Metallo-beta-lactamase Producers Research Articles

Detection of NDM-1 in Metallo-Beta-Lactamase-Producing Strains of Pseudomonas aeruginosa isolates from a Tertiary Hospital in Southwest Nigeria

Background: Metallo-beta-lactamase producingPseudomonas aeruginosa (MBL-PA) infections pose a major healthcare concern globally due to the associated extended morbidity, increased cost of treatment and high mortality.
 Objectives: This study aimed to determine the prevalence of circulating MBL-PA strains and evaluate probable risk factors associated with the carriage of MBL-PA among patients in a selected South-western tertiary hospital in Nigeria.
 Methods: One hundred and forty-four isolates recovered from diverse clinical specimens were identified as Pseudomonas aeruginosa by conventional methods. Eight antibiotics were tested on the isolates using Kirby-Bauer disc diffusion technique. All carbapenem resistant isolates were phenotypically screened for MBL-production using the combined disc synergy test. The MBL-producing strains were evaluated for the presence of three MBL genes blaIMP, blaVIM-2 and blaNDM1 by Polymerase chain reaction (PCR).
 Results: Fourteen (9.7%) isolates were positive for metallobeta-lactamase production by combined disc synergy test. The MBL-producing strains were more commonly resistant to all the tested antibiotics except to piperacillin tazobactam and imipenem compared with the non MBL producing strains (p<0.05). Antibiotic use and the occurrence of multidrug resistance phenotype were significantly associated with MBLcarriage (p<0.05). Four (28.6%) MBL-PA isolates carried NDM-1 gene, while IMP and VIM-2 genes were not detected in any of the isolates.
 Conclusion: The study demonstrated a low prevalence of NDM-1 in P. aeruginosa circulating among patients in this environment. This may be because carbapenems are seldom prescribed in our hospital or probably due to the existence of other MBL genes and non-enzymatic resistance mechanisms that we did not investigate. Studies that evaluate the level of carbapenem resistance via non-MBL production route could assist and improve future surveillance of these fast spreading genes.
 Keywords: IMP; MBL-PA; Metallo-beta-lactamase; NDM1; VIM.

Phenotypic Methods for the Detection of Metallo-Beta-Lactamase Production by Gram-negative Bacterial Isolates from Hospitalized Patients in A Tertiary Care Hospital in India

Drug-resistant bacteria are a global health concern owing to the high morbidity and mortality they can cause, especially in countries such as India. Gram-negative bacteria, including Enterobacteriaceae, Pseudomonas, and Acinetobacter, are primarily responsible for expanding the scope of drug resistance. These antibiotic-resistant pathogens are particularly associated with serious infections in hospitals. The production of carbapenemase by gram-negative bacteria appears to be the major reason for their resistance to carbapenems. The study was a prospective study done from March 2018 to December 2020. All the carbapenem-resistant isolates from various clinical samples were further tested for the production of carbapenemases/metallo-beta-lactamases production by various phenotypic tests like carbaNp, Imipenem–EDTA combined disc synergy test, Double-disc synergy test and E-test methods. Of all carbapenem-resistant gram-negative bacteria isolated from patients in a hospital in India, 237 (88.1%) carbapenemase producers were identified, among which 217 (91.5%) were metallo-beta-lactamase (MBL) producers. Therefore, the detection of MBL producers is important for preventing their infectious spread. The present study revealed that most MBL producers were isolated from patients of 0–9 to years of age (63.9%). The double-disc synergy test (DDST) and E-test MBL strips were more sensitive than the combined disc test in detecting MBLs. Because the DDST was the simplest and most effective method, it can be used for the routine laboratory screening of MBL producers in hospitals.

Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Klebsiella spp. Isolated at the National Reference Laboratory, Kathmandu, Nepal

Klebsiella spp. are associated with several nosocomial and opportunistic infections. Increasing antimicrobial resistance of Klebsiella species is aggravated by a number of intrinsic and extrinsic factors. The main aim of this study is to determine antimicrobial resistance due to production of β-lactamase enzymes, extended spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC and Klebsiella pneumoniae carbapenemase (KPC) and biofilm formation in Klebsiella isolates. A total of 2197 non-duplicate specimens of urine, sputum and pus were obtained from the National Public Health Laboratory (NPHL), Kathmandu, Nepal, between February and August 2019. Klebsiella species were isolated, identified and screened for antimicrobial susceptibility testing with the disk diffusion method. Phenotypic detection of ESBL, MBL, KPC and AmpC production was observed and biofilm production was detected by the microtiter plate method. Out of a total of 2197 clinical specimens, bacterial growth was detected in 8% (175/2197) of the specimens. Of the total isolates, 86.3% (151/175) were Gram-negative bacteria and 37.7% (57/151) were Klebsiella spp. Of the total Klebsiella spp., 56% (32/57) were multi drug resistant (MDR), 16% (9/57) were ESBL, 26% (15/57) were MBL, 4% (2/57) were KPC (class A carbapenemase), 16% (9/57) were AmpC producers and 95% (54/57) were biofilm producers. Gentamicin was the most effective antibiotic, followed by cotrimoxazole, as 68% (39/57) and 47% (27/57) of the Klebsiella isolates were susceptible towards these drugs, respectively. The study results show evidence of β-lactamase production, high prevalence of MDR and biofilm producing Klebsiella species. Integrating the test parameters for phenotypic confirmation of ESBL, MBL, AmpC β lactamase and KPC in routine diagnostic procedures can help in the early detection and management of these resistant strains.

High prevalence of multidrug-resistant non-fermentative Gram-negative bacilli harboring bla IMP-1 and bla VIM-1 metallo-beta-lactamase genes in Birjand, south-east Iran.

Background and Objectives:Non-fermentative Gram-negative Bacilli (NFGNB) is known as a major cause of healthcare-associated infections with high levels of antibiotic resistance. The aim of this study was to investigate the antibiotic resistance profiles and molecular characteristics of metallo-beta-lactamase (MBL)-producing NFGNB.Materials and Methods:In this cross-sectional study, the antibiotic resistance profile of 122 clinical NFGNB isolates was determined by the Kirby-Bauer disk diffusion and microdilution broth methods. Bacterial isolates were investigated for the detection of MBLs production using the combination disk diffusion Test (CDDT). The existence of blaIMP, blaVIM, and blaNDM genes in all carbapenem-resistant isolates was determined employing polymerase chain reaction (PCR) assays.Results:High resistance in Pseudomonas aeruginosa was reported to cefotaxime and minocycline, whereas Acinetobacter baumannii isolates were highly resistant to all antibiotics except colistin. Multidrug resistance (MDR)-NFGNB (66% vs. 12.5%, P=0.0004) and extensively drug resistant (XDR)-NFGNB (55.7% vs. 12.5%, P=0.001) isolates were significantly more common in hospitalized patients than in outpatients. The production of MBL was seen in 40% of P. aeruginosa and 93.3% of A. baumannii isolates. It was found that 33.3% and 46.7% of carbapenem-resistant P. aeruginosa isolates, and 13.3% and 28.9% of carbapenem-resistant A. baumannii isolates were harboring blaIMP-1 and blaVIM-1 genes, respectively. The incidence of MDR (98.2% vs. 28.3%, P<0.001) and XDR (96.4% vs. 11.7%, P<0.001) in MBL-producing NFGNB isolates was significantly higher than non-MBL-producing isolates.Conclusion:This study demonstrated a higher rate of resistance among NFGNB isolates with an additional burden of MBL production within them, warranting a need for robust microbiological surveillance and accurate detection of MBL producers among the NFGNB.

Identification, Speciation and Antibiogram along with Detection of Metallo Beta-lactamase Production in Acinetobacter Isolated from Clinical Samples in a Tertiary Care Hospital

