Imipenem-EDTA Combined Disc Test Research Articles

Characterization of carbapenemase-producing Gram-negative bacilli: first report of blaNDM-1 in Enterobacter cloacae.

The spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacilli (CR-GNB), has become a serious challenge for clinicians due to limited therapeutic options. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates recovered from 352 samples collected in Tebessa hospital, Algeria. Bacterial isolates were identified by 16S RNA gene sequencing and susceptibility to antibiotics was determined by disk diffusion method. Carbapenem-resistant isolates were screened for carbapenemase production using modified carba Nordmann-Poirel test, modified Hodge test and imipenem-EDTA combined disc test. Extended-spectrum β-lactamases (ESBL) were detected using double-disk synergy test. Molecular characterization of carbapenemases and ESBL genes was performed by polymerase chain reaction (PCR) and sequencing. A total of 85 Gram-negative bacilli isolates were recovered mainly from urine samples and were identified as: Klebsiella pneumoniae (17.65%), Serratia odorifera (15.29%), Escherichia coli (12.94%), Raoultella ornithinolytica, Enterobacter cloacae (11.76%), Serratia marcescens (10.59%), Morganella morganii (7.06%), Proteus mirabilis (5.88%), Acinetobacter baumannii (4.70%) and Pseudomonas aeruginosa (2.35%). All strains were resistant or intermediate to imipenem and/or ertapenem. ESBL, carbapenemase and metallo-beta-lactamases (MBL) phenotypes were detected in 19 (22.35%), 9 (10.59%) and 2 (2.35%) GNB isolates, respectively. PCR results in nine carbapenemase-producing GNB strains chosen showed the presence of one to four carbapenemase genes (blaGES, blaSME, blaNDM-1, blaVIM, blaGIM, blaSPM, blaOXA-48) in four strains; however, seven strains had at least one ESBL gene (blaTEM-1, blaCTXM-15, blaSHV). In this study, we report the first incidence of blaNDM-1 gene in Enterobacter cloacae isolated from urine sample in Algeria.

Antibiogram and Detection of Metallo-β-Lactamase Producing Escherichia coli Isolated From Cow Dung in Owo Metropolis

This study was carried out to determine the antibiotic susceptibility profile and the production of metallo-β-lactamase (MBL) by Escherichia coli isolated from cow dung in Owo metropolis. The isolation of Escherichia coli was done using MacConkey agar and Eosin Methylene Blue Agar and was conventionally characterized. Antimicrobial susceptibility test of the isolates were by disc diffusion method against ceftazidime (30 µg), cefuroxime (30 µg), gentamicin (10 µg), cefixime (5 µg), ofloxacin (5 µg), augmentin (30 µg), nitrofurantoin (300 µg) and ciprofloxacin (5 µg). Detection of MBL producing isolates was by imipenem-EDTA combined disc test. The isolates showed highest resistance to augmentin (97.8%) and least resistance to nitrofurantoin (20.0%). Out of the 45 Escherichia coli isolated from cow dung, 8 (17.8%) produced MBL and were all multidrug resistant. The production of MBL and the high prevalence of antibiotic resistance observed among the Escherichia coli in this study infer that cow dung does not only serve as a reservoir for MBL-producers but also as source for the growth and dissemination of clinically significant antibiotic resistant species. Hence, the use of antibiotics as growth enhancers in cow production should be discouraged to help prevent the spread of antibiotic resistant bacteria and thus, preserve the efficacy of available antibiotics.

