Antibiotic Potentiation as a Promising Strategy to Combat Macrolide Resistance in Bacterial Pathogens.

Antibiotics, which hit the market with astounding impact, were once called miracle drugs, as these were considered the ultimate cure for infectious diseases in the mid-20th century. However, today, nearly all bacteria that afflict humankind have become resistant to these wonder drugs once developed to stop them, imperiling the foundation of modern medicine. During the COVID-19 pandemic, there was a surge in macrolide use to treat secondary infections and this persistent use of macrolide antibiotics has provoked the emergence of macrolide resistance. In view of the current dearth of new antibiotics in the pipeline, it is essential to find an alternative way to combat drug resistance. Antibiotic potentiators or adjuvants are non-antibacterial active molecules that, when combined with antibiotics, increase their activity. Thus, potentiating the existing antibiotics is one of the promising approaches to tackle and minimize the impact of antimicrobial resistance (AMR). Several natural and synthetic compounds have demonstrated effectiveness in potentiating macrolide antibiotics against multidrug-resistant (MDR) pathogens. The present review summarizes the different resistance mechanisms adapted by bacteria to resist macrolides and further emphasizes the major macrolide potentiators identified which could serve to revive the antibiotic and can be used for the reversal of macrolide resistance.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Imipenem and meropenem influence the Las/Rhl quorum-sensing systems in clinical isolates of Pseudomonas aeruginosa.

The present study was conducted to study the influence of imipenem and meropenem at subinhibitory concentration on the transcriptional response of Las/Rhl quorum-sensing systems in isolates of Pseudomonas aeruginosa. In the present study, six representative carbapenem nonsusceptible clinical isolates of P. aeruginosa were obtained. The agar dilution method was used to determine the minimum inhibitory concentration against imipenem and meropenem. The bacterial isolates were then cultured up to the early log phase in fresh Luria Bertani (LB) broths at 37°C with and without 2 µg mL-1 imipenem and meropenem, respectively. mRNA was then isolated from the bacterial isolates and was immediately reverse-transcribed to cDNA. The relative quantity of the expression of the lasI, lasR, rhlI, and rhlR genes was assessed by quantitative real-time Polymerase Chain Reaction (PCR) using the ΔΔCt method. The transcriptional response of the lasI and lasR genes was upregulated at subinhibitory concentration of meropenem. In contrast, the transcriptional response of the lasI, lasR, and rhlR genes was downregulated at subinhibitory concentration of imipenem as compared to the expression in untreated isolates. The data obtained in the current study showcased the ability of imipenem and meropenem to influence the response of the quorum-sensing genes at subinhibitory concentration.

Gut microbiome in the emergence of antibiotic-resistant bacterial pathogens.

The human gastrointestinal tract is home to a complex and dynamic community of microorganisms known as gut microbiota, which provide the host with important metabolic, signaling, and immunomodulatory functions. Both the commensal and pathogenic members of the gut microbiome serve as reservoirs of antimicrobial-resistance genes (ARG), which can cause potential health threats to the host and can transfer the ARGs to the susceptible microbes and into the environment. Antimicrobial resistance is becoming a major burden on human health and is widely recognized as a global challenge. The diversity and abundance of ARGs in the gut microbiome are variable and depend on the exposure to healthcare-associated antibiotics, usage of antibiotics in veterinary and agriculture, and the migration of the population. The transfer frequency of the ARGs through horizontal gene transfer (HGT) with the help of mobile genetic elements (MGEs) like plasmids, transposons, or phages is much higher among bacteria living in the GI tract compared to other microbial ecosystems. HGT in gut bacteria is facilitated through multiple gene transfer mechanisms, including transformation, conjugation, transduction, and vesicle fusion. It is the need of the hour to implement strict policies to limit indiscriminate antibiotic usage when needed. Developing rapid diagnostic tests for resistance determination and alternatives to antibiotics like vaccination, probiotics, and bacteriophage therapy should have the highest priority in the research and development sectors. Collective actions for sustainable development against resistant pathogens by promoting endogenous gut microbial growth and diversity through interdisciplinary research and findings are key to overcoming the current antimicrobial resistance crisis.

Elevated expression of rsmI can act as a reporter of aminoglycoside resistance in Escherichia coli using kanamycin as signal molecule.