Acinetobacter species are gram negative non fermenters, which are important nosocomial pathogens involved in various outbreaks in hospitals due to widespread resistance to majority antibiotics. The aim of this study is to speciate Acinetobacter isolated from clinical samples, to assess the antibiotic sensitivity pattern and to detect the production of metallo-β-lactamase by double disc synergy test. The study was conducted in the department of microbiology, A. J Institute of Medical Sciences. All clinical samples were subjected to gram stain &amp; cultured; the Acinetobacter isolates obtained were subjected to antibiogram. Those isolates that showed Imipenem resistant were further tested for production of metallo-β-lactamase by double disc synergy test. Out of 6625 culture positive isolates, 414 (36.1%) were identified biochemically to belong to Acinetobacter species. Of the 414 cases, 393 (94.9%) were further identified to be Acinetobacter baumannii and the remaining 21 (5.1%) to be Acinetobacter lwoffi. Acinetobacter lwoffii showed 100% sensitivity to all the drugs. Of the 393 Acinetobacter baumannii isolates 109 (27.7%) showed resistant to Imipenem. Out of these 109 isolates, 65 (59.63%) were positive for metallo-β-lactamase production by double disk synergy test. The speciation is highly demanding and laborious but it’s important to be demonstrated due to difference in the antibiotic susceptibility pattern. Carbapenem resistant Acinetobacter nosocomial strains in ICUs are detected to be more resistant to antibiotics. As shown in this study the metallo-β-lactamase producing A.baumannii isolates were 59.63% and therapeutic options were limited. Therefore early identification of metallo-β-lactamase producers is of great importance to start appropriate treatment and to control the spread.

Identification of bacteriological quality and antimicrobial resistance of microorganisms isolated from animal foods collected from the abattoir, butcher shops and local seafood market

The current study aimed to isolate bacteria that harbour various animal food products like meat, chicken and seafoods collected from the abattoir, butcher shops and local seafood market and to determine the antimicrobial resistance pattern of isolated pathogens which are responsible for various foodborne illnesses in human beings. A total of forty raw animal product samples were collected from the abattoir, butcher shops and local seafood market of Visakhapatnam. The samples selected for the study include raw chicken, meat, crab, prawns and different varieties of fish. A classic random sampling technique was employed to collect the study samples. All the samples were processed immediately using standard microbiological protocols. The bacteria isolation and characterization were done by studying morphological characteristics with staining methods, cultural characteristics by isolating and growing the pathogenic microorganisms in various selective and differential culture media. Antimicrobial susceptibility testing was performed by the Kirby -Bauer method by following Clinical and Laboratory Standards Institute (CLSI) guidelines. EDTA-Disc Potentiation Test and Imipenem-EDTA Double disc synergy test are used to detect the metallo beta-lactamase production of isolated pathogens. The highest number of isolates belong to Salmonella species (18), Pseudomonas aeruginosa (18) followed by Vibrio species (14) and few isolates belong to Enterobacter species (4). Majority of the microbial isolates obtained in the current study were multidrug resistant. The isolates from the abattoir environments, slaughterhouses, fish markets were found to exhibit variable resistance pattern to aminoglycosides, macrolides, β-lactams, cephalosporins, quinolone antibiotics used in the present study and at the same time most of them were sensitive to carbapenem antibiotic imipenem. Antimicrobial resistance (AMR) prevents the designing and assessment of effective interventions. If such a link can be established, then the tracking of antibiotic use and consumption data could be furthermore used as a surrogate indicator for the risk of potential antibiotic resistance (ABR) emergence.

Molecular Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Isolates Producing blaVIM, blaNDM, and blaIMP in Clinical Centers in Isfahan, Iran

