EDTA Combined Disc Test Research Articles

Evaluation of the Expression of SIM and NDM Genes in Pseudomonas Aeruginosa Isolated from Clinical Sources

Pseudomonas aeruginosa has recently been labeled a major threat to public health due to its resistance to almost all commonly used antibiotics. Many factors have been suggested for P. aeruginosa's antibiotic resistance. The goal of this study is to find out what role SIM and NDM, which are related to carbapenem resistance, play in P. aeruginosa isolates from local clinical sources. In this study, out of 110 different clinical specimens, 50 were identified as P. aeruginosa from hospitalized patients. All of the isolates were characterized based on the biochemical test and confirmed using the VitekII compact system. P. aeruginosa isolates were tested for antibiotic susceptibility using 8 antibiotics, including: amikacin, tobramycin, ciprofloxacin, levofloxacin, imipenem, meropenem, piperacillin/tazobactam, ticarcillin, and clavulanate. Susceptibility testing results revealed that every isolate was highly resistant to Piperacillin/Tazobactam, with lower resistance to Ticarcillin/clavulanate (TCC). Using the broth dilution method, the minimum inhibitory concentration (MIC) of P. aeruginosa isolates resistant to meropenem ranged from 32µg/100µl to 128 µg/100µl. The EDTA combined disc test was used to detect the ability of P. aeruginosa isolates to produce carbapenemase, and the results showed that all isolates were carbapenemase producers. Additionally, conventional PCR confirmed the identification of P. aeruginosa using 16S. Real-time PCR was adopted to assess the expression of the NDM and SIM genes in 25 of the identified P. aeruginosa isolates. The mean of gene expression results for NDM showed increased expression compared to the control sample of 1.74, while the SIM gene showed expression of 0.95. These genes, SIM and NDM in class B (which are important for resistance in Pseudomonas aeruginosa), result from chromosomal changes that mutate the membrane permeability flow pump, causing excessive expression.

Molecular characterization of metallo-beta lactamase geneblaimp-1 in imipenem resistant Pseudomonas aeruginosa isolates from patients of chronic suppurative otitis media at a tertiary care hospital

Chronic suppurative otitis media (CSOM) is a chronic inflammation of the middle ear and mastoid cavity, persisting for more than 2 weeks. is one of the most common organisms to cause CSOM. Carbapenems are among the most effective antibiotics used against infections resistant to carbapenems is often associated with production of metallo-ß-lactamases strains. Detection of MBLs producing strains can be effective for optimal treatment of patients for the control and spread of resistance. The main purpose of the study is to determine the imipenem resistant carrying metallo-ß-lactamase (MBL) genebla in Patients of Chronic Suppurative Otitis Media at a Tertiary care hospital”This was a cross sectional study carried out in the Department of Microbiology and ENT Department for a period of 1 year between July 2021 to July 2022. A total of 185 patients clinically suspected cases for CSOM were studied. Swabs taken from discharging ears were sent for Gram’s staining, culture and antibiotic sensitivity test as per the latest CLSI guidelines. The isolates were further tested for MBL by screening test, by Imipenem – EDTA combined disc test, and MBL E test (Imipenem). The DNA was extracted by using Qiagen DNA Extraction kit and gene for isolates was detected by conventional PCR.In our study the number of cases clinically diagnosed of having CSOM was 185, out of which 63 (34%) was found to be culture positive for CSOM infection. Males were 38 (60.3%) as compared to that of female 25 (39.6%), Both the ears affected were almost in equal distribution, with the left ear being 32 (50.7%) and the right ear being 28 (44.4%) while in 3 cases (4.7%) it was bilateral. Gram negative bacilli were (74.6%), being the most common isolate with 47.6% followed by 19% and among the Gram positive isolates (25.3%). (12.6%) was the most common isolate. Colistin (96.6%), Piperacillin‑tazobactam (73.3%), Amikacin (76.6.3%), and Cefepime (73.3%) were found to be the most effective Antibiotics. The resistance to ciprofloxacin was (56.6%), Levofloxacin (50%), Piperacillin (46.6%), Gentamicin(36.6%), Imipenem (36.6%), Tobramycin (30%), Ceftazidime (30%). Out of 30 isolated 11(36.6%) were screening test-positives for MBL by Imipenem – EDTA combined disc test, and MBL E test (Imipenem). The bla gene was detected in all 11 (36.6%) of the isolates of In the present study being the most common isolate with Colistin, Piperacillin‑tazobactam, Amikacin, and cefepime to be the most effective antibiotics. Resistance of isolates to Imipenem due to MBL enzymes is increasing in the Kanpur region (36.6%). Judicial use of broad spectrum antibiotics, like Imipenem is the need of the hour, hence knowledge of the etiological agents of CSOM and their antibiogram is of paramount importance for an efficient treatment and prevention of both disease complications and antimicrobial resistance.

