Disk Potentiation Test Research Articles

Detection of Metallo β-Lactamase in Acinetobacter Species Using Phenotypic Methods with their Antibiotic Resistance Profile in Tertiary Care Hospital

Background: Acinetobacter species are commonly found in nature, in water, and in soil, and also, and they have been isolated from humans and animals. In the hospital setting, Acinetobacter species has emerged as a major opportunistic pathogen, being able to colonize and develop infections in patients. In Acinetobacter species, β-lactamases and outer membrane alterations are likely to function together to impart resistance. Understanding the epidemiology, and resistance mechanism, and for infection management and preventing a potential global health crisis, techniques for identifying Carbapenem-Resistant-Acinetobacter species are essential. Aim: To detect Metallo β-Lactamase (MBL) in Acinetobacter species with their antimicrobial susceptibility pattern. Material and Methods: 100 Acinetobacter species isolates from various clinical samples such as blood, cerebrospinal fluid, sputum, bronchoalveolar lavage, pus, urine, wound swab, and body fluids were included in the study. Phenotypic tests such as, Disc Potentiation test & Modified Hodge test were used to detect the presence of Metallo β-lactamase in imipenem & meropenem-resistant isolates. Results: The highest prevalence of Acinetobacter was found in blood (37%), followed by sputum (31%) and others [pus, urine, ascitic fluid, cerebrospinal fluid (CSF), bronchoalveolar Lavage (BAL), and pleural fluid]. A maximum number of Acinetobacter were isolated from MICU followed by medicine and surgery. The Modified Hodge test yielded the largest percentage of MBL-positive Acinetobacter isolates (67.26%) when compared to the Disc Potentiation test method (50.45%). Conclusion: Early detection of Metallo β-lactamase production is essential for planning appropriate treatment according to the resistance mechanisms of the multi-drug resistant strains, as well as for efficient infection control procedures to prevent the spread of infection.

Phenotypic methods for detection of metallo-β-lactamase (MBL) production in multidrug resistant gram negative bacilli – comparative study

Introduction: One of the most common mechanism of resistance of bacterial pathogens to β-lactam antibiotics is production of β-lactamase, there are different types of Beta lactamases, which are expressed by drug resistant gram-negative bacteria. Carbapenemases (Metallo beta lactamases/MBL) are the β-lactamases with the widest spectrum of activity. Early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their interhospital and intrahospital dissemination. Several phenotypic methods are available for the detection of MBL producing bacteria. As there is no standardized method present study was done to screen MDR gram negative bacilli isolated from clinical samples for MBL-production by a low cost, convenient and sensitive procedure. Methods: All non-duplicate MDR gram negative isolates obtained from various clinical samples were screened for carbapenam resistance. All carbapenam resistant bacteria were screened for production of MBL by 3 phenotypic tests (Double disc synergy, combined disk test, Hodge test). The results were compared and analyzed on the basis of results obtained by E test. Results: During Study Period, 988 non duplicate gram negative bacilli were isolated, 70.64% (698) were multi drug resistant. Amongst Total number of MDR Isolates to carbapenam resistance was seen in 62(9.28%). These 62 isolates that were resistant to carbapenam were tested for MBL production. 54 (87%) of these 62 isolates showed MBL production by disc potentiation test whereas 41 isolates (66%) gave positive result by DDST. By Modified Hodge test, out of 62, 48 isolates (77.4%) were MBL positive. Compared to E- test, the Sensitivity Specificity and Accuracy for Disc potentiation test was 90%,100% and 90.32%, for Modified Hodge test was 80%,100% and 80.6% and for Double disc synergy test was 68.3%,100% and 69.3%. Conclusion: In our study, in comparison to MBL E test, disc potentiation test is more sensitive than double disc synergy test and Modified Hodge test for detection of MBL phenotypically

Extended-Spectrum Beta-lactamase and Metallo-beta-lactamase Production among Escherichia coli and Klebsiella pneumoniae Strains from Urine of Pregnant Women in Afikpo, Ebonyi State, Nigeria