Background: The emergence and spread of metallo-beta-lactamase (MBL)-producing Klebsiella pneumoniae are growing global public health concerns. One of the most common mechanisms of carbapenem resistance is the production of MBLs, including Verona integron-encoded metallo-beta-lactamase (VIM), New Delhi metallo-beta-lactamase (NDM) and imipenemase (IMP). Objectives: This study aimed to investigate MBLs production among K. pneumoniae isolates. Methods: In this study, 240 K. pneumoniae isolates were collected from clinical samples in three clinical centers of Isfahan, Iran, during February 2017 and November 2018. All isolates were identified using biochemical, microbiological, and molecular methods, and then antimicrobial susceptibility tests were performed to find MBL-producing isolates via phenotypic and genotypic detection methods. Moreover, the minimum inhibitory concentration (MIC) of antibiotics against MBL-positive strains was determined by E-test. Eventually, the clonal relatedness of the MBL-positive strains was analyzed using both multilocus sequence typing (MLST) and rep-PCR. Results: Overall, 33.7% (81/240) of the isolates were resistant to carbapenems, among which 25 (30.8%) were considered MBL-positive. Among 81 strains resistant to carbapenems, genes encoding FimH, rmpA, and mrkD were detected in 87.6% (71/81), 11.1% (9/81), and 67.9% (55/81) of the isolates, respectively. Besides, TEM and SHV as antibiotic resistance genes were detected in 49.3% (40/81) and 80.2% (65/81) of the isolates. But, magA was not detected in any of the tested isolates. The PCR results revealed that blaVIM-1 was the most prevalent gene (13.6%; 11/81), while both blaIMP-1 and blaNDM-1 were only detected in two isolates. Multilocus sequence typing demonstrated that 15 MBL producers belonged to three sequence types (ST): 11 to ST23, two to ST1147, and two to ST15. Finally, rep-PCR typing showed similar fingerprints with MLST, except for ST23, such that ST23 was discriminated in two clonal groups, suggesting the greater discriminatory power of rep-PCR. Conclusions: Here, we reported the emergence of MBL-producing K. pneumoniae in clinical centers of Isfahan, Iran. The findings are alarming and represent the urgent need for the application of infection control programs.

Prevalence of Extended-Spectrum Beta-Lactamases (ESBLs) and Metallo-BetaLactamases (MBLs) Among Healthy and Hospitalized Children in Abraka and Eku Communities, Delta State, Nigeria

This study was conducted to detect the prevalence of Metallo-Beta-Lactamases (MBLs) and Extended Spectrum Beta-Lactamases (ESBLs) in bacterial isolates from clinical and community settings of two communities in Delta State, Nigeria. Eighty four isolates obtained from blood, urine, wound and stool of patients and community subjects were analyzed and identified by standard microbiological methods. Carbapenemase detection was carried out using modified Hodge test. EDTA-disc synergy test was used todetect MBLs production in 26.2% of the isolates. ESBLs production determined by double disc synergy test (DDST) was detected in 36.7% isolates of Escherichia coli, 37.5% of Pseudomonas aeruginosa and 75% of Klebsiella pneumoniae. Co-production of MBL and ESBL was observed in 31.8% of the isolates. The study observed 2 major troubling findings. The first is the prevalence and co-production of MBLs and ESBLs both in hospitalized patients and in isolates of healthy community children. Second, antibiotic resistantbacteria may be able to persist in the community free from the high selective pressure pervading in clinical settings. There is therefore the need for increased surveillance.&#x0D; Keywords: Antibiotic resistance, Multidrug resistance, Healthy children, Beta-lactamases, Plasmids

Determination rates of antibiotic resistance, inducible beta-lactamase, and metallo beta-lactamase ratios in Pseudomonas aeruginosa isolates in a university hospital in Turkey

Objective: This study aimed to determine the antibiotic resistance, inducible beta-lactamase (IBL), and Metallo beta-lactamase (MBL) rates in P. aeruginosa isolates.&#x0D; Material and Methods: In our study, 100 P. aeruginosa isolates obtained from various clinical samples were used. Antibiotic susceptibility was performed by using the Kirby-Bauer disk diffusion method. Carbapenem resistance to imipenem and meropenem was verified by the E test. The disk induction method was used to determine the IBL production while the Modified Hodge test, MBL E test, and combined imipenem/ EDTA disk were used to determine the production of MBL.&#x0D; Results: According to the results of antibiotic susceptibility tests, 58% of P. aeruginosa isolates were susceptible to all antipseudomonal drugs, while resistance rates to other drugs were as follows; ceftazidime 7%, cefoperazone sulbactam 8%, cefepime 13%, piperacillin 14%, piperacillin-tazobactam 12%, imipenem 9%, meropenem 11%, aztreonam 8%, amikacin 8%, gentamicin 13%, tobramycin 12%, netilmicin 19%, There was a 10% resistance to ciprofloxacin. 8% of the isolates were resistant to at least three drugs, of which two isolates were positive for MBL enzyme production. IBL production was detected in 86% of the isolates with the disk induction method.&#x0D; Conclusion: The results we obtained in our study are consistent with other researchers globally and in Turkey. It was concluded that there is a need for well-standardized phenotypic tests with defined evaluation criteria and further studies to verify these tests genotypically.