We aimed to design and analyse expressional response of endogenous and exogenous 16S rRNA methyl transferase genes under sub inhibitory concentration stress of different clinically relevant aminoglycoside antibiotics in Escherichia coli to identify an endogenous marker. One hundred twenty nine aminoglycoside resistant E. coli of clinical origin were collected for detection of 16S rRNA methyl transferase genes by PCR assay and each gene type was cloned within E. coli JM107. Parent isolates were subjected to plasmid elimination by SDS treatment. Expression analysis of both acquired and endogenous 16S rRNA methyl transferase genes were performed by quantitative real-time PCR in clones and parent isolates under aminoglycoside stress (4 mg/L). Majority of the isolates were harbouring rmtC (35/129), followed by rmtB (32/129), rmtA (21/129), rmtE (13/129), armA (11/129) rmtF (9/129) and rmtH (8/129). Plasmid was successfully eliminated for all the isolates with 6% of SDS. Expression analysis indicates that kanamycin, tobramycin and netilmicin stress could increase the expression of 16S rRNA methyltransferese genes. In the presence of kanamycin stress the expression of rsmI was consistently elevated for all the wild type isolates and clones tested. Except for isolates harbouring rmtB and rmtC expression of rsmE and rsmF was increased in the presence of all aminoglycosides. For all the cured mutants it was apparently observed that expression of endogenous methyl transferases were marginally increased. Elevated expression of constitutive rsmI can be used as a potential biomarker for detection of acquired 16S rRNA methyl transferase mediated aminoglycoside resistance by using sub inhibitory concentration of kanamycin as signal molecule.

ß-Lactam potentiators to re-sensitize resistant pathogens: Discovery, development, clinical use and the way forward.

ß-lactam antibiotics are one of the most widely used and diverse classes of antimicrobial agents for treating both Gram-negative and Gram-positive bacterial infections. The ß-lactam antibiotics, which include penicillins, cephalosporins, monobactams and carbapenems, exert their antibacterial activity by inhibiting the bacterial cell wall synthesis and have a global positive impact in treating serious bacterial infections. Today, ß-lactam antibiotics are the most frequently prescribed antimicrobial across the globe. However, due to the widespread use and misapplication of ß-lactam antibiotics in fields such as human medicine and animal agriculture, resistance to this superlative drug class has emerged in the majority of clinically important bacterial pathogens. This heightened antibiotic resistance prompted researchers to explore novel strategies to restore the activity of ß-lactam antibiotics, which led to the discovery of ß-lactamase inhibitors (BLIs) and other ß-lactam potentiators. Although there are several successful ß-lactam-ß-lactamase inhibitor combinations in use, the emergence of novel resistance mechanisms and variants of ß-lactamases have put the quest of new ß-lactam potentiators beyond precedence. This review summarizes the success stories of ß-lactamase inhibitors in use, prospective ß-lactam potentiators in various phases of clinical trials and the different strategies used to identify novel ß-lactam potentiators. Furthermore, this review discusses the various challenges in taking these ß-lactam potentiators from bench to bedside and expounds other mechanisms that could be investigated to reduce the global antimicrobial resistance (AMR) burden.

Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, India.

OBJECTIVES: We investigated the prevalence and diversity of antimicrobial resistance in bacteria isolated from urine samples of community-onset urinary tract infection (UTI) patients in southern Assam, India. METHODS: Freshly voided midstream urine samples were collected from patients attending primary healthcare centres, with the patients' epidemiological data also recorded. Species identification was confirmed using a VITEK 2 compact automated system. Phenotypic confirmation of ESBLs was performed using the combined disc diffusion method (CLSI 2017) and carbapenemase production was phenotypically characterized using a modified Hodge test. Common ESBLs and carbapenem-resistance mechanisms were determined in Escherichia coli isolates using PCR assays. Incompatibility typing of the conjugable plasmids was determined by PCR-based replicon typing; the phylotypes and MLSTs were also analysed. RESULTS: A total of 301 (59.7%) samples showed significant bacteriuria along with symptoms of UTI and among them 103 isolates were identified as E. coli of multiple STs (ST3268, ST3430, ST4671 and others). Among them, 26.2% (27/103) were phenotypically ESBL producers whereas 12.6% (13/103) were carbapenemase producers. This study describes the occurrence of diverse ESBL genes-bla CTX-M-15, bla SHV-148, bla PER-1 and bla TEM-and two E. coli isolates carrying the bla NDM-1 carbapenemase gene. ESBL genes were located within transconjugable plasmids of IncP and IncF type whereas bla NDM-1 was carried in an IncFrepB type plasmid. CONCLUSIONS: This study illustrates the high rate of MDR in E. coli causing UTI in primary care in rural Assam. UTIs caused by ESBL- or MBL-producing bacteria are very difficult to treat and can often lead to treatment failure. Thus, future research should focus on rapid diagnostics to enable targeted treatment options and reduce the treatment failure likely to occur with commonly prescribed antibiotics, which will help to combat antimicrobial resistance and the burden of UTIs.