THE PREVALENCE OF BLANDM, BLAVIM GENES AMONG ENTEROBACTER CLOACAE BACTERIA

This study was aimed to detect the prevalence of blaVIM and blaNDM genes among Enterobacter cloacae isolates that were isolated from Iraqi patients. About 50 bacterial isolates were collected from different hospitals in Baghdad city and all these isolates were diagnosed using biochemical tests and CHROMagar culture media and conformed using the Vitek II system. The antibiotic susceptibility for E.cloacae isolates was determined using the disk diffusion (Kirby Bauer) method and the results showed that these bacteria were showed resistance to Cefepime and Meropenem antibiotics in percentage (50%), (40%) respectively. In addition it is resistant to Amikacin, ampicillin/sulbactam, Piperacillin, and aztreonam was (20%), (50%), (40%), and (55%) respectively. Also it has been noted that all isolates showed resistance to Cefixime in percentage (100%). EDTA combined disc test was used to detect the ability of E. cloacae isolates to produce carbapenemase and the results were showed that only 8 isolates were gave positive results , While the prevalence of blaVIM and blaNDM was determined using the conventional Polymerase chain reaction technique. And the result showed that only four isolates harbored blaNDM while blaVIM was present in 3 isolates

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.

<p>Antibiogram and Genetic Characterization of Carbapenem-Resistant Gram-Negative Pathogens Incriminated in Healthcare-Associated Infections</p>

PurposeCarbapenems are considered the most efficient antibiotic used in the treatment of nosocomial infections. Carbapenem-resistant Gram-negative rods are becoming a serious hazard in hospitals threatening public health. The aim of the current study was to investigate the prevalence of carbapenem-resistant Gram-negative pathogens incriminated in healthcare-associated infections, along with antimicrobial resistance profiles, carbapenemase and metallo-β-lactamase production, and their molecular characterization.MethodsA total of 186 clinical specimens were collected from 133 patients at various hospitals in Cairo, Egypt. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, detection of carbapenemase production using the modified Hodge test (MHT), the metallo-β-lactamase production using the EDTA combined disc test (CDT), and PCR-based detection of the blaKPC and blaGES resistance genes. The identification of the highly resistant retrieved isolates was then confirmed by 16S rRNA gene sequencing.ResultsThe most common isolated Gram-negative species was Klebsiella pneumoniae (40.9%), followed by Acinetobacter baumannii (18.8%), Pseudomonas aeruginosa (17.3%), Escherichia coli (15.4%), Enterobacter aerogenes (5.3%), and Proteus mirabilis (2.4%). The prevalence of carbapenem-resistant isolates was 36.1% (n=75). However, 86.5% of the recovered clinical isolates were susceptible to colistin. The MHT revealed that 33.6% (n=70) of the tested strains were positive for carbapenemase production, while the CDT showed that 33.17% (n=69) of the examined strains were metallo-β-lactamase producers. The PCR revealed that 98.6% (74/75) of the tested strains possessed the blaKPC gene; moreover, 97.3% (73/75) of the examined strains harbored the blaGES gene.ConclusionThis study displayed the emergence of carbapenem-resistant Gram-negative pathogens incriminated in healthcare-associated infections. The accurate identification of carbapenem-resistant bacterial pathogens is pivotal for the treatment of patients, in addition to propelling appropriate contamination control measures to restrain the fast spread of such pathogens. Colistin showed a potent in vitro antimicrobial activity against the carbapenem-resistant strains.