Extended-spectrum and Metallo-beta lactamase enzymes mediated resistance in Pseudomonas aeruginosa in clinically isolated specimens

Pseudomonas aeruginosa is one of the leading opportunistic pathogens, frequently highlighted for the production of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-beta Lactamase (MBL) enzymes. This is believed to be the primary inhabitant of soil due to its adaptive nature and can survive in aquatic and even in a toxic environment. The current study aimed to screen ESBL, MBL producing, and Multiple Drug Resistant (MDR) strains of P. aeruginosa. Clinical specimens collected from patients were screened for the presence of P. aeruginosa. After identification, all the isolates were tested for the sensitivity pattern following the Kirby-Bauer disc diffusion method. The presence of ESBL and MBL enzymes were detected following DDST and IMP-EDTA detection tests, respectively. The sample size used in the study was 1369, and colonies for P. aeruginosa were obtained from 126 (9.20%) samples of culture media. 54.76% (69/126) of the positive cases were detected in the female population, whereas 45.24% (57/126) in the male population. High-frequency rate (n=43/126) was detected in the age group ≥31 year, followed by 21-30 (n=35/126), and 11-20 (n=34/126) age group, the minimal frequency (n=14/126) was detected in age group 0-10. The sensitivity pattern showed that the majority of the isolates were resistant, but class carbapenem (imipenem, meropenem), aminoglycosides (amikacin, gentamicin, tobramycin), and other antibiotics like sulbactam- Cefoperazone, tazobactam-piperacillin, tigecycline, and Fosfomycin exhibited the best efficacy against P. aeruginosa. It is concluded that the emergence of resistance due to ESBL and MBL enzymes in P. aeruginosa is directly linked with a public health concern because these strains are almost resistant to a wide range of antibiotics, and only limited antibiotics are potent against these organisms.

Isolation of Metallo Beta Lactamases from Clinical Sample (MBL)

The rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is an emerging threat and a matter of particular concern worldwide. Carbapenems are among the few useful antibiotics against gram negative bacteria particularly those with extended spectrum beta-lactamase. Resistant to carbapenem is mediated by loss of outer membrane proteins and production of beta lactamase that is capable of hydrolyzing carbapenems.During this study period, 80 different clinical samples were collected from various hospitals of Raipur. All clinical samples were processed according to standard microbiological methods. Isolated GNRs were subjected to susceptibility testing against antibiotics by disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Carbapenem-resistant isolates were subjected to the detection of MBL production by different methods. Out of 80 samples, 28 (35%) samples showed significant growth. MBL production was observed in 20 samples.

Metallo-Beta-Lactamase-Producing Multidrug-Resistant Acinetobacter Isolates in Patients with Ventilator-Associated Pneumonia

Background: Acinetobacter, a nonfermenting Gram-negative coccobacilli, have emerged as significant pathogens causing multidrug-resistant (MDR) ventilator-associated pneumonia (VAP). Metallo-beta-lactamase (MBL)-producing Acinetobacter spp. have become an emerging therapeutic concern worldwide due to the MDR isolates. Aim and Objectives: Phenotypic detection of MBL producing MDR Acinetobacter isolates in patients with VAP and to study the antibiotic susceptibility pattern of MBL-producing isolates. Materials and Methods: This was a prospective observational and noninterventional study conducted on patients with VAP over a period of 2 years. This study was conducted at a tertiary care teaching hospital in the intensive care unit. A total of 164 MBL-producing MDR AB isolates were included in the study. MBL was detected by imipenem-EDTA double-disc synergy test (DDST), imipenem-EDTA combined disc synergy test (CDST-IPM), and MBL-E test. Results: A total of 188 samples were enrolled for the study, fulfilling the inclusion criteria of VAP. Total MDR Acinetobacter spp. isolates were 188 (76.42%) of them, 164 (87.23%) were MBL producing and 24 (12.76%) were nonMBL (P