Aim: The aim of this study was to determine the occurrence of Extended-spectrum beta-lactamase (ESBL) and Metallo-beta-lactamase (MBL) among Escherichia coli and Klebsiella pneumoniae strains from pregnant women attending Mater Misericordia Hospital Afikpo, Ebonyi state, Nigeria.
 Study Design: This is a laboratory based prospective study carried out on pregnant women suspected of having urinary tract infection and was requested to undergo diagnosis at microbiology laboratory of the hospital.
 Place and Duration of Study: The study was conducted in the Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Afikpo, Ebonyi State, Nigeria from October, 2022 to January, 2023.
 Methodology: Clean-catch midstream urine samples were collected from 206 pregnant women suspected of having urinary tract infection and were requested to undergo medical diagnosis at microbiology laboratory of the hospital. The urine samples were processed following standard microbiological procedure. Antimicrobial susceptibility testing was determined using the disc diffusion method, while ESBL phenotypes were determine by the Double-Disc Synergy Test (DDST). Disc potentiation test was performed to check for MBL production.
 Results: Out of the 206 urine samples processed, 24 (11.7 %) E. coli and 12 (5.8 %) K. pneumoniae were isolated. The antimicrobial susceptibility of the isolates recorded a 100 % resistance with Amoxicillin/Clavulanic acid and Cotrimoxazole. The Gram-negative isolates showed a high sensitivity of 100 % to Netilmicin, Meropenem and Ofloxacin. Overall, 35 (97.2 %) multidrug resistance (MDR) was observed of the bacteria isolates. A total of 9 (37.5 %) E. coli and 4 (33.3 %) K. pneumoniae was found positive for ESBL production whereas, 5 (20.8 %) E. coli and 2 (16.7 %) K. pneumoniae were MBL positive.
 Conclusion: The level of drug resistance in this study underscores the need for regular surveillance for effective management of urinary tract infection in pregnancy.

Detection of extended spectrum β-Lactamase (ESBL) and Metallo β-Lactamase (MBL) in Pseudomonas aeruginosa isolated from various clinical samples

Introduction: β-Lactamase production is considered one of the most important resistance mechanisms among virulent Pseudomonas aeruginosa isolates. The aim of the study was to detect Extended Spectrum β-Lactamase (ESBL) and Metallo β-Lactamase (MBL) in Pseudomonas aeruginosa obtained from various clinical samples. Materials and Methods: The study was conducted in the Department of Microbiology, Govt. Medical College, Shahdol. All clinical specimens were processed and P. aeruginosa isolates were identified by standard protocols. Antibiotic sensitivity testing for all isolates was done using Kirby-Bauer disc diffusion method. Detection of ESBL and MBL was done by Disc potentiation test. Results: Among 347(27.2%) isolates of P. aeruginosa, 284(88.2%) were MDR, 139(40%) and 73(21%) were ESBL and MBL producers respectively where 39(11.2%) isolates produced both ESBL and MBL. Conclusion: A high prevalence of MDR, ESBL, MBL positive isolates among P. aeruginosa is increasing at an alarming rate. Thus, proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this MDRPA.

Phenotypic and molecular characterization of extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Klebsiella pneumoniae in Iran.

The objective of the current study is to evaluate the phenotypic and molecular characterization of ESBL/AmpC- and carbapenemase-producing K. pneumoniae isolates in Iran. From October 2018 until the end of April 2020, different clinical samples were collected and K. pneumoniae isolates were identified using conventional biochemical tests and PCR assay. Antibiotic susceptibility pattern was determined using the Kirby-Bauer disk diffusion method. Modified Hedge Test (MHT) was applied to the identification of carbapenemase-producing K. pneumoniae. ESBL and AmpC-producing K. pneumoniae were detected using Double Disc Test (DDT) and Disc Potentiation Test (DPT), respectively. The presence of carbapenemase, ESBL, and AmpC encoding genes was screened by Polymerase Chain Reaction (PCR) assay. A total of 100 K. pneumoniae isolates were collected. K. pneumoniae isolates had the highest resistance rate to cefazolin (66%) and cefotaxime (66%). Meropenem and amikacin with sensitivity rates of 76% and 69% were the most effective antimicrobial agents on K. pneumoniae isolates. It was found that 12 (12%), 27 (27%), and 9 (9%) K. pneumoniae isolates were positive in MHT, DDT, and DPT tests, respectively. Among the carbapenemase-encoding genes, blaOXA-48 (24%) and blaIMP (13%) genes had the highest frequency, while blaKPC and blaGIM genes were not detected among K. pneumoniae isolates. blaTEM (48%) and blaCMY (8%) genes had the highest frequency among ESBL and AmpC β-lactamase-encoding genes, respectively. It is vital to adopt effective control strategies for K. pneumoniae infections and ensure rapid identification of antibiotic resistance profile.