A STUDY OF PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF NON FERMENTER GRAM NEGATIVE BACILLI ISOLATED FROM VARIOUS CLINICAL SAMPLES AT A TERTIARY CARE CENTER, JAIPUR

Introduction: Non-fermentative Gram-negative bacilli (NFGNB) are occasionally involved in infectious diseases pathology, but have shown resistance to multiple antibiotics and the capability to gain new resistance factors in the hospital environment. The present study was aimed to investigate the prevalence of Metallo-beta-lactamase (MBL) producing non-fermenters among the carbapenem-resistant isolates. AMethods: total of 200 NFGNB isolates from various clinical samples were analyzed by using different standard microbiological techniques and antimicrobial susceptibility using Kirby-Bauer method. MBL detection was performed by Modied Hodge test (MHT), imipenem-EDTA combined disc test, and modied Carbapenemase inactivation method (mCIM). In our study, 200 NFGNB isolates recovered from various clinical samples.Results: Pseudomonas aeruginosa was the most common NFGNB, accounting 112 (56%), followed by Acinetobacter baumannii 56 (28%), Burkholderia species 26 (13%) and other rare nonfermenter Gram negative bacilli 6 (3%). NFGNB isolates were maximally isolated from blood samples 73 (36.5%) followed by respiratory samples 46 (23%), urine 40 (20%), pus 37 (18.5%) and CSF 4 (2%). All the NFGNB isolates were susceptible to polymyxin B, colistin (100%), and cefoperazone + sulbactam (45.53%). Out of 112 Pseudomonas isolates, 77(68.75%) were imipenem resistant and of 56 Acinetobacter isolates, 35(62.5%) were imipenem resistant. Out of 77 imipenem resistant Pseudomonas 70 (90.9%) were MBL producers and out of 35 imipinem resistant Acinetobacter spp 32 (91.42%) were MBL producers. NFGNB are now emerging asConclusion: organisms of nosocomial infections. The use of broad-spectrum antibiotics should be avoided and quick detection and efcient infection control measures are essential to prevent the further spread of MBLs to other Gram-negative bacilli. Detection of MBL production and rationale antibiotic usage are the most important factors which control the gradually increasing NFGNB related infections

Clinical Conditions and Risk Factors of Acinetobacter Baumannii Producing Metallo Beta-Lactamases Among Hospitalized Patients

Purpose: The study aimed to determine the clinical conditions and risk factors associated with MBL produced by A.bumannii among hospitalized patients. Subjects and Methods: The clinical samples were collected from inpatients and subcultured on routine culture media for growth. Identification of bacteria along with antimicrobial sensitivity testing was done by VITEK®2 compact (bioMerieux). Isolates that were resistant to Meropenem and/or Imipenem were followed to detection of MBL by using metallo-β-lactamases by imipenem EDTA combined disc test (IMP-EDTA CDT) method. Demographic and clinical data of each patient were collected in terms of the type of infection, hospital-stay, associated factors, and outcome till discharge. Results: A number of 73(86.9%) isolates of A.baumannii were resistant to carbapenem. out of 73 carbapenem-resistant isolates, 64(87.7%) were found to be MBL positive. The patients with age more than 60 years i.e. 35.9% (23/64) were found to be more common in MBL positive isolates of A. baumannii. The difference in the distribution of MBL positive and MBL negative cases with endotracheal intubation and in Surgery during the last 30 days of incubation was found to be statistically significant. The mortality rate of patients infected by MBL positive isolates of A.baumannii was 12.5%. Conclusion: The MBL positive strains among carbapenem-resistant isolates of A.baumannii were high. endotracheal intubation and Surgery during the last 30 days were independently associated with MBL positive cases.

Screening of Clostridium difficile Isolates for Metallo-Resistant Gene and Extended Spectrum Beta-Lactamases

Clostridium difficile naturally inhabits intestinal tract of animals including humans. Spores of C. difficile are known contaminant in the Environment. Indiscriminate use of antibiotics has led to the emergence of a powerful resistance determinant (known as Metallo resistant genes) in many bacteria worldwide. Multidrug resistant bacteria (MRB) pose threats to health globally. This study aims to determine the distribution of Metallo resistant Clostridium difficile in hospital environment within Yola. A total of 150 surface samples from the hospital wards were collected from Federal Medical Centre, Yola (FMC) and Specialist hospital, Yola (SHY) Adamawa state, Nigeria. Clostridium difficile were recovered by inoculating samples in modified Cycloserine-cefoxitin fructose broth (CCFB) and Cycloserine-cefoxitin fructose agar (CCFA). Screening of isolates for extended spectrum β-lactamase (ESBL) and metallo-β-lactamase enzymes was done by double disc diffusion method and Imipenem-EDTA combined disc test respectively. A total of 18 C. difficile isolates were obtained and found to be extended spectrum β-lactamase (ESBL) positive. 16 (88.89 %) of the Clostridium difficile were found to be metallo-β-lactamase positive. Polymerase chain reaction showed that 11(61.1 %) of the isolates possess bla-IMP metallo resistant gene. This study suggests that multidrug resistant C. difficile with metallo resistant genes are wide spread in hospital settings and there is need for relevant authorities to improve on the sanitization of environment as well as continuous surveillance to hinder C. difficile transmission.