Comparative evaluation of phenotypic methods for detection of MBL producing Pseudomonas aeruginosa strains isolated from burn wound infections

Introduction: Pseudomonas aeruginosa is a notorious nosocomial pathogen with a resistant drug profile and a predominant isolate in burn wound infections. Recently Metallo beta lactamase (MBL) producing isolates have emerged particularly in P.aeruginosa leading to failure of therapy with carbapenems with increased mortality and morbidity in burn units. Objectives: To detect MBL production among the imipenem resistant isolates of P.aeruginosa by different phenotypic methods – Modified Hodge test (MHT), Combined disc test (CDT), Imipenem-EDTA Double disc synergy test (DDST) and MBL-E-test. Materials and Methods: A total of 108 clinical isolates of P.aeruginosa obtained in 189 pus samples collected from different burn wound sites, were subjected to antimicrobial susceptibility testing by modified Kirby-Bauer disc diffusion method. All the imipenem resistant isolates were further tested for MBL-production by MHT, CDT, DDST and MBL-E-test. Results: Out of 108 isolates of P.aeruginosa 26 (24.07%) ware imipenem resistant. Among 26 imipenem resistant isolates 16(61.54%) were detected as MBL producers by MBL-E-test, 15(57.69%) by DDST, 14(53.85%) by CDT and 11(42.31%) by MHT. Considering MBL-E-test as standard, maximum sensitivity was shown with DDST (93.75%) followed by CDT (87.5%) and MHT (68.75%). Conclusion: A comparative evaluation of MHT, CDT and DDST against MBL-E-test proved DDST to be most effective, with higher sensitivity and specificity. DDST is observed to be an economical feasible alternative compared to MBL-E-test as a regular screening test in MBL detection in critically ill. Keywords: Pseudomonas aeruginosa, Metallo beta lactamases (MBLs), Modified Hodge test (MHT), Combined disc test (CDT), Imipenem -EDTA Double disc synergy test (DDST), MBL-E-test.

Detection of metallobetalactamase producing imipenem resistant acinetobacter species in intensive care unit patient in a Tertiary Care Centre

Introduction: Metallobetalactamase producing Acinetobacter species has been reported to be an important cause of nosocomial infection and is a critical therapeutic problem worldwide, especially in the intensive care unit. Objectives: To determine the frequency of metallo‐β‐lactamases among imipenem‐resistant Acinetobacter species and to compare different phenotypic methods. Materials and Methods: 59 imipenem‐resistant Acinetobacter species isolated from various clinical samples were tested for metallo‐β‐lactamase production using different phenotypic methods. Minimal Inhibitory Concentratrion (MIC) to meropenem was determined by E test. Results: Of all the imipenem resistant isolates, 50.8% of Acinetobacter species were MBL producers. MIC of all those MBL producing isolates were ? 16?g/ml. Among the Acinetobacter species MBL production were detected in 30 isolates (50.8%) by E test, 29 isolates (49.2%) by CDT and in 15 isolates (25.4%) by DDST and DPT. The sensitivity and specificity of CDT, DDST and DPT compared to E test was 98%, 48.1%, 48.1% and 100%, respectively. Conclusion: Prevalence of MBL producing Acinetobacter species is being increasingly reported in ICU patients. The MIC of all the MBL producing isolates for meropenem were >16?g/ml (Resistant). E test and CDT were more reliable for MBL detection. CDT was cost effective, easy to perform and interpretation also straightforward. MBL producing isolates were multidrug resistant making therapeutic choices limited. Continuous antibiotic surveillance, infection control practices and an effective antibiotic policy are required to address the problem of MBL – associated infections. Keywords: Acinetobacter species, MBL detection.