Biofilm Production and its Association with Multi Drug Resistance in Pseudomonas aeruginosa among ICU Patients with Special Reference to ESBL, AmpC and MBL production

Multidrug resistant Pseudomonas aeruginosa is an alarming and emerging public health problem globally across the developing countries. Pseudomonas aeruginosa is still a major cause for nosocomial infection and approx 10-20% of these patients are admitted to the ICU’s. Bacterial isolates those are biofilm producers are more drug resistant than biofilm Non-producers. The aim of the present study was to evaluate the production of biofilm and β-lactamases (ESBL, MBL, AmpC) in multi drug resistant Pseudomonas aeruginosa isolated from ICU patients. The present cross-sectional prospective study was carried out in the Department of Microbiology, Santosh Medical College & Hospital, Ghaziabad, Uttar-Pradesh, India. A total of 115 isolates of P. aeruginosa were isolated from 502 clinical samples. After confirmation of MDR status of P. aeruginosa further processing for biofilm and beta lactamases was performed accordingly. Biofilm production was done by test tube method and tissue culture plate method along with phenotypic profiling of ESBL, MBL and AmpC was performed by disc potentiation test; IMP-EDTA combined disc test and Cefoxitin Cloxacillin Double Disc synergy test (CC-DDST) respectively. Out of 502 total human clinical samples 115 isolates were Pseudomonas aeruginosa giving the prevalence rate of 23%. Among 115 isolates of P. aeruginosa 60 (52%) were MDR phenotypes, Out of 60 MDR isolates 23 (38.3%) were ESBL producers, 22 (36.6%) were MBL producers, and 3(5%) were AmpC producers. Out of total 115 isolates 68(59%) isolates were biofilm producers and 47 (40.8%) were biofilm non-producers. Strict antibiotic policies with early detection of beta lactamases and detection of biofilm production should be performed regularly for all clinical isolates of Pseudomonas aeruginosa so as to guide antibiotic selection along with better management of severe infection in ICU patients.

Early Detection of Drug Resistant Enterobacteriaceae in Urinary Tract Infections using Chromogenic Agar Medium in a Tertiary Care Hospital, Kakinada, Andhra Pradesh, India

Introduction: The irrational and inappropriate use of beta lactam antimicrobial drugs has led to the advent of Extended Spectrum Beta-Lactamase (ESBL) resistant strains. ESBL producing Enterobacteriaceae strains are frequent causative agents both in community and in acquired nosocomial infections and Urinary Tract Infections (UTI). The phenotypic confirmatory tests rarely identify all ESBLs. Chrom ID (Chromogenic identification Media) ESBL – Bx (bioMerieux) is a completely new and innovative chromogenic medium designed specifically for the screening of ESBL producing Enterobacteria directly from urine samples. It is a ready to use selective media which is sensitive and specific for rapid and presumptive identification of ESBL producing Enterobacteriaceae. Aim: Early detection of ESBL producing Enterobacteriaceae directly from urine samples on chromogenic medium (Chrom ID- ESBL- Bx) and confirmation of ESBL producing Enterobacteria using Disc Potentiation Test (DPT). Materials and Methods: The present cross-sectional study was conducted in the Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India from November 2019 to March 2020 (five months duration). The study was done on 70 urine samples from patients with UTI. All samples were subjected to wet mount, inoculated directly for culture on Chrom ID ESBL- Bx agar and MacConkey agar. Antibiotic Susceptibility testing of ceftazidime and cefotaxime was done by Kirby-Bauer disc diffusion method and conformation of ESBL production by DPT using Clinical and Laboratory Standards Institute (CLSI) method. The Statistical Package for the Social Sciences (SPSS) Statistical package version (18.0) was used. Results: A total of 56 (80%) isolates were obtained from 70 urine samples, out of them 28 (50%) were Escherichia coli, 21 (37.5%) were Klebsiella spp., 7 (12.5%) were Proteus spp., 23 (82.14%) isolates of Escherichia coli, 15 (71.43%) of Klebsiella spp., 6 (85.71%) of Proteus spp., isolated were screened positive using Chrom ID ESBL-Bx agar. About 44 (78.57%) of total Enterobacteria (56) were screened for ESBL production. 20 (86.96%) of Escherichia coli, 11 (73.33%) of Klebsiella spp., and 5 (83.33%) of Proteus spp., that were screened positive using Chrom ID ESBL agar were confirmed (by DPT) as ESBL producers and 2 (16.6%) of total (12) isolates that were screened negative by Chrom ID ESBL agar were confirmed as ESBL producers when screened and confirmed by DPT. So sensitivity and specificity CHRO Magar was 94.73% and 55.5%. Conclusion: ESBL continues to become a serious public health threat. Results from present study showed that CHROMagar ESBL has a high sensitivity and a convenient method for making provisional diagnosis of drug resistant Enterobacterial infections in 24 hours. Chrom ID ESBL- Bx agar medium allows easy differentiation of different bacteria based on colony colouration.