Detection of VIM and NDM-1 metallo-beta-lactamase genes in carbapenem-resistant Pseudomonas aeruginosa clinical strains in Bahrain.

INTRODUCTION:Carbapenem-resistant Pseudomonas aeruginosa has emerged as a life-threatening infectious agent worldwide. Carbapenemase genes are reported to be some of the most common mechanisms for carbapenem resistance in P. aeruginosa. No reports are available from the Kingdom of Bahrain about carbapenem resistance and the underlying cause. In this study, we determined to study the presence of the metallo-beta-lactamase (MβL) genes of VIM family and NDM-1 in carbapenem-resistant P. aeruginosa strains.METHODOLOGY:Fifty carbapenem-resistant P. aeruginosa isolates were obtained from three main hospitals of Bahrain. They were subjected to antimicrobial susceptibility testing by disc diffusion test. Subsequently, MβL was detected by imipenem-ethylene diamine tetraacetic acid (EDTA) combined disc test and conventional polymerase chain reaction.RESULTS:Among 50 P. aeruginosa strains, 40 (80%) were imipenem resistant. Among the 40 imipenem-resistant strains, 35 (87.5%) strains were positive for the imipenem-EDTA combined disc test, and 21 (52%) were carrying MβL genes. Nineteen (47.5%) strains were positive for the VIM gene; one (2.5%) strain was carrying the NDM-1 gene, while one strain was carrying both the VIM and NDM-1 genes. None of the imipenem sensitive strains carried the VIM or NDM-1 gene.CONCLUSION:This is the first study to report the presence of the VIM family gene and NDM-1 genes in imipenem-resistant P. aeruginosa isolates in the Kingdom of Bahrain. The study also confirms the multiple drug resistance by the MβL strains, attention should therefore from now on, be focused on prevention of further spread of such isolates by firm infection control measures, and to reduce its threat to public health.

Molecular detection of carbapenem-resistant genes in clinical isolates of Klebsiella pneumoniae.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a considerable threat to public health worldwide. The aim of this study was to determine the prevalence of CRKP isolated from clinical specimens by phenotypic and genotypic methods. In total, 110 consecutive non-repetitive isolates of K. pneumoniae were analyzed. Minimal inhibitory concentrations (MICs) of imipenem were determined. The mechanism of resistance was evaluated by imipenem-EDTA combined disk test and modified Hodge test. PCR method was used for the detection of blaVIM, blaIMP, blaNDM-1, blaOXA-48 and KPC genes. Totally, 8 (7.3 %) isolates were resistant to imipenem, showing MIC ≥4 µg/mL. Based on imipenem-EDTA combined disk test, all imipenem-resistant isolates were metallo-beta-lactamase (MBL) positive. PCR confirmed that 6 (75%) isolates were blaNDM-1 positive. Other resistance genes (blaVIM, blaIMP, blaOXA-48 and KPC) were not detected. Based on this study, the prevalence of CRKP strains was not at a high level, however, continuous monitoring of antibiotic resistance should be performed to control dissemination of CRKP infections.