Comparative Study of Phenotypic Detection Methods of Metallo-beta-lactamase (MβL) Among Carbapenem Resistant Gram-Negative Bacilli (GNB) Isolated from Intensive Care Units (IUC) Patients

Background: Metallo-beta-lactamase (MβL) mediated resistance is an emergency threat in health care settings, and its identification is essential for treatment and infection control. Objectives: this study aimed to detect the MβL prevalence in Gram negative bacilli (GNB) and to compare its phenotypic detection methods. Methodology: Ninety six (96) isolates of GNB were isolated from different clinical specimens collected from patients admitted to intensive care units (ICU) of Ain Shams Hospitals, from February 2018 to June 2018. Isolates were screened for carbapenem resistance with imipenem 10 µg and meropenem 10 µg discs. The resistant isolates were tested for antibiotic susceptibility by disc diffusion method, and Meropenem minimum inhibitory concentration (MIC) were determinated, then the production of MβL was detected by imipenem-ethylene diamine tetra-acetic acid (EDTA) combined disc test (IPM-EDTA CDT), ceftazidime -EDTA combined disc test (CAZ-EDTA CDT) and Imipenem - EDTA double disc synergy test (IPM-EDTA DDST).Results: Forty three (43) isolates (44.7%) were resistant to carbapenem. Klebsiella pneumoniae (K.pneumoniae) was the most common isolated species; 29 (67.4%) isolates. Forty (40) isolates (93%) were positive for MβL by IPM-EDTA CDT method, whereas 36 (83.7%) were positive by CAZ -EDTA CDT method and 19 isolates (44.2%) were positive for MβL by IPM-EDTA DDST. Conclusion: High prevalence of MβL was detected among our isolates and IMP-EDTA CDT can be used as a phenotypic test in detection of MβL production.

Phenotypic detection of Metallo Beta Lactamase Producing Pseudomonas aeruginosa among Clinical Isolates from the intensive care unit of a tertiary care centre

Background: Pseudomonas aeruginosa is an opportunistic pathogenic bacterium responsible for both, acute and chronic infection, causing serious infections in patients who are mechanically ventilated individuals, who are immunocompromised, and patients with malignancies or HIV infection. Objectives: The phenotypic screening of metallo beta-lactamases (MBL) in strains of Pseudomonas aeruginosa resistant to imipenem. Material and Methods: The study was carried out in the department of Microbiology, in a medical hospital, Bareilly, over a period 18 months from January 2013 to June 2014. A total of 103 Pseudomonas aeruginosa isolated from sputum, broncho alveolar lavage (BAL), urine, pus and blood from critically ill patients admitted in the intensive care unit of a tertiary care centre. Strain of P. aeruginosa resistant to imipenem, were tested for metallo beta-lactamase production by phenotypic methods. Result: Out of 103 Pseudomonas aeruginosa, 48 strains were found imipenem resistant, of which 43(89.6%) were MBL positive by imipenem -EDTA combined disc test and the rest 35(72.9%) were by modified Hodge test. Conclusion: Imipenem – EDTA combined disc test found to be the best phenotypic method for detection of MBL. The appearance of the MBL and their spread among bacterial pathogens is a matter of concern with regard to the future of antimicrobial therapy. Both the methods are simple and one of these phenotypic methods can be easily incorporated in routine lab procedures to detect MBL.