Bacteriological Profile of Gram-Negative Organisms from Cases of Septicemia and Their Antibiotic Sensitivity Pattern with Special Reference to Beta-Lactamase and Carbapenemase Production

Background: Blood stream infections are amongst the most important causes of morbidity and mortality throughout the world. Septicemia is a serious bloodstream infection and a biggest challenge for the clinicians in selection of appropriate antimicrobial agents. Presence of intravascular catheters, immunosuppression and use of cytotoxic drugs are some precipitating factors for antibiotic resistance which is a global problem. Reports of multi-resistant bacteria causing septicemia in developing countries are increasing. Extended-spectrum beta-lactamases (ESBL) and Carbapenemase-producing Enterobacteriaceae (CRE) has caused a major public health concern. Aim and Objectives: To determine the bacteriological profile of septicemia and their antibiotic susceptibility profile with special references to β-lactamase and carbapemase productionin gram negative bacilli. Material and Method: During the 3 month period of study total 100 blood samples from suspected cases of septicaemia are collected. Isolates were processed and identified by standard protocols. Detection of extended spectrum β-lactamase (ESBL), inducible AmpC and MBL (Metallo β-lactamases) was done by using screening test and then confirmatory tests. Results: Out of 100 samples 68 gram negative organisms were isolated. Maximum growth was of Pseudomonas aeruginosa i.e. 34, followed by Klebsiella pnemoniae i.e. 19 and Acinetobacter sps and E.coli. From all isolates 4.4% were ESBL, Metallo –beta-lactamases (MBL) production were seen in 5.9 %. 40% and 10.5% were MBL-E.coli and MBL-Klebseilla pnuemoniae respectively. Conclusion: Many of the isolates from cases of septicaemia were resistant to most of the recently used antibiotics. So, Detection of the etiological agents and their antibiotic sensitivity will definitely help in effective prophylactic measures, prompt and accurate diagnosis of septicaemia. Simple methods like disc potentiation test for ESBL, disk antagonism test for inducible AmpC producers and Combined disk potentiation test for MBL production are relatively inexpensive and less cumbersome, so it can also be performed as a part of routine sensitivity testing. High degree of antibiotic resistance is an alarming sign for development of antibiotic policies and protocols for treatment of septicaemia.

Prevalence of extended beta-lactamase-producing Pseudomonas aeruginosa isolated from human immunodeficiency virus patients in Ibadan, Nigeria

BackgroundThe aim of this study was to determine the occurrence and production of Extended-Spectrum Beta-Lactamases (ESBLs) and Metallo Beta-Lactamases (MBLs) in P. aeruginosa isolated from HIV-infected patients from a hospital in Ibadan, Nigeria using currently available phenotypic tests and real-time PCR of selected reference genes. MethodsThis was a cross sectional study and samples were aseptically collected from consenting HIV infected patients between April and May 2018 at a tertiary hospital in Ibadan, Nigeria. Pseudomonas species were isolated from stool samples using Pseudomonas Base Agar and were identified by standard protocol. Phenotypic detection of ESBL and MBL was carried out using the disk potentiation test. Antibiotics susceptibility testing was done using Kirby-Bauer disk diffusion technique against ceftazidime and cefepime, amoxicillin-clavulanate, aztreonam, trimethoprim, gentamicin, ciprofloxacin and imipenem. Using Real-Time PCR, ESBL and beta-lactamase encoding genes such as blaTEM, blaSHV, AmpC, blaCTX and blaKPC in the ESBL-producing Pseudomonas aeruginosa isolated from the HIV-infected patients stool samples. Data were analyzed using descriptive statistics and chi-square test. ResultsThe Pseudomonas species comprised 95.3% P. aeruginosa and 4.7% P. fluorescens, of which 29.1% produced ESBL, but none produced MBL. All ESBL-producers showed resistance to trimethoprim and ciprofloxacin while, 20.0% were resistant to aztreonam, but none resist imipenem. In the ESBL-producing isolates, AmpC and blaTEM genes were detected while, blaSHV, blaCTX and blaKPC genes were not detected. ConclusionThe ESBL-production was only detected in Pseudomonas aeruginosa isolates out of which only AmpC and blaTEM genes were detected. The ESBL and ESBL genes detection in the Pseudomonas aeruginosa and multidrug-resistant strains portend public health threat among the studied patients. Hence, it is recommended that indiscriminate use of antibiotics among people living with HIV should be discouraged. Secondly, because of the AmpC significance in the provision of high levels of beta-lactams antibiotic resistance, especially the third generation cephalosporins commonly used for treatments, further research is hereby recommended.