English

Acquired metallo-β-lactamases (MβL) are emerging determinants of resistance in Pseudomonas aeruginosa . The objectives of this study were to phenotypically detect MβL in P. aeruginosa collected in urology ward from Skikda hospital Algeria. A total of seventeen P. aeruginosa isolates were identified using API 20NE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOFMS). Antibiotic susceptibility was performed using disk diffusion method on Muller-Hinton agar. The minimum inhibitory concentrations (MIC) of imipenem were determined by Etest method. Positively screened isolates were further subjected to four different methods phenotypic; Modified Hodge test (MHT), Imipenem-EDTA combined disk test (CDT), Imipenem-EDTA double-disk synergy test (DDST) and new biochemical method Modified Carba NP test (MCNP).  Out of 32 (45.71%) isolates were resistant to imipenem; 20 (62,5%) isolates were MβL producing, 5 (15.62%) were carbapenemase class A or D producing, and 7 (21.87%) isolates were detected as negative test. Rapid detection of MβL-producing P. aeruginosa may help inappropriate antimicrobial therapy and avoid the development and dissemination of these strains. Thus far, the validation of a simple and accurate MβL detection method such as CDT, DDST and MCNP test, can be easily incorporated into the daily routine of a clinical laboratory. Key words: Phenotypic detection, Pseudomonas aeruginosa, Metallo-β-lactamases. &nbsp

Detection of Metallo-β-lactamase producing Pseudomonas aeruginosa in clinical isolates from hospitals in Erbil city, Iraqi Kurdistan

Multidrug resistance (MDR) became a great concern in P. aeruginosa has appeared as issue of great concern with the emergence of Metallo-β-lactamases (MBL)-producing isolates, which showed to be the primary resistance mechanism in carbapenem-resistant P. aeruginosa . The aim of the present study was to detect MBL-producing P. aeruginosa isolates from patients and evaluating antibiotic guide for clinicians in prescribing accurate antibiotic and to further controlling hospital infections. A total of 98 consecutive isolates of P. aeruginosa causing infections were isolated from various clinical samples. We applied Vitek-2 automated system as a panel of antimicrobial agents. Imipenem-EDTA combined disk test (CDT) and modified Hodge test (MHT) for phenotypic detection of MBL-producing isolates were followed. Out of 98 isolates of P.aeruginosa , only 72 isolates (73%) showed IMP-resistance and 26 (26%) were sensitive to imipenem. Around 63 (87.5%) of the IMP-resistant isolates were able to produce MBLs as determined by CDT, while 30 (41.6%) showed positive results in MHT. The resistance to tested antibiotics was significantly higher (P <0.001) with MBL producing P. aeruginosa isolates compared to non-MBL producers. The prevalence of MDR to the antibiotics among MBL-producing P. aeruginosa isolates was established. CDT is a reliable screening test for the detection of MBL producer

ESBL and MBL in Cefepime Resistant Pseudomonas aeruginosa: An Update from a Rural Area in Northern India.

Cefepime, a fourth generation cephalosporin, is widely used for the empirical treatment of serious infections in critically ill hospitalized patients. Pseudomonas aeruginosa (P. aeruginosa), one of the commonest bacteria causing nosocomial infections has a propensity to develop antibiotic resistance quite promptly. We undertook this study to assess the efficacy of cefepime against current clinical isolates of P. aeruginosa and to study existence of different beta-lactamase enzymes among cefepime resistant P. aeruginosa isolates. Total of 618 isolates of P. aeruginosa recovered consecutively from various clinical samples of a tertiary care hospital were analysed. Their Antimicrobial sensitivity profile against piperacilin (100μg), piperacillin/tazobactam (100μg/10μg), ceftazidime (30μg), cefoperazone (75μg), cefepime (30μg), ciprofloxacin (5μg), gentamycin (10μg), amikacin (30μg) and imipenem (10μg) (Himedia) was tested by Kirby-Bauer disc diffusion method (Clinical and Laboratory Standards Institute guidelines). We further looked for ESBL, MBL and ESBL + MBL co producers among the cefepime resistant isolates by two different methods (combined double disc synergy test, imipenem-EDTA combined disc test and vitek2). Among 618 consecutive clinical isolates of P. aeruginosa, we observed resistance to cefepime in 457 (74%) isolates. We observed resistance to ciprofloxacin (n=506, 82%) in maximum number of isolates followed by that to Gentamycin (n=475, 77%), amikacin (n=366, 60%), and cefoperazone (n=350, 56.6%). Among all our cefepime resistant P. aeruginosa isolates only 27(6%) were ESBL producers, 18(4%) MBL producers and 2(0.4%) were ESBL+ MBL co-producers. All the ESBL and MBL isolates were also tested by VITEK 2 advanced expert system (bioMırieux Vitek Systems Inc, Hazelwood, MO, France) which revealed a 100% concordance with the phenotypic method tested. This paper highlights the need to reconsider prescribing empirical antibiotics for Pseudomonas infections in this region and formulate a strong antibiotic policy to curb the menace of spread of multidrug resistant strains.