English

Pseudomonas aeruginosa is a Gram negative aerobic rod shaped bacterium and is an opportunistic pathogen that usually causes nosocomial infection in immunocompromised patient with various infections and affects normal healthy human as well. P. aeruginosa is also an omnipresent pathogen that can be inhabited in soil, water, vegetable, human and animal. Metallo-β-lactamases (MBL) producing P. aeruginosa are known to be resistant to almost the entire anti-pseudomonas agent via mechanism of low outer membrane permeability, β-lactamases synthesis and the efflux systems. This study was conducted to detect the potential of metallo-β-lactamases producing P. aeruginosa presence in water samples from various parts of Malaysia. In this study, 52 water samples were collected from various parts of Malaysia. These P. aeruginosa isolates were processed to these phenotypic methods, Hodge test which is used to detect the carbapenemase production, Imipenem- EDTA combined disc test (CDT). Imipenem-EDTA double disc synergy test (DDST) was used to determine metallo-beta-lactamases producing P. aeruginosa. Among 52 various sources of water samples, 13 water samples had positive P. aeruginosa isolates. 6 out of 13 positive isolates have shown positive results in Hodge test, CDT and DDST. All metallo-β-lactamases producing isolates are multi-drug resistant. Among water samples from various parts of Malaysia, MBL producing P. aeruginosa are highly found in Selangor followed by KL, and this P. aeruginosa are detected from drain followed by river. Although some advantages and disadvantages exist among these phenotyping methods, still CDT and DDST are optimal to detect MBL. Key words: Metallo-β-lactamases, multi-drug resistance, phenotyping method, Pseudomonas aeruginosa.

Effective in Vitro Synergy of Piperacillin/Tazobactam Plus either Netilmicin or Aztreonam against Metallo-β-lactamase-producing Pseudomonas Aeruginosa

This study was designed to compare the activity of piperacillin/tazobactam (TZP) in combination with either netilmicin (NET) or aztreonam (ATM) against metallo-β lactamases (MβL) Pseudomonas aeruginosa. Out of 46 nosocomial Ps. aeruginosa isolates, 38 (82.6%) isolates were carbapenem-resistant by disc diffusion susceptibility testing. These were then screened by imipenem + EDTA combined disc test for MβL production. Thirty (78.9%) MβL-producing isolates detected were further tested for antibiotic synergy against NET-TZP and ATM-TZP combinations by Etest synergy and Etest/agar dilution tests. Neither combination showed antagonism. The synergistic effect of NET-TZP combination was detected in (27 (90%) and 28 (93.3%), respectively) and ATM-TZP combination in (24 (80%) and 25 (83.3%), respectively) of the tested isolates by Etest synergy and Etest/agar dilution tests, respectively. Thus, we have determined the synergistic effect of these two combinations for treating MβL-producing Ps. aeruginosa. Significance and Impact of the Study: The present study has shown valuable information on the combination treatment of Ps. aeruginosa and recommends their use for treatment of resistant Ps. aeruginosa.

English

Dissemination of carbapenem resistant Enterobacteriaceae (CRE) poses a considerable threat to public health. The aim of the present work was to estimate the prevalence of metallo beta lactamases (MBL) among CRE isolated from Alexandria Main University Hospital, Egypt, to evaluate the performance of different phenotypic methods for the detection of MBL, and to investigate the local antimicrobial sensitivity profile of these isolates. Eighty CRE were tested for MBL production by Etest® MBL MP/MPI, EDTA double disc synergy test, and EDTA combined disc test. All isolates were confirmed as MBL producers by polymerase chain reaction (PCR). Antibiotic sensitivity testing was performed using disc diffusion method. Among the 80 CRE, 56 isolates (70%) were MBL by PCR. Fifty-four isolates were positive for NDM (96.4%). Meropenem EDTA CDT was the most sensitive test (94.6%). Blood was the most frequent sample from which MBL were isolated (51.7%). Majority of the isolates were isolated from intensive care units (82.1%). All MBL were multidrug resistant; Colistin and polymyxin B showed the lowest resistance rate (26.8 and 19.6%, respectively). The EDTA-CDT will provide a reliable, convenient, and cost-effective approach for detection of MBL in laboratories, which cannot afford to perform molecular tests. Key words: Metallo beta lactamases, carbapenem resistant Enterobacteriaceae, New Delhi lactamase.

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.

Description of IMP-31, a novel metallo-β-lactamase found in an ST235 Pseudomonas aeruginosa strain in Western Germany.