Report of a carbapenemase gene blaIMP-4 in multi-drug resistant Escherichia coli from sewage water: A threat on clinical-environmental interphase.

Acquired carbapenemases pose a significant role in the dissemination of antimicrobial-resistant Enterobacteriaceae and in this study we have identified the occurrence of blaIMP-4 in E. coli isolate from a sewage outfall located nearby a secondary health Centre. It was found to co-existed with blaCTX-M-15 located within a self-conjugable plasmid of IncF type. The current study underscores environment as a potential reservoir of carbapenem resistance and the need of the hour is to track and check dissemination of resistance in environment, human and agricultural settings.

Transcriptional Analysis of IncFrepB-Mediated blaOXA-48-Positive Plasmid Characterized from Escherichia coli ST448.

Objectives: To investigate the transcriptional response of blaOXA-48 and the copy number alteration of IncFrepB plasmid carrying blaOXA-48 under an antibiotic concentration gradient. Methods: Escherichia coli strains harboring blaOXA-48 on an IncFrepB plasmid were isolated from Silchar Medical College and Hospital, Silchar, India. Sequence type and common resistance determinants were determined by PCR assay. Plasmid copy number alteration and the transcriptional expression of blaOXA-48 under different antibiotic pressures were determined by quantitative real-time PCR, and the relative fold change was measured by the ΔΔCT method. Results and Conclusion: The plasmid that carried blaOXA-48 in E. coli ST448 was characterized as IncFrepB and found to be conjugatively transferable. The isolates were found to coexist with blaNDM-1 within the IncX3-type plasmid. It was observed that the copy number and transcriptional response of blaOXA-48 were directly proportional to the increasing concentration of meropenem and ertapenem, whereas in the case of imipenem, it was reversed. The identification of blaOXA-48 through IncFrepB-type plasmid in this study indicates the potential route of spread of this resistance determinant in this area and also the insights we gained from the transcriptional changes of blaOXA-48 in response to different antibiotic pressures could also facilitate the development of novel or alternative therapeutic options needed for multidrug-resistant infections.

Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa.

Purpose: Pseudomonas aeruginosa is an opportunistic pathogen with biofilm-forming ability, by the virtue of which they can evade the immune response and antimicrobial chemotherapy. Several methods have been designed for the detection of biofilms but require sophisticated instrumentation and expertise. The present study, therefore, used an improvised device, 'fluorescence foldscope' which is an origami-based fluorescence microscope as an easy and effective tool to detect biofilm formation. Methodology: Three representatives of P. aeruginosa of clinical origin were taken for the study along with two reference strains PA01 and ATCC27853. The strains were cultured in Luria Bertani (LB) broth with and without carbapenem (imipenem and meropenem) and cephalosporin (ceftazidime, cefotaxime and ceftriaxone) pressure, respectively. The cultures were diluted to 1:100 in LB; seeded with sterile glass slides at 90° angle and incubated for 5 consecutive days. The slides were observed with fluorescence foldscope. Results: Fluorescence emission was observed in two test isolates CD1 and CD2 at 48 and 72 h, respectively, whereas no fluorescence was observed in CD3. The fluorescence observed in the isolates was not affected by 2 µg/ml carbapenem pressure, while with 2 µg/ml ceftazidime stress, a change in fluorescence was observed in CD2 in comparison to the fluorescence observed under normal growth condition. Conclusion: Fluorescence foldscopy is an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa under different laboratory conditions. Biofilm-forming P. aeruginosa worsens the medical condition and is difficult to eradicate. The present study came up with an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa.

Distribution of carbapenem resistant Acinetobacter baumannii with blaADC-30 and induction of ADC-30 in response to beta-lactam antibiotics.

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other ß-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 µg/ml (170 fold), ceftriaxone at 8 µg/ml (136 fold), ceftazidime at 4 µg/ml (65 fold), cefepime at 8 µg/ml (77 fold) and aztreonam at 8 µg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 µg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.