Approach to Carbapenemase Detection in Klebsiella pneumoniae in Routine Diagnostic Laboratories.

Resistance to Carbapenems in Klebsiella may be due to Carbapenem hydrolysing enzymes. Accurate detection of carbapenemase must be done for patient treatment and epidemiological purposes. To detect carbapenemase production by performing Modified Hodge Test (MHT), Combined Disk Test (CDT) for Metallo-β-Lactamases (MBL) and PCR for blaKPC gene, to evaluate the performance of MHT using MacConkey Agar (MCA) and to access the value of MHT for carbapenemase detection. Using a prospective laboratory study design, 153 Extended Spectrum Beta-Lactamases (ESBL) producing Klebsiella pneumoniae from clinical samples of patients admitted in the Kasturba Medical College were collected from January 2014 to December 2015. Isolates resistant to carbapenems by disk diffusion were subjected to MHT on MCA and Mueller Hinton agar (MHA). All isolates were tested for (MBL) production by Imipenem and Imipenem-EDTA CDT and subjected to PCR for the presence of blaKPC gene. Out of 153 isolates, 54 were resistant to one of the carbapenems. Among these, 13 were positive for MHT on MHA, while 23 were positive by MHT on MCA. Number of MBL producers was 23 (42.5%), while blaKPC was detected in 2 out of the 54 isolates. Though detection of drug resistance gene remains the method of choice, it can be performed only in centers with adequate resources. Hence, for most laboratories in resource poor countries, the MHT performed on MCA with concomitant CDT for MBL detection seem to be a better option for detection of Carbapenem resistance.

English

Resistance to broad spectrum β-lactams mediated by AmpC and metallo beta-lactamases (MBLs) enzymes is a rising problem worldwide. The wide dissemination of Gram negative bacteria harboring these enzymes represents a significant clinical threat during the last decade, which is mainly due to treatment failure and restriction of therapeutic options. This problem should be really estimated in our locality with special emphasis on immunocompromised patients. The aim of this study was to isolate Gram negative bacteria from differrent sites of infection among patients with hematological malignancy, and to examine those isolates for AmpC and MBLs production by phenotypic and genotypic methods. Seventy four Gram negative bacterial strains were isolated from 387 clinical samples collected from different infection sites. Those isolates were screened for the presence of AmpC and MBLs by modified three dimensional test and Imipenem–EDTA combined disc test, respectively. Multiplex PCR was done as a confirmatory step for detection of AmpC and MBLs production by these isolates. Pseudomonas aeroginosa was the most common isolated Gram negative strain that was found to be positive for AmpC and MBL production. DHA gene was the most frequently detected AmpC β-lactamase gene, whereas VIM was the only detected MBL gene among the Gram negative bacterial isolates by multiplex PCR. The strong association found between AmpC production and MBL gene carriage is alarming which necessitate continuous surveillance of such resistance mechanisms among the Gram negative bacteria, especially in patients with hematological malignancy. Key words: AmpC, metallo beta-lactamase (MBL), multiplex polymerase chain reaction, Pseudomonas aeroginosa.

