Gut resident Escherichia coli profile predicts the eighteen-month probability and antimicrobial susceptibility of urinary tract infections.

Background: Community-acquired UTI is the most common bacterial infection managed in general medical practice that can lead to life-threatening outcomes. While UTIs are primarily caused by Escherichia coli colonizing the patient's gut, it is unclear whether the gut resident E. coli profiles can predict the person's risks for UTI and optimal antimicrobial treatments. Thus, we conducted an eighteen-month long community-based observational study of fecal E. coli colonization and UTI in women aged 50 years and above. Methods and Findings: We enrolled a total of 1,804 women distributed among age groups 50-59 yo (437 participants), 60-69 yo (632), 70-79 yo (532), and above 80 yo (203), lacking antibiotic prescriptions for at least one year. The provided fecal samples were plated for the presence of E. coli and other enterobacteria resistant to trimethoprim/sulfamethoxazole (TMP/STX), ciprofloxacin (CIP) and 3rd generation cephalosporins (3GC). E. coli was also characterized as belonging to the pandemic multi-drug resistant clonal groups ST131 (subclone H30) and ST1193. Following sample collection, the women were monitored for 18 months for occurrence of UTI.E. coli was cultured from 90.8% fecal samples, with 24.1% containing bacteria resistant to TMP/STX, 19.4% to CIP, and 7.9% to 3GC. In 62.5% samples, only all-susceptible E. coli were present. Overall, there were no age-related differences in resistance prevalence. However, while the total E. coli H30 and ST1193 carriage rates were similar (4.3% and 4.2%, respectively), there was a notable increase of H30 carriage with age (P = .001), while carriage decreased with age for ST1193 (P = .057).Within 18 months, 184 women (10.2%) experienced at least one episode of UTI - 10.9% among the gut E. coli carriers and 3.0% among the non-carriers (P=.0013). The UTI risk among carriers of E. coli H30 but not ST1193 was significantly above average (24.3%, P = .0004). The UTI probability increased with age, occurring in 6.4% of 50-59 yo and 19.7% of 80+ yo (P<.001), with the latter group being especially at high risk for UTI, if they were colonized by E. coli H30 (40.0%, P<.001).E. coli was identified in 88.1% of urine samples, with 16.1% resistant to TMP/STX, 16.1% to CIP, 4.2% to 3GC and 73.1% to none of the antibiotics. Among tested urinary E. coli resistant to antibiotics, 86.1% matched the resistance profile of E. coli in the fecal samples, with the clonotyping and whole genome sequencing confirming the matching strains' identity. Positive predictive value (PPV) of using gut resistance profiles to predict UTI pathogens' susceptibility to TMP/STX, CIP, 3GC and all three antibiotics were 98.4%, 98.3%, 96.6% and 95.3%, respectively. Corresponding negative predictive values (NPV) were 63.0%, 54.8%, 44.4% and 75.8%, respectively. The AUC ROC curve values for the accuracy of fecal diagnostic testing for the prediction of UTI resistance ranged .86-.89. The fecal test-guided drug-bug mismatch rate for empirical (pre-culture) prescription of TMP-SXT or CIP is reduced to ≤2% in 89.6% of patients and 94.8% of patients with an optional 3GC prescription. Conclusion: The resistance profile and clonal identity of gut colonizing E. coli, along with the carrier's age, can inform personalized prediction of a patients' UTI risk and the UTI pathogen's antibiotic susceptibility within an 18-month period.

Increase in the community circulation of ciprofloxacin-resistant Escherichia coli despite reduction in antibiotic prescriptions.