The Rising Threat of Drug Resistant Pseudomonas aeruginosa- A Nightmare for Intensive Care Unit Patients

Introduction: Multidrug Resistant (MDR), Extensively Drug Resistant (XDR) and Pan Drug Resistant (PDR) variants manifest a high level of intrinsic resistance to antimicrobial drugs by the help of efflux pump, biofilm formation and aminoglycoside modifying enzymes. The potentiality of Pseudomonas spp. to produce variety of drug resistance mechanism has led to evolution of drug resistant phenotypes this poses a challenge for clinicians in the treatment of severe infection among Intensive Care Unit (ICU) patients. Aim: To determine the phenotypic profiling of b-lactamases and burden of MDR, XDR and PDR Pseudomonas aeruginosa (P. aeruginosa) in ICU patients. Materials and Methods: The present cross-sectional prospective study was carried in the Department of Microbiology, Santosh Medical College and Hospital, Ghaziabad, Uttar Pradesh, India, after permission from Institutional Ethics Committee (IEC). A total of 115 isolates of P. aeruginosa were isolated from 502 human clinical samples from January 2019 to February 2021 and all the clinical samples were non duplicate. Antimicrobial Susceptibility Testing (AST) was performed for all isolates by standard Kirby- Bauer disc diffusion method on Mueller Hinton Agar (MHA). Phenotypic profiling of Extended Spectrum b-Lactamase (ESBL), Metallo b-Lactamase (MBL) and Ampicillinase C (AmpC) was performed by disc potentiation test; Imipenemase (IMP) - Ethylenediamine Tetraacetic Acid (EDTA) combined disc test and Cefoxitin Cloxacillin Double Disc Synergy Test (CC-DDST), respectively. The obtained results were statistically analysed in numbers and percentages using MS Excel 2013 version. Results: Out of 502 total human clinical samples, 115 isolates were P. aeruginosa giving the prevalence rate of 23%. Among 115 Pseudomonas isolates, 60 (52%) were MDR phenotypes, 8 (7%) were XDR phenotypes and there was no PDR phenotypes isolated in present study as all isolates were sensitive to Ticarcillin/Clavulanic acid, Colistin and Polymyxin B. Out of 115 isolates, 59 (51%) were ESBL producers, 26 (23%) were MBL producers, and 6 (5%) were AmpC producers. Conclusion: Strict antibiotic policies and regular surveillance programme of antimicrobial resistance must be tailored to fend off the emergence of drug resistant Pseudomonas aeruginosa.

Antimicrobial resistance profile in clinical isolates of Pseudomonas aeruginosa producing Extended-spectrum beta-lactamase (ESBL) at a teaching tertiary care hospital, Tamilnadu

The spread of extended-spectrum betalactamase (ESBL)-producing clinical isolates of P. aeruginosa has dramatically increased worldwide and frequently reported in India. The present study was undertaken to identify the prevalence of ESBL producing Pseudomonas aeruginosa and its antimicrobial resistance profile. This prospective study was conducted at Clinical microbiology Laboratory of Sri Muthukumaran Medical college hospital and research Institute for a period of 9 months.. P. aeruginosa isolates from all samples were identified by standard protocols. Antibiotic sensitivity testing for all isolates was done using Kirby-Bauer disc diffusion method. Disc potentiation test was performed to detect ESBL production. ESBL producing pseudomonas aeruginosa(16/69) in this study was 23.18%. Prevalence rate of ESBL isolates among the MDR strains was 41.17%.We observed ESBL isolates with high resistance rate to tested antipseudomonal antibiotics than Non ESBL. All ESBL isolates were sensitive to Polymyxin and Colistin. This study gives an alarming sign towards high prevalence of cephalosporins due to ESBL emphasizes the need for early detection to prevent therapeutic failure. In addition ,our study results would be useful to formulate antibiotic policy and its compliances for effective infection control practices.