The objective of this study was to characterize a novel IMP-type metallo-β-lactamase (MBL) found in an MDR clinical isolate of Pseudomonas aeruginosa. The P. aeruginosa isolate NRZ-00156 was recovered from an inguinal swab from a patient hospitalized in Western Germany and showed high MICs of carbapenems. MBL production was analysed by Etest for MBLs, an EDTA combined disc test and an EDTA bioassay. Typing of the isolate was performed by MLST. Genetic characterization of the new blaIMP gene was performed by sequencing the PCR products. A phylogenetic tree was constructed. The novel blaIMP gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization. The P. aeruginosa isolate NRZ-00156 expressed the ST235 allelic profile and was resistant to all the β-lactams tested except aztreonam. The isolate was positive for MBL production and harboured a new IMP allele, blaIMP-31, located on a disrupted class I integron [also carrying the blaOXA-35, aac(6')-Ib, aac(3)-Ic and aphA15 genes]. Its closest relative was IMP-35, with 96.7% amino acid identity. Expression of blaIMP-31 demonstrated that E. coli TOP10 producing IMP-31 had elevated resistance to all the β-lactams tested except aztreonam. Kinetic data were obtained for both IMP-31 and IMP-1. In comparison with IMP-1, IMP-31 showed weaker hydrolytic activity against all the β-lactams tested, which resulted from lower kcat values. The characterization of the new IMP-type gene blaIMP-31 from an ST235 P. aeruginosa isolate indicates an ongoing spread of highly divergent IMP-type carbapenemases in clinical P. aeruginosa strains and highlights the continuous need for the prevention of nosocomial infections caused by MDR Gram-negative bacteria.

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.

Molecular characterization of VIM1 isolates of Pseudomonas aeruginosa isolated from cases of sinusitis

Background Acquired metallo-b-lactamases (MBLs) are emerging resistance determinants in clinically relevant Gram negative species. These enzymes confer broad spectrum b lactam resistance, including resistance to carbapenems. Their potential for rapid and wide dissemination makes them of great concern. Methods This study describes the molecular characterization of 15 Pseudomonas aeruginosa strains, isolated from endoscopic pus specimens, obtained from sinusitis cases. Susceptibility testing was performed by disk diffusion assay. The MIC of ceftazidime was determined by E test. Ceftazidime (CDM) - EDTA combined disc test was used to screen for class B b lactamase production. Plasmid DNA extraction was performed by alkali lysis method. The carriage of the MBL gene, blaVIM and IMP1 gene were screened by PCR. PCR products of 7 positive strains for MBL were sequenced and analysed using bioinformatics tools. Results Metallo-beta-lactamase was present in 9 strains. These results correlated with the presence of VIM1 gene by PCR. IMP1 gene was absent in all the 15 strains. Plasmid was present in 10 strains only. The nucleotide and deduced protein sequences were analyzed with the software available over the Internet. The nucleotide sequences reported in the study has been submitted to the Genbank and accession numbers were obtained (KF975367, KF975368, KF975369, KF975370, KF975371, KF975372, KF975373). A Phylogenetic tree was constructed and it consisted of 2 clades. Both the clades had evolved from the common ancestor VIM-28. Conclusion Direct detection of MBL genes may circumvent the problems of the phenotypic detection of carbapenemase producing microorganisms especially those exhibiting low carbapenem MICs.

Incidence of metallo-betalactamase producing pseudomonas aeruginosa in Kesar SAL Medical College and Hospital, Ahmedabad

Metallo-betalactamase producing pseudomonas aeruginosa have been reported to show resistance to carbepenem drugs. Detection of metallo-betalactamases producing pseudomonas is now important to prevent their spread as dissemination of these bacteria could be fatal to the patients. The present study was undertaken over period of six months from November-2010 to May-2011 in Kesar SAL Medical College & Research institute, Ahmedabad to study the incidence of MBL producing pseudomonas aeruginosa from various clinical specimens. 100 isolates were obtained from different clinical samples of patients. These isolates were subjected to susceptibility testing to antipseudomonal drugs as per CLSI guidelines they were further screened for production of MBL by two methods i.e. EDTA impregnated imipenem double disc synergy method and Imipenem – EDTA combined disc test. Out of hundred isolates 55 imipenem resistant isolates were screened for MBL production. 38 isolates showed MBL production. MBL production was found to be 9.09%. This presents therapeutic challenge to the clinicians and also needs proper selection of antibiotics especially carbapenems.