BACKGROUND: Community circulating gut microbiota is the main reservoir for uropathogenic Escherichia coli, including those resistant to antibiotics. Ciprofloxacin had been the primary antibiotic prescribed for urinary tract infections, but its broad use has been discouraged and steadily declined since 2015. How this change in prescriptions affected the community circulation of ciprofloxacin-resistant E. coli is unknown. METHODS: We determined the frequency of isolation and other characteristics of E. coli resistant to ciprofloxacin in 515 and 1604 E. coli-positive fecal samples collected in 2015 and 2021, respectively. The samples were obtained from non-antibiotic-taking women of age 50+ receiving care in the Kaiser Permanente Washington healthcare system. RESULTS: Here we show that despite a nearly three-fold drop in the prescription of ciprofloxacin between 2015 and 2021, the rates of gut carriage of ciprofloxacin-resistant E. coli increased from 14.2 % to 19.8% (P = .004). This is driven by a significant increase of isolates from the pandemic multi-drug resistant clonal group ST1193 (1.7% to 4.2%; P = .009) and isolates with relatively few ciprofloxacin-resistance determining chromosomal mutations (2.3% to 7.4%; P = .00003). Though prevalence of isolates with the plasmid-associated ciprofloxacin resistance dropped (59.0% to 30.9%; P = 2.7E-06), the isolates co-resistance to third generation cephalosporins has increased from 14.1% to 31.5% (P = .002). CONCLUSIONS: Despite reduction in ciprofloxacin prescriptions, community circulation of the resistant uropathogenic E. coli increased with a rise of co-resistance to third generation cephalosporins. Thus, to reduce the rates of urinary tract infections refractory to antibiotic treatment, greater focus should be on controlling the resistant bacteria in gut microbiota.

The alarming rise of bacteria causing infections that are difficult to treat with antibiotics, known as multidrug-resistant bacteria, is a major problem in medicine. The reduction in the use of antibiotics has been encouraged to control the spread of antibiotic-resistant bacteria. Some multidrug-resistant bacteria reside in the gut of healthy individuals and can cause various forms of urinary tract infections (UTIs). Ciprofloxacin is an antibiotic that was widely used to treat UTIs, but strong recommendations to reduce its prescription have been recently introduced. We compared the presence of bacteria in the gut that could not be killed by ciprofloxacin in women aged 50 and above who do not use antibiotics and reside in the Seattle area. Despite a nearly three-fold drop in the prescription of ciprofloxacin between 2015 and 2021, antibiotic-resistant bacteria in the gut were found more frequently, affecting one in five women. Our study demonstrates that antibiotic-resistant bacteria continue to be present even when antibiotic prescriptions are reduced, demonstrating the need to undertake further similar studies.

Increase in the Rate of Gut Carriage of Fluoroquinolone-Resistant Escherichia coli despite a Reduction in Antibiotic Prescriptions.

Background : Fluoroquinolone use for urinary tract infections has been steadily declining. Gut microbiota is the main reservoir for uropathogenic Escherichia coli but whether the carriage of fluoroquinolone-resistant E. coli has been changing is unknown. Methods . We determined the frequency of isolation and other characteristics of E. coli nonsuceptible to fluoroquinolones (at ³0.5 mg/L of ciprofloxacin) in 515 and 1605 E. coli -positive fecal samples collected in 2015 and 2021, respectively, from non-antibiotic- taking women of age 50+ receiving care in the Seattle area Kaiser Permanente Washington healthcare system. Results . Between 2015 and 2021 the prescription of fluoroquinolones dropped nearly three-fold in the study population. During the same period, the rates of gut carriage of fluoroquinolone-resistant E. coli increased from 14.4 % to 19.9% (P=.005), driven by a significant increase of isolates from the recently emerged, pandemic multi-drug resistant clonal group ST1193 (1.7% to 4.3%; P=.007) and those with an incomplete set of or no fluoroquinolone-resistance determining mutations (2.3% to 7.5%; P<.001). While prevalence of the resistance-associated mobile genes among the isolates dropped from 64.1% to 32.6% (P<.001), co-resistance to third generation cephalosporins has increased 21.5% to 33.1%, P=.044). Conclusion . Despite reduction in fluoroquinolone prescriptions, gut carriage of fluoroquinolone-resistant uropathogenic E. coli increased with a rise of previously sporadic lineages and co-resistance to third generation cephalosporins. Thus, to reduce the rates of antibiotic resistant urinary tract infections, greater focus should be on controlling the gut carriage of resistant bacteria.

Acquisition of the L452R Mutation in the ACE2-Binding Interface of Spike Protein Triggers Recent Massive Expansion of SARS-CoV-2 Variants.