English

BACKGROUND: Metallo-β-Lactamase (MBL) mediated resistance to various antibiotics is an emerging threat in Pseudomonas aeruginosa isolates. The present study was undertaken to detect MBL producing isolates of Pseudomonas aeruginosa from various clinical specimen by four different phenotypic methods. OBJECTIVE: To evaluate the various methods for the detection of Metallo-β-Lactamase in clinical isolates of Pseudomonas aeruginosa in a teaching hospital of rural Gujarat - India. STUDY SETTING: Department of Microbiology, P.S. Medical College & Shree Krishna Hospital, Karamsad, Gujarat. STUDY DESIGN: Prospective observational. MATERIALS AND METHODS: Total 50 isolates of Pseudomonas aeruginosa isolated & identified from different clinical specimens as per the standard guidelines. Screening for MBL production was done by Imipenem- EDTA combined disc test, Imipenem-EDTA double disc synergy test (DDST), EDTA disc potentiation using four cephalosporins, MBL E-test. RESULT: Of 50 imipenem sensitive or resistant isolates, 24 were MBL positive, Of which 12(24%) were MBL positive by Imipenem-EDTA combined disc test and Imipenem-EDTA double disc synergy test (DDST), 2(4%) were MBL positive by EDTA disc potentiation using four cephalosporins and 19(63.33%) were MBL positive by MBL E-test method. CONCLUSION: Imipenem-EDTA combined disc test and Imipenem-EDTA double disc synergy test (DDST) are equally effective for MBL detection while MBL E-test is more effective for MBL detection but given the cost-constraints, combined disc test and DDST can be used as a convenient screening method in the clinical microbiology laboratory.

Comparative Evaluation of Four Phenotypic Tests for Detection of Metallo-β-Lactamase and Carbapenemase Production in Acinetobacter baumannii.

Acinetobacter baumannii is an emerging multi-drug resistant opportunistic pathogen that causes a variety of nosocomial infections. In recent years, carbapenem resistance in A.baumannii has increased due to Ambler class B Metallo β-lactamases or class D OXA Carbapenemases. The present study was undertaken to detect and compare the various phenotypic methods for MBL production in nosocomial A.baumannii isolates. One hundred sixty eight A.baumannii isolates were subjected to disc diffusion assay. Imipenem resistant isolates were subjected to 4 different phenotypic tests. MBL screening was done by Imipenem-EDTA double disc synergy test, Imipenem-EDTA combined disc test, Modified Hodge test and MBL E-test. Out of 168 A.baumannii isolates, 85 (50.59%) were imipenem resistant. Among these 85 isolates, 57 (67.05%) were MBL positive by DDST, 69 (81.18%) by CDT, 85 (100%) by MHT and all these 85 isolates were confirmed to be MBL positive by MBL E-test method. Combined disc test, Modified Hodge test & E-test are equally effective to detect MBL production. However, considering the cost constraints of E-test, simple MHT and CDT can be used. They are easy, economical and can be incorporated into routine testing in laboratories to monitor the emergence of MBLs in MDR A.baumannii.

Occurrence of VIM-2 Metallo-ß- Lactamases in imipenem resistant and susceptible Pseudomonas aeruginosa clinical isolates from Egypt

