. The first detection of this integron mediated blaKPC-2 coexisting with blaNDM-1 in P. aeruginosa from India is worrisome, and further investigation is required to track the gene cassette mediated blaKPC-2 in terms of infection control and to prevent the spread of this gene in hospitals as well as in the community.

Background This study aimed to identify ten different 16S rRNA methyltransferase genes ( rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. Methods A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with β-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. Results A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only bla CTX-M-15 , whereas combination of genes were observed in three isolates ( bla VEB + bla CTX-M-15 in 2 isolates and bla PER  +  bla CTX-M-15 in 1 isolate). bla NDM and bla OXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coli ST4410, ST1341 and ST3906. Conclusion The present study identified emergence of three clones of E.coli , resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.