We report that there is a recent global expansion of numerous independent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with mutation L452R in the receptor-binding domain (RBD) of the spike protein. The massive emergence of L452R variants was first linked to lineage B.1.427/B.1.429 (clade 21C) that has been spreading in California since November and December 2020, originally named CAL.20C and currently variant of interest epsilon. By PCR amplification and Sanger sequencing of a 541-base fragment coding for amino acids 414 to 583 of the RBD from a collection of clinical specimens, we identified a separate L452R variant that also recently emerged in California but derives from the lineage B.1.232, clade 20A (named CAL.20A). Notably, CAL.20A caused an infection in gorillas in the San Diego Zoo, reported in January 2021. Unlike the epsilon variant that carries two additional mutations in the N-terminal domain of spike protein, L452R is the only mutation found in the spike proteins of CAL.20A. Based on genome-wide phylogenetic analysis, emergence of both viral variants was specifically triggered by acquisition of L452R, suggesting a strong positive selection for this mutation. Global analysis revealed that L452R is nearly omnipresent in a dozen independently emerged lineages, including the most recent variants of concern/interest delta, kappa, epsilon and iota, with the lambda variant carrying L452Q. L452 is in immediate proximity to the angiotensin-converting enzyme 2 (ACE2) interaction interface of RBD. It was reported that the L452R mutation is associated with immune escape and could result in a stronger cell attachment of the virus, with both factors likely increasing viral transmissibility, infectivity, and pathogenicity.

Acquisition of the L452R mutation in the ACE2-binding interface of Spike protein triggers recent massive expansion of SARS-Cov-2 variants.

The recent rise in mutational variants of SARS-CoV-2, especially with changes in the Spike protein, is of significant concern due to the potential ability for these mutations to increase viral infectivity, virulence and/or ability to escape protective antibodies. Here, we investigated genetic variations in a 414-583 amino acid region of the Spike protein, partially encompassing the ACE2 receptor-binding domain (RBD), across a subset of 570 nasopharyngeal samples isolated between April 2020 and February 2021, from Washington, California, Arizona, Colorado, Minnesota and Illinois. We found that samples isolated since November have an increased number of amino acid mutations in the region, with L452R being the dominant mutation. This mutation is associated with a recently discovered CAL.20C viral variant from clade 20C, lineage B.1.429, that since November-December 2020 is associated with multiple outbreaks and is undergoing massive expansion across California. In some samples, however, we found a distinct L452R-carrying variant of the virus that, upon detailed analysis of the GISAID database genomes, is also circulating primarily in California, but emerged even more recently. The newly identified variant derives from the clade 20A (lineage B.1.232) and is named CAL.20A. We also found that the SARS-CoV-2 strain that caused the only recorded case of infection in an ape - gorillas in the San Diego Zoo, reported in January 2021 - is CAL.20A. In contrast to CAL.20C that carries two additional to L452R mutations in the Spike protein, L452R is the only mutation found in CAL.20A. According to the phylogenetic analysis, however, emergence of CAL.20C was also specifically triggered by acquisition of the L452R mutation. Further analysis of GISAID-deposited genomes revealed that several independent L452R-carrying lineages have recently emerged across the globe, with over 90% of the isolates reported between December 2020 - February 2021. Taken together, these results indicate that the L452R mutation alone is of significant adaptive value to SARS-CoV-2 and, apparently, the positive selection for this mutation became particularly strong only recently, possibly reflecting viral adaptation to the containment measures or increasing population immunity. While the functional impact of L452R has not yet been extensively evaluated, leucine-452 is positioned in the receptor-binding motif of RBD, in the interface of direct contact with the ACE2 receptor. Its replacement with arginine is predicted to result in both a much stronger binding to the receptor and escape from neutralizing antibodies. If true, this in turn might lead to significantly increased infectivity of the L452R variants, warranting their close surveillance and in-depth functional studies.

Cyathea gigantea (Cyatheaceae) as an antimicrobial agent against multidrug resistant organisms.