Different metallo-β-lactamases (MBL) have been increasingly recognized among imipenem (IMP)-resistant Pseudomonas aeruginosa (P. aeruginosa) isolates. The existence of MBL IMP-susceptible isolates has been lately reported bearing the risk of unnoticed spread in hospitals. Early detection of MBL-producing organisms is critical to stop their uncontrolled spread and to allow for the prompt use of appropriate antibiotic. The current study aimed to determine MBL frequency among IMP-resistant and susceptible P. aeruginosa isolates by phenotypic and molecular testing in a medical hospital setting in Cairo. A total of 50 P. aeruginosa isolates from Theodor Bilharz Research Institute (TBRI) hospital were identified and examined for the phenotypic expression of MBL by Imipenem-EDTA com-bined disk test (CDT) and the MBL E test. MBL coding genes, blaVIM-2 and blaIMP-1, were detected by PCR. Of all P. aeruginosaisolates, 41 (82%) were MBL producers by phenotypic methods while PCR assay detected VIM-2 type of MBL gene in 35 (70%) and all test isolates were negative for IMP-1 gene. Based on data from the combined use of the three methods, 32 (64%) were confirmed to be MBL producers. MBLs were detected in 64% (23/36) IMP-resistant, 58.3% (7/12) IMP-sensitive and 100% (2/2) IMP-intermediate isolates by both phenotypic tests and gene amplification of blaVIM-2. All MBL carrying IMP-sensitive isolates had zone diameter between 16 to 20 mm by the Kirby-Bauer disk diffusion method. This study reports the occurrence of VIM-2 in IMP-susceptible P. aeruginosa isolates which may repre-sent a risk for therapeutic failure. We propose that isolates having an IMP zone diameter ≤20mm should be screened for the presence of MBL. The CDT is an alternative phenotypic assay to detect MBL production in our population in circumstances where PCR is not a feasible option. The high prevalence of isolates possessing MBL activity in the present study represents an emerging threat of complete resistance to carbapenemes among P. aeruginosa in Egypt. Key words: Pseudomonas aeruginosa, imipenem-resistance, metallo-β-lactamase (MBL) detection, VIM, imipenem (IMP).

Metallo– β – lactamase producing nonfermentative gram–negative bacteria: An increasing clinical threat among hospitalized patients

ObjectiveTo detect and evaluate the various methods for metallo- β -lactamases (MBL) production in Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter species. MethodsA total of 109 P. aeruginosa and 85 Acinetobacter species were screened for imipenem resistance by Kirby-Bauer disc diffusion methods. Detection of MBL production was done by imipenem-EDTA combined disc test, double disc synerygy test (DDST) and imipenem-EDTA MBL E test. ResultsA total of 63 (57.8%) strains of P. aeruginosa and 46 (54.1%) strains of Acinetobacter spp. were found to be resistant to imipenem. Of the 63 imipenem resistant P. aeruginosa tested for MBL production, 44 (69.8%) were found to be positive and among 46 imipenem resistant Acinetobacter, 19 (41.3%) were shown to be the MBL producers. ConclusionsImipenem-EDTA combined disc test and MBL E test are equally effective for MBL detection in both P. aeruginosa and Acinetobacter spp., but given the cost-constraints, combined disc can be used as a convenient screening method in the clinical microbiology laboratory.

Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates

The objectives of the study were to investigate the distribution of cphA-related genes (cphA) encoding a CphA metallo-β-lactamase (MBL) among 51 consecutive Aeromonas blood isolates and to compare different phenotypic methods for detecting CphA. The presence of cphA was detected by PCR. Four phenotypic methods, the imipenem-EDTA combined disc test, imipenem-EDTA MBL Etest, agar dilution test and modified Hodge test (MHT), were used to detect imipenem susceptibility and MBL production. The results showed that 35 (69%) blood isolates had cphA. All (100%) of 16 Aeromonas aquariorum isolates and 12 Aeromonas veronii isolates, and 4 (80%) of 5 Aeromonas hydrophila isolates, carried cphA, but none of 15 Aeromonas caviae isolates did. With the standard inocula, irrespective of the presence or absence of cphA, all but one (50, 98%) isolates were susceptible to imipenem tested by disc diffusion, Etest and agar dilution (10(4) c.f.u. spot inocula), and did not exhibit MBL production by the imipenem-EDTA combined disc test and MBL Etest. By the agar dilution test using large inocula (10(7) c.f.u.), 34 (97%) of 35 cphA(+) isolates had imipenem MICs of ≥16 µg ml(-1), higher than the susceptible breakpoint (4 µg ml(-1)), and demonstrated positive results for the MHT, while one cphA(+) and all 17 cphA(-) isolates had imipenem MICs of ≤4 µg ml(-1). In conclusion, the distribution of cphA among aeromonads is species-specific, found in A. aquariorum, A. veronii and A. hydrophila, and the MHT may be a phenotypic screening test for CphA production.