Phenotypic methods for detection of metallobetalactamase in Meropenem resistant Pseudomonas aeruginosa and molecular detection of blaIMP & blaVIM genes carrying strains

Introduction and Objectives: Pseudomonas aeruginosa is one of the major organisms responsible for nosocomial infections such as pneumonia, UTIs, surgical site infections, and bloodstream infections.1 High intrinsic and acquired resistance to many antimicrobial agents have allowed P. aeruginosa to persist in both community and hospital settings. This study was done to find the prevalence rate of Meropenem resistant Pseudomonas aeruginosa carrying metallobeta lactamase producing genes. Materials and Methods: The study included a total of 100 non-duplicate clinically significant, random bacterial isolates obtained from a North Indian rural tertiary care hospital (Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, India) from various clinical specimens, over a period of one year (January2012 to December 2012) . Identification of organisms was done using the standard microbiological techniques as described by Colle et al 1996.7 The antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method. To detect MBL producing isolates phenotypicaly, Disc potentiation test (DPT) and Double disc synergy test (DDST) were performed. PCR was performed on Meropenem resistant isolates to detect blaIMP and blaVIM genes. Results: Out of 100 examined isolates, 36% isolates were resistant to Meropenem. Out of these Meropenem resistant strains, 44.4%(16) P. aeruginosa isolates were MBL producers as detected by DDST method and 69.44%(25) isolates were MBL producers phenotypically by DPT method . PCR revealed prescence of bla VIM gene in 16.66%(6) of Meropenem resistant Pseudomonas aeruginosa isolates and 38.88% (14) isolates were found to be carrying bla IMP gene . Three isolate were shown to carry both blaIMP and blaVIM genes. Conclusion: Resistance of P. aeruginosa isolates to Meropenem due to MBL enzymes is increasing in rural area. Resistance due to MBL has clinical significance, rapid detection of MBL producing strains followed by

Antimicrobial resistance pattern and molecular epidemiology of ESBL and MBL producing Acinetobacter baumannii isolated from hospitals in Minia, Egypt

ABSTRACT Introduction: Multidrug resistant (MDR) Acinetobacter baumanii (A. baumannii) strains have emerged as novel nosocomial pathogens threatening patients’ lives, especially in intensive-care units (ICUs). This study aims to determine the prevalence of carbapenemase genes and CTX-M-15 and the resistance pattern of carbapenemase producing isolates.Methods: A total of 530 clinical specimens were collected from patients suffering from different infections, antibiotic susceptibility test was performed using kirby-bauer disk diffusion method. ESβL production was detected phenotypically by double-disc synergy test (DDST). Carbapenemase production was tested by Modified Hodge Test (MHT). Then, these isolates were tested for MBL detection by disc potentiation test. Carbapenemase encoding genes (VIM, IMP, GIM and SPM, OXA-51, OXA-23 and OXA-143) and CTX-M-15 were tested by polymerase chain reaction (PCR).Results: Out of 530 samples, 20 bacterial isolates were identified as A. baumannii from different infectious cases, 35% of isolates were ESBL-producers. Eleven isolates were resistant to imipenem (4 isolates) and meropenem (7 isolates). All carbapenem resistant isolates were MHT positive. Nine (45%) isolates were confirmed as A. baumannii by OXA-51 (all were carbapenem resistant). Distribution of IMP, VIM, GIM and SPM, OXA-23, OXA-143 and CTX-M-15 by PCR were 55, 50, 50, 25, 35, 45 and 33% respectively.Conclusion: The high prevalence of resistance genes and the resistance pattern of the isolates indicate that the detection of ESBLs and MBLs phenotypically and genotypically with the study of the resistance pattern of the isolates is critically important for the surveillance of drug resistance in the hospital environment.

Antimicrobial resistance pattern and molecular genetic distribution of metallo-β-lactamases producing Pseudomonas aeruginosa isolated from hospitals in Minia, Egypt.