BACKGROUND: Rapid emergence of multidrug resistant (MDR) organisms in hospital and community settings often result into treatment failure, thus leading the clinicians with fewer treatment options. Cyathea gigantea, an ethnomedicinally important fern used in cuts and wound infections. So, if this medicinal plant is used in treating the MDR infections then it might bring certain relief in future treatment options. METHODS: Antibacterial activity of C. gigantea against MDR bacteria was assed using well diffusion and broth microdilution methods to determine the diameters of growth inhibition zones, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Synergistic activity was also determined with the conventional antibiotics by disc diffusion method followed by FIC index of each of the tested antibiotic was calculated. The active extract was then subjected to fractionation by column chromatography and antibacterial activity was done with each of the collected fractions. RESULTS: Crude extract of C. gigantea was found to be active against all the tested organisms. The MIC was 200 µg/ml against Gram-positive i.e., Staphylococcus aureus ATCC 25923 and 400 µg/ml against Gram-negative i.e., Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PAO1, while the MBC was 400 µg/ml in case of Gram-positive and 800 µg/ml for Gram-negative. The synergistic activity revealed that the plant extract increased the antibacterial property of the studied antibiotics and the FIC index showed that significant synergistic activity was shown by ciprofloxacin followed by tetracycline, ampicillin and oxacillin. Antibacterial activity with the fractionated extract showed that the FR II, FR III and FR IV were active against both Gram-positive and Gram-negative bacteria, whereas FR I, FR V and FR VI did not show antibacterial property against any of the tested bacteria. CONCLUSIONS: Extracts of C. gigantea was found active against both selected Gram-positive and Gram-negative organisms and thus offers the scientific basis for the traditional use of the fern. The present study also provides the basis for future study to validate the possible use against multidrug resistant organisms.

Expansion of highly stable bla OXA-10 ß-lactamase family within diverse host range among nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of Northeast India.

BACKGROUND: The current study reports dissemination of highly stable bla OXA-10 family of beta lactamases among diverse group of nosocomial isolates of Gram-negative bacilli within a tertiary referral hospital of the northern part of India. METHODS: In the current study, a total number of 590 Gram negative isolates were selected for a period of 1 year (i.e. 1st November 2011-31st October 2012). Members of Enterobacteriaceae and non fermenting Gram negative rods were obtained from Silchar Medical College and Hospital, Silchar, India. Screening and molecular characterization of ß-lactamase genes was done. Integrase gene PCR was performed for detection and characterization of integrons and cassette PCR was performed for study of the variable regions of integron gene cassettes carrying bla OXA-10. Gene transferability, stability and replicon typing was also carried out. Isolates were typed by ERIC as well as REP PCR. RESULTS: Twenty-four isolates of Gram-negative bacilli that were harboring bla OXA-10 family (OXA-14, and OXA16) with fact that resistance was to the extended cephalosporins. The resistance determinant was located within class I integron in five diverse genetic contexts and horizontally transferable in Enterobacteriaceae, was carried through IncY type plasmid. MIC values were above break point for all the tested cephalosporins. Furthermore, co-carriage of bla CMY-2 was also observed. CONCLUSION: Multiple genetic environment of bla OXA-10 in this geographical region must be investigated to prevent dissemination of these gene cassettes within bacterial population within hospital settings.

Carbapenem nonsusceptibility with modified OprD in clinical isolates of Pseudomonas aeruginosa from India.

This study was undertaken to investigate OprD porin-mediated carbapenem nonsusceptibility in clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital of Northeast India. A total of 267 nonduplicate, consecutive clinical isolates of P. aeruginosa were obtained. Mutation and expression levels of OprD gene were determined in carbapenem-nonsusceptible carbapenemase-nonproducing isolates. Among 19 carbapenem-nonsusceptible carbapenemase-nonproducing isolates, 11 of them demonstrated variable band pattern while performing denaturing gradient gel electrophoresis with amplified products of OprD gene. Sequencing of variable band products revealed three mutation patterns in three isolates. Relevant decrease in expression of OprD gene could also be observed in them. All the three isolates exhibited a higher minimum inhibitory concentration for imipenem (64-128 µg/mL) compared to meropenem (16-64 µg/mL). Inactivating mutation and decreased expression of OprD contribute mainly to imipenem resistance as well as to meropenem.