An insight into analysis and elimination of plasmids encoding metallo-ß-lactamases in Pseudomonas aeruginosa.

OBJECTIVES: The aim of this study was to characterise metallo-ß-lactamase (MBL)-harbouring plasmids, their change in copy number in respect to different antibiotic pressure, and the efficiency of different curing agents in eliminating these resistance plasmids from nosocomial Pseudomonas aeruginosa isolates. METHODS: Plasmids were extracted from four isolates harbouring blaNDM-1 or blaVIM-2 under four different concentrations of imipenem, meropenem, ertapenem, aztreonam and cefotaxime. Quantitative real-time PCR was performed to analyse the change in plasmid copy number under these different conditions. The effect of different physical and chemical curing agents in elimination of plasmids carrying blaNDM-1 and blaVIM-2 was examined, with meropenem resistance used as a selectable marker. RESULTS: Conjugatively transferable MBL genes (blaNDM-1 and blaVIM-2) carried on plasmids were found to be highly stable. Sodium dodecyl sulfate (SDS) was the most effective agent in eliminating these resistance plasmids. The change in copy number of the blaNDM-1-encoding plasmid was found to be similar to the blaVIM-2-encoding plasmid, with a single exception under cefotaxime pressure. CONCLUSION: The spread of multidrug resistance plasmids has been noted as a key factor associated with increasing carbapenem resistance. Successful curing of resistance plasmids can reverse the bacterial phenotype back to susceptible. This study revealed that different antibiotic pressure induces a change in copy number of MBL-encoding plasmids. SDS can be successfully used as an eliminating agent for these resistance determinants, although therapeutic application of this agent is not possible due to its high toxicity and mutagenic nature.

High prevalence of carbapenemase, AmpC ß-lactamase and aminoglycoside resistance genes in extended-spectrum ß-lactamase-positive uropathogens from Northern India.

OBJECTIVES: This study investigated the occurrence of extended-spectrum ß-lactamase (ESBL) genes coexisting with carbapenemase, AmpC and aminoglycoside resistance gene in uropathogens in India. METHODS: Antimicrobial susceptibility testing was performed by disk diffusion. Antimicrobial resistance genes were detected by multiplex PCR. RESULTS: Of 1516 consecutive urine samples, 454 (29.9%) showed significant bacteriuria with a single micro-organism, predominantly Escherichia coli (n=343), followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the uropathogens, 61 ESBL-producers were identified containing blaCTX-M-15 (n=32), blaCTX-M-15+blaOXA-2 (n=15), blaCTX-M-15+blaOXA-2+blaTEM-1 (n=6), blaOXA-2 (n=5), blaOXA-2+blaSHV-76 (n=1), blaTEM-1+blaSHV-76 (n=1) and blaTEM-1 (n=1). All ESBL genes were located on horizontally transferable plasmids of incompatibility types HI1, I1, FIA+FIB, FIA and Y. Among the 61 ESBL-producers, 59 harboured carbapenemase genes, including blaNDM-5 (n=48), blaNDM-5+blaOXA-48 (n=5), blaNDM-5+blaIMP (n=5) and blaNDM-5+blaIMP+blaVIM (n=1). ESBL-producing uropathogens also harboured 16S rRNA methylase genes, including rmtB (n=9), rmtA (n=4), rmtC (n=1) and armA (n=1). ESBL-positive isolates also contained AmpC genes, including blaCIT (n=8) and blaDHA-1 (n=1). Imipenem and gentamicin had the lowest resistance rates against the uropathogens. CONCLUSION: This is the first report showing the high prevalence of carbapenemases in ESBL-positive isolates in this area. Regular surveillance for such resistance mechanisms will be useful for health personnel to treat infections by these multidrug-resistant pathogens.

Occurrence of diverse aminoglycoside modifying enzymes with co-existing extended-spectrum-ß-lactamases within Enterobacteriaceae isolated in India.