Background: Pseudomonas aeruginosa (P. aeruginosa) represents a great threat to public health worldwide, due to its high ability to acquire resistance to different antibiotic classes. Carbapenems are effective against multidrug resistant (MDR) P. aeruginosa, but their widespread use has resulted in the emergence of carbapenem-resistant strains, which is considered a major global concern. This study aimed to determine the prevalence of carbapenem resistance among P. aeruginosa strains isolated from different sites of infection.Methods: Between October 2016 and February 2018, a total of 530 clinical specimens were collected from patients suffering from different infections, then processed and cultured. Isolates were tested for extended spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) production using double-disk synergy test, modified Hodge tests, and disc potentiation test. PCR was used for the detection of selected OXA carbapenemases encoding genes.Results: Of 530 samples, 150 (28.3%) P. aeruginosa isolates were obtained. MDR strains were found in 66.6% (100 of 150) of isolates. Of 100 MDR P. aeruginosa isolates, 54 (54%) were ESBL producers and 21 (21%) carbapenem resistant P. aeruginosa. MBL production was found in 52.3% (eleven) carbapenem-resistant isolates. CTX-M15 was found among 55.5% of ESBL- producing P. aeruginosa. Carbapenemase genes detected were blaIMP (42.8%, nine of 21), blaVIM (52.3%, eleven of 21), blaGIM (52.3%, eleven of 21), blaSPM (38%, 8/21). In addition, isolates that were positive for the tested genes showed high resistance to other antimicrobials, such as colistin sulfate and tigecycline.Conclusion: Our study indicates that P. aeruginosa harboring ESBL and MBL with limited sensitivity to antibiotics are common among the isolated strains, which indicates the great problem facing the treatment of serious infectious diseases. As such, there is a need to study the resistance patterns of isolates and carry out screening for the presence of ESBL and MBL enzymes, in order to choose the proper antibiotic.

Detection of different β- lactamases and their co-existence in gram negative bacteria isolated from clinical samples at a Tertiary care centre

Detection of different β- lactamases and their co-existence in gram negative bacteria isolated from clinical samples at a Tertiary care centre - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2019.0013, Indian Journal of Microbiology Research-Indian J Microbiol Res

Prevalence and antibiotic resistance pattern of Metallo-β-lactamase-producing Pseudomonas aeruginosa isolates from clinical specimens in a tertiary care hospital

Background: Pseudomonas aeruginosa is emerging as a nosocomial pathogen by producing metallo-β-lactamases (MBLs) and acquiring resistance to many antimicrobial agents. The infections caused by metallo-beta-lactamases producing P. aeruginosa (MBL-PA) are associated with higher rates of mortality, morbidity, and overall healthcare costs. Aim: The aim of the study was to find the incidence of MBL in P. aeruginosa isolates and their antimicrobial resistance pattern. Material and Methods: A total of 180 non-duplicate P. aeruginosa isolates from various clinical specimens between April 2016 and March 2017 were subjected to susceptibility testing by disc diffusion test as per the Clinical and Laboratory Standards Institute guidelines 2015. Imipenem and meropenem resistant isolates were selected for the detection of MBL production by disc potentiation test and modified Hodge test. Results: Out of 180 isolates of P. aeruginosa, MBL was detected in 36 (20.00%) isolates. Resistance was significantly higher in the MBL-PA with 94.44% resistance to aztreonam followed by cefoxitin (91.66%), piperacilline/tazobactam and cefepime (80.55%). The prevalence of multidrug-resistant and possible extensively drug-resistant isolates was significantly higher among the MBL group as compared to that in the non-MBL group [50.00% vs. 11.11% and 5.55% vs. 0.69% (P =

Molecular characterisation and antibiogram of water quality indicator bacteria in water samples from Klang river

Abstract Klang River is a 120km long extremely polluted River with 11 major tributaries. The pollution is due to the urbanization and industrialization. The aim of this research was to phenotypically identify multidrug resistant (MDR) induced traits of Pseudomonas aeruginosa and Enterobacter aerogenes, and to determine the molecular characterisation of these strains. In this research, 30 water samples were collected from different sites along Klang River. Their physical properties and biological oxygen demand were estimated. The Gram negative MDR strains were isolated and followed by Hodge test, Double Disk Synergy Test and Disk Potentiation Test, and plasmid DNA profiling was carried out. One MDR P. aeruginosa strain was isolated from the 30 samples; it was a non-carbapenemase and metallo-β-lactamase (MBL) producing strain. Nine E. aerogenes carbapenemase and MBL producing strains were identified, six of them were MDR, and the plasmid DNA profiling shows similarity among the isolates.

Prevalence of Extended Spectrum Betalactamase (ESBL) and Metallobetalactamase (MBL) Producing Pseudomonas aeruginosa and Acinetobacter baumannii Isolated from Various Clinical Samples.