CO-PRODUCTION OF EXTENDED-SPECTRUM BETA-LACTAMASES AND METALLO BETA-LACTAMASES AMONG MULTI-DRUG RESISTANT GRAM-NEGATIVE BACTERIA ISOLATES COLLECTED FROM TERTIARY HOSPITALS IN OYO STATE, NIGERIA

Extended-spectrum beta-lactamases (ESBLs) and metallo beta-lactamases (MBLs) are compromising the chemotherapeutic use of cephalosporins and carbapenems respectively. This study investigated the burden of ESBLs and MBLs co-production among multi-drug resistant (MDR) Gram-negative bacteria collected from two tertiary hospitals in Oyo State. A total of 240 non-duplicated clinical isolates of Escherichia coli, Klebsiella spp. and Pseudomonas spp. were collected from the Microbiology units of two tertiary hospitals in Oyo State and their identities authenticated using standard identification techniques. Antimicrobial susceptibility testing was carried out by disc-diffusion method and isolates exhibiting resistance to ≥3 classes of antibiotics selected as MDR strains. ESBL and MBL production was detected by double-disc synergy test (DDST) and combined-disc-diffusion test (CDDT) respectively. Selected beta-lactamase genes were detected by PCR, amplicons sent out for sequencing and phylogenetic tree of the sequences constructed using Mega X software. MDR was exhibited by 43.8% of the isolates. ESBLs and MBLs were produced by 32.4% and 7.6% of the MDR isolates respectively. Co-production of ESBL and MBL was observed in 6.7% of the MDR isolates. BlaCTX-M-15 (67.7%), blaTEM-1 (55.9%), blaSHV-1 (47.1%), co-existing blaTEM + blaSHV, blaTEM + blaCTX-M, blaCTX-M + blaSHV (each in 5.9%) and blaCTX-M +blaTEM + blaSHV (26.5%) were detected among the ESBL-producers. MBL genes were not detected among the MBL-producers. Only blaTEM-1 sequences showed two different claudes on the phylogenetic tree. The occurrence of MDR isolates co-harbouring different classes of beta-lactamse genes observed in this study is of public health concern and hence, requires stricter control of antibiotic use.

Prevalence of Community Acquired Uropathogens and their Antimicrobial Susceptibility in Patients from the Urology Unit of A Tertiary Care Medical Center

Urinary tract infections (UTI) are one of the most common community acquired infections and can also present with similar lower urinary tract symptoms (LUTS). Moreover, UTI can be a complication of these urological diseases. Thus, this study was conducted in patients with LUTS to find out the prevalence of uro-pathogens and their antibiotic susceptibility pattern so that appropriate antibiotics can be started on clinical suspicion of UTI. The study was conducted over a period of 12 months. Culture and susceptibility of urine specimen was done as per standard microbiological guidelines. Apart from the growth of common bacteria with their antibiogram, Methicillin-resistant Staphylococcus aureus (MRSA), high level aminoglycoside resistance (HLAR) was investigated among Enterococci, and metallo beta-lactamases(MBL) production was production was investigated among gram-negative pathogens. Out of 407 urine samples included in the study, 80 (19.6%) samples showed bacterial growth. The commonest isolate was E. coli 44 (55 %). Majority of the isolates were multidrug resistant with two E.coli strains showing pan-resistance to the first line drugs tested. Carbapenem resistance was seen in 67.2% of all gram negative isolates tested. Metallo beta-lactamases production was found to be highest among the Klebsiella isolates. Among the Enterobacterales, highest susceptibility was noted to Fosfomycin (87.7%) followed by Nitrofurantoin (62.7%). Similarly in gram positive group, highest susceptibility was again to Fosfomycin followed by Nitrofurantoin. Overall the prevalence of MDR is increasing however Fosfomycin or Nitrofurantoin, both oral antibiotics, can be the considered for starting empirical antibiotic therapy.

Molecular investigation of antibiotic resistant bacterial strains isolated from wastewater streams in Pakistan.

Antibiotic resistance is a global public health issue and it is even more daunting in developing countries. The main objective of present study was to investigate molecular responses of antibiotic-resistant bacteria. The 48 bacterial strains, which were previously isolated and identified were subjected to disc diffusion and MIC (minimum inhibitory concentration) determination, followed by investigating the production of the three beta-lactamases (ESBLs (Extended-spectrum Beta-lactamases), MBLs (Metallo Beta-lactamases), AmpCs) and exploring prevalence of the two antibiotic-resistant genes (ARGs); blaTEM and qnrS. Higher MIC values were observed for penicillin(s) than that for fluoroquinolones (ampicillin > amoxicillin > ofloxacin