Effect of single-dose carbapenem exposure on transcriptional expression of blaNDM-1 and mexA in Pseudomonas aeruginosa.

The therapeutic option of a carbapenem antibiotic is compromised in Pseudomonas aeruginosa owing both to acquired and intrinsic resistance mechanisms. In recent years, New Delhi metallo-ß-lactamase has been the focus as a predominant carbapenem resistance determinant. However, it is unclear which of the mechanisms might be adopted by a P. aeruginosa strain possessing both blaNDM-1 and an overexpressed MexAB-OprM system during carbapenem therapy. This study investigated the interplay of both mechanisms in clinical isolates of P. aeruginosa when exposed to meropenem. Five strains were used: (i) strain overexpressing MexAB-OprM but with no blaNDM-1; (ii) strain harbouring blaNDM-1 but expressing MexAB-OprM at basal level; (iii) strain possessing blaNDM-1 and overexpressing MexAB-OprM; (iv) P. aeruginosa PAO1; and (v) P. aeruginosa K2733-PAO1 (ΔMexAB-OprMΔMexCD-OprJΔMexEF-OprNΔMexXY-OprM) into which blaNDM-1 was cloned. Strains were incubated in Luria-Bertani broth with and without 1µg/mL meropenem. Total RNA was isolated at 45-min intervals and was immediately reverse transcribed to cDNA. This was repeated for 6h. Quantitative real-time PCR was performed for both resistance mechanisms. Meropenem exposure did not significantly elevate transcription of either the blaNDM-1 or mexA gene. However, an interesting finding was that upon single-dose exposure to carbapenem, the efflux pump system played a major role in bacterial survival compared with NDM-1. This study gives an insight into the bacterial response to carbapenem antibiotic when two different resistance mechanisms coexist. This type of study would be helpful in designing future antimicrobials.

Screening of Natural Products and Derivatives for the Identification of RND Efflux Pump Inhibitors.

AIM AND OBJECTIVE: Overexpression of efflux pumps belonging to the Resistance Nodulation cell Division (RND) family is the most important intrinsic resistance mechanism of Pseudomonas aeruginosa. Hence, it is imperative to identify suitable efflux pump inhibitors (EPI) that can lead to increased intracellular concentration of antibiotics by blocking the pump. This study was undertaken to identify a putative plant based efflux pump inhibitor for RND efflux pump of P. aeruginosa. MATERIAL AND METHOD: Using molecular docking approach, 328 secondary plant metabolites have been screened for their inhibitory activity against cytoplasmic exporter protein MexB of MexAB-OprM efflux pump of P. aeruginosa. After the initial in silico screening, the shortlisted compounds were subjected to in vitro test for efflux pump inhibitory activity using double disc synergy test. A combinatorial library of 1000 molecules was generated from active p-coumaric acid and docked with MexB protein to find a suitable EPI with better binding efficacy compared to the p-coumaric acid. RESULTS: Preliminary screening resulted in five plant-based natural products with significant docking score and were subsequently subjected to double disc synergy test. p-Coumaric acid , amongst the five, was found to potentiate activity of ciprofloxacin in MexAB-OprM overexpressing P. aeruginosa strain. Library compound 482, i.e 4-(4-((Z)-2-carboxy-2-((Z)-2,3-dihydrobenzo[e][1,4]diazepin-1-yl)-1-(4- hydroxyphenyl)vinylamino) phenylsulfonamido)-2-hydroxybenzoic acid, a derivative of p-coumaric acid exhibited the highest docking score of -42.1030 Kcal/mol, which was much higher than parent compound (-17.9403 Kcal/mol) and also known EPI, MC-207,110 (-28.0960 Kcal/mol). CONCLUSION: p-Coumaric acid and its derivative, 4-(4-((Z)-2-carboxy-2-((Z)-2,3-dihydrobenzo[e][1,4] diazepin-1-yl)-1-(4-hydroxyphenyl)vinylamino)phenylsulfonamido)-2-hydroxybenzoic acid may be used as potential lead molecules for effective RND efflux pump inhibition in P. aeruginosa.