This study was conducted with an objective to find the prevalence of extended spectrum betalactamase (ESBL) and metallobetalactamase (MBL) in P. aeruginosa and A. baumannii isolates obtained from various clinical samples. It was conducted in the Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda, over a period of two years from July 2014 to June 2016. Clinical specimens including urine, pus, blood, high vaginal swabs, respiratory samples, and various body fluids were processed and P. aeruginosa and A. baumannii isolates were identified by standard protocols. Antibiotic sensitivity testing for all isolates was done using Kirby-Bauer disc diffusion method. Disc potentiation test was performed to check ESBL and MBL production in these bacteria. Maximum ESBL positive isolates of P. aeruginosa were observed among pus samples and maximum MBL positive isolates were detected in tracheal aspirates. A. baumannii showed maximum positivity for ESBL and MBL production in endotracheal secretions. This study gives an alarming sign towards high prevalence of cephalosporin and carbapenem resistance due to production of extended spectrum betalactamases and metallobetalactamases, respectively. Early detection, stringent antibiotic policies, and compliance towards infection control practices are the best defenses against these organisms.

Drug Resistance and Molecular Epidemiology of Carbapenem Resistant Gram-negative Bacilli Isolates.

Objectives:Detection and comparison of metallo-β-lactamase (MBL) production in clinical isolates by phenotypic and genotypic measures. The objective of this study is to evaluate clinical characteristics and risk factors in patients infected with MBLs.Materials and Methods:Study was conducted by the Department of Microbiology, Jawaharlal Nehru Medical College from February 2014 to December 2015. Bacterial culture, identification, and antibiotic susceptibility were carried out according to standard guidelines. MBL production was detected both phenotypically (Modified Hodge test [MHT], imipenem-ethylene diamine tetraacetic acid double disk potentiation test [IMP-EDTA DDPT], IMP-EDTA combined disk synergy test [IMP-EDTA CDST]), and genotypically (blaNDM-1, blaVIM and blaIMP).Results:Among 116 carbapenem-resistant Gram-negative Bacilli (CRGNB), Citrobacter species 28 (24.1%) was the most common pathogen. Phenotypically, MHT, IMP-EDTA DDPT, and IMP-EDTA CDST detected MBL production in 105 (90.5%), 96 (81%), and 87 (75%) CRGNB, respectively. BlaNDM-1 genes were detected in 6 6 (56.8%) isolates, however, very few blaVIM (16, 15.2%) and blaIMP (1, 1.2%) were identified. Considering polymerase chain reaction (PCR) as the gold standard, it was observed that IMP-EDTA CDST was most specific (78.3%) while MHT was most sensitive (97.4%). Results of blaNDM-1 gene by PCR were further confirmed by sequencing (Triyat genomics, Nagpur). All the 11 representative strains were confirmed to be NDM-1 gene. Major risk factors in patients infected with MBLs were in-dwelling devices (68%), prolonged hospital stay (72%) and prior antibiotic treatment (86%). However, on tracing their outcome, it was interesting to note that mortality was relatively low 5 (4.3%).Conclusion:The present study shows a rising trend of blaNDM-1 in CRGNB, an ominous sign heralding the post antibiotic era. It is essential to assess the prevalence of various MBLs so that infection control measures can be reinforced. We recommend three phenotypic tests in tandem for the detection of MBL. While phenotypic tests are easy and cost-effective to perform, quick, effective molecular diagnostic techniques can tailor treatment guidelines to optimize patient's management.

A Modified Boronic Acid (MBA) Disc Potentiation Assay for Detection of Extended Spectrum ?-Lactamase (ESBL) Producing Bacteria

In this study, a modified boronic acid (MBA) disc potentiation assay has been used to detect extended spectrum â-lactamase (ESBL) producing isolates from clinical liquid waste (CLW) in Bangladesh. The method is modified from the existing boronic acid (BA) disc potentiation assay in that, in our protocol only one type of antibiotic disc is required in comparison to the requirement of different types of antibiotic discs for the BA test. Clavulanic acid (CA) and Boronic acid (BA) were used to phenotypically identify ESBL and AmpC producers, respectively. A total of 181 multidrug-resistant (MDR) isolates were screened for ESBL production by both the conventional double disc diffusion synergy test (DDST) and the MBA disc potentiation test. Among these two methods, 38% of the isolates showed positive ESBL phenotype in MBA test in comparison to the 24% of the isolates in DDST, hence showed higher efficiency. The MBA method described here is efficient, easy to perform, and cost effective to detect ESBL producing bacteria.Bangladesh J Microbiol, Volume 31, Number 1-2,June-Dec 2014, pp 29-33