AN EVALUATION OF FOUR DIFFERENT PHENOTYPIC TECHNIQUES FOR DETECTION OF METALLO-β-LACTAMASE PRODUCING PSEUDOMONAS AERUGINOSA

Purpose: The present study was undertaken to detect metallo-β-lactamase (MBL) in nosocomial isolates of Pseudomonas aeruginosa by four different phenotypic methods. Methods: Ninety-one consecutive P. aeruginosa isolates were subjected to susceptibility testing by disc-diffusion assay and Vitek 2. Imipenem resistance was determined by three different methods (disc-diffusion, Vitek 2 and E test). Screening for MBL production was done by imipenem-EDTA combined disc test, imipenem-EDTA double-disc synergy test, imipenem-EDTA MBL E test and EDTA disc potentiation using four cephalosporins. Results: Of 63 imipenem resistant isolates, MBL screening could be done in 56 isolates, of which 48 were MBL positive by combined disc test and 36 by the double disc synergy test. For confirmation of MBL production, MBL E test was done in 30 isolates. All the 30 isolates were confirmed to be MBL positive by the MBL E test method. EDTA disc potentiation using four cephalosporins was not very useful for MBL detection. Conclusions: Imipenem-EDTA combined disc test and imipenem-EDTA MBL E test are equally effective for MBL detection, but given the cost-constraints, combined disc test can be used as a convenient screening method in the clinical microbiology laboratory.

An evaluation of four different phenotypic techniques for detection of metallo-β-lactamase producing &lt;i&gt; Pseudomonas aeruginosa&lt;/i&gt;

The present study was undertaken to detect metallo-beta-lactamase (MBL) in nosocomial isolates of Pseudomonas aeruginosa by four different phenotypic methods. Ninety-one consecutive P. aeruginosa isolates were subjected to susceptibility testing by disc-diffusion assay and Vitek 2. Imipenem resistance was determined by three different methods (disc-diffusion, Vitek 2 and E test). Screening for MBL production was done by imipenem-EDTA combined disc test, imipenem-EDTA double-disc synergy test, imipenem-EDTA MBL E test and EDTA disc potentiation using four cephalosporins. Of 63 imipenem resistant isolates, MBL screening could be done in 56 isolates, of which 48 were MBL positive by combined disc test and 36 by the double disc synergy test. For confirmation of MBL production, MBL E test was done in 30 isolates. All the 30 isolates were confirmed to be MBL positive by the MBL E test method. EDTA disc potentiation using four cephalosporins was not very useful for MBL detection. Imipenem-EDTA combined disc test and imipenem-EDTA MBL E test are equally effective for MBL detection, but given the cost-constraints, combined disc test can be used as a convenient screening method in the clinical microbiology laboratory.

Imipenem Resistant Pseudomonas aeruginosa: The fall of the final quarterback.

Objective:To isolate, determine the frequency, and study the demographic trends of MBL positive Pseudomonas aeruginosa from imipenem resistant isolates collected from clinical samples in a tertiary care hospital of Pakistan.Methods:In this cross sectional study a total of 230 strains of Pseudomonas were isolated from various clinical specimens on the basis of culture and biochemical tests. Imipenem resistant isolates were selected by Kirby Bauer Diffusion technique, followed by screening for MBL production by Imipenem EDTA Combined Disk Test. Demographic details of each patient were recorded on a separate questionnaire. Chi-Square goodness-of-fit test was computed to review the isolation of MBL positive isolates (P-value ≤ 0.05) in different specimen.Results:Out of 230 strains of P. aeruginosa 49.5% were imipenem resistant; MBL production was confirmed in 64.9% of the resistant isolates. Resistance to polymyxin B (12.5%) was notable. Majority of the MBL positive strains were isolated from patients aged between 20-39 years (45.9%) and the predominant source was pus (43.24%) which was found to be statistically significant (P-value=0.04). Outpatient departments (24.3%) and burn unit (21.6%) were the major places for resistant isolates.Conclusion:MBL production is one of the major causes of IRPA. Increasing resistance to polymyxin B is grave. Due to acquisition of MBL strains MDR P. aeruginosa has become endemic in tertiary setups.