OBJECTIVE: The present study describes aminoglycoside modifying enzymes (AMEs) among clinical isolates with coexisting extended spectrum beta-lactamases. METHODOLOGY: A total of 227 non duplicate enterobacterial isolates were collected and identified from patients who were admitted to different wards or attended OPD of a tertiary referral hospital of North-East India. Isolates were initially screened for antimicrobial susceptibility testing followed by PCR based screening of aminoglycosides modifying enzymes and co-existing ESBLs and carbapenemases. Horizontal transferability, incompatibility typing and stability of plasmids were also analyzed. RESULTS: Diverse types of AMEs were observed namely; ant(3″)-I, ant(4')-Ia, aac(3)-IIc, ant(3')-I, aac(6')-Ib, ant(2″)-Ia and aac(6'). Majority of the AME positive isolates harboured blaTEM followed by blaCTX-M-15 and a combination of blaTEM and blaCTX-M-15 were also observed. Nine isolates were found to harbour carbapenemases genes. AME genes were found to be located within a self conjugative plasmid of Inc FIA, IncY, IncN, IncFIB and IncA/C incompatibility types. It was observed that most AME genes were stable over 50 days of serial passages whereas aph(3')-Via and aph(3')-IIb were completely lost within 50 days. CONCLUSION: This study underscores the co-existence of AMEs and ESBLs within enterobacteriaceae which emphasize a reassessment of combination therapy in the health settings.

Human carriage of cefotaxime-resistant Escherichia coli in North-East India: an analysis of STs and associated resistance mechanisms.

OBJECTIVES: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India. METHODS: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes. RESULTS AND CONCLUSIONS: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15.

AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.

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.

Detection and molecular typing of methicillin-resistant Staphylococcus aureus from northeastern part of India.

BACKGROUND: In Staphylococcus aureus, methicillin resistance is exhibited by modifications in penicillin-binding protein that minimises the binding affinity to beta-lactam antibiotics. The present study investigated the occurrence of methicillin-resistant S. aureus (MRSA) in community-acquired infections, that is, community-acquired MRSA (CA-MRSA) and in-hospital-acquired infections, that is, hospital-acquired MRSA (HA-MRSA) from Northeast India. METHODS: A total of 197 consecutive non-duplicate isolates were collected from Silchar Medical College and Hospital and other private diagnostic laboratories. The isolates were confirmed to be S. aureus at our centre. All isolates were subjected to antibiotic susceptibility testing and were screened for methicillin resistance using cefoxitin disc test. All MRSA were subjected to Polymerase Chain Reaction (PCR) assay for detection of mecA and mecC genes. DNA fingerprinting was performed for determining clonal diversity. RESULTS: Seventy-one isolates of 127 confirmed S. aureus were found to be methicillin resistant by screening test. mecA gene was detected in 43 isolates, and none of the isolates were positive for mecC gene. Linezolid and teicoplanin showed better activity with susceptibility pattern being 83.6% and 72.44%, respectively, whereas 66.14% were sensitive to vancomycin. Other antibiotic showed low level of activity. Pulsed Field Gel Electrophoresis (PFGE) showed 14 different banding patterns that suggest isolates were of different clonal types. CONCLUSION: mecA was responsible for methicillin resistance in majority of strains. Polyclonal spread of MRSA infection in the study area indicates its diverse origin and possible lateral transfer. Thus, this study is of clinical interest in terms of selection of proper antimicrobial chemotherapy and infection control management.

Adaptation of blaNDMthrough IncP plasmid within broad host range.

Introduction: It was also known that the IncP-1 plasmids are ubiquitous in environmental bacteria and those reside in soil, sewage, marine sediments and in manure. The blaNDMis associated with resistance determinants along with various mobile elements such as plasmid, insertion sequences and transposons, which facilitates its horizontal dissemination. These plasmids, if tracked, can be a starting point for the control of infection due to multidrug-resistant pathogens. The aim of the study was to investigate that IncP-type plasmids carrying blaNDMis adapted in different hosts. Materials and Methods: Thirteen of the isolates were harbouring IncP-type plasmid and they all were Escherichia coli isolated from hospitalised patients of Silchar Medical College and Hospital, India. The isolates were checked for susceptibility test, and the stability was assessed by a serial passage. These isolates were further subjected to transcriptional analysis of NDM gene as well as plasmid copy number alteration. Results: The study isolates were highly stable, and the resistance gene (blaNDM) was retained within isolates till 55th subsequent serial passages. Plasmid copy number alteration was random in isolates when exposed to carbapenem antibiotics, whereas increasing trend in transcriptional expression was observed with the increase in imipenem concentration. Conclusion: This study was able to underscore the presence of IncP plasmid that was harbouring blaNDMand was maintained within diverse host. The finding also highlights the adaptation of the broad-host-range plasmid that responds in terms of transcriptional expression under antibiotic exposure.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli.

BACKGROUND: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. OBJECTIVE: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. MATERIALS AND METHODS: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. RESULTS: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. CONCLUSION: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance.

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.