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India.

OBJECTIVES: The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. METHODS: 76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. RESULTS: Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. CONCLUSIONS: This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.

Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India.

Carbapenem resistance presents severe threat to the treatment of multidrug resistant Pseudomonas aeruginosa infections. The study was undertaken to investigate the role of efflux pumps in conferring meropenem resistance and effect of single dose exposure of meropenem on transcription level of mexA gene in clinical isolates of P. aeruginosa from a tertiary referral hospital of India. Further, in this investigation an effort was made to assess whether different components of MexAB-OprM operon expresses in the same manner and the extent of contributions of those components in meropenem resistance in its natural host (P. aeruginosa) and in a heterologous host (E. coli). Out of 83 meropenem nonsusceptible isolates, 22 isolates were found to possess efflux pump activity phenotypically. Modified hodge test and multiplex PCR confirmed the absence of carbapenemase genes in those isolates. All of them were of multidrug resistant phenotype and were resistant to all the carbepenem drug tested. MexAB-OprM efflux pump was found to be overexpressed in all the study isolates. It could be observed that single dose exposure meropenem could give rise to trivial increase in transcription of mexA gene. Different constructs of MexAB-OprM (mexR-mexA-mexB-OprM; mexA-mexB-OprM; mexA-mexB) could be expressed in both its natural (P. aeruginosa PAO1) and heterologous host (E. coli JM107) but transcription level of mexA gene varied in both the hosts before and after single dose exposure of meropenem. Different components of the operon failed to enhance meropenem resistance in E. coli JM107 and P. aeruginosa PAO1. This study could prove that MexAB-OprM efflux pump can significantly contribute to meropenem resistance in hospital isolates of P. aeruginosa where an acquired resistant mechanism is absent. Thus, equal importance should be given for diagnosis of intrinsic resistance mechanism so as to minimize treatment failure. As meropenem could not enhance mexA transcriptions significantly, there might be a possibility that the increase in expression of efflux pump genes does not mediated by single antibiotic but rather by a combination of antipseudomonal drugs which are used during treatments. Early detection of efflux genes will help in selection of proper therapeutic options.

Emergence of integron borne PER-1 mediated extended spectrum cephalosporin resistance among nosocomial isolates of Gram-negative bacilli.

BACKGROUND & OBJECTIVES: Pseudomonas extended resistant (PER) enzymes are rare type of extended-spectrum beta lactamases (ESBLs) that confer third generation cephalosporin resistance. These are often integron borne and laterally transmitted. The aim of the present study was to investigate the emergence of integron borne cephalosporin resistant PER-1 gene in diverse incompatibility (Inc) group plasmids among Gram-negative bacteria. METHODS: A total of 613 consecutive, non-duplicate, Gram-negative bacteria of Enterobacteriaceae family and non-fermenting Gram-negative bacteria were isolated from different clinical specimens during a period of 18 months. For amplification and detection of blaPER, multiplex PCR was done. For understanding the genetic environment of blaPER-1, integrase gene PCR and cassette PCR (59 be) was performed. Gene transferability experiment was carried out and PCR based replicon typing was performed for incompatibility group typing of plasmids using 18 pairs of primers. An inhibitor based method was used for phenotypic detection of intrinsic resistance. RESULTS: Multiplex PCR and sequencing confirmed that 45 isolates were harbouring blaPER-1. Both class 1 and class 2 integrons were observed among them. Integrase and cassette PCR (59 be) PCR results confirmed that the resistant determinant was located within class 1 integron. Transformation and conjugation experiments revealed that PER-1 was laterally transferable and disseminated through diverse Inc plasmid type. Efflux pump mediated carbapenem resistance was observed in all isolates. All isolates belonged to heterogenous groups. INTERPRETATION & CONCLUSIONS: This study demonstrates the dissemination of cephalosporins resistant, integron borne blaPER-1 in hospital setting in this part of the country and emphasizes on the rational use of third generation cephalosporins to slow down the expansion of this rare type of ESBL gene.

Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa in clinical settings.

New Delhi metallo ß-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of blaNDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with blaNDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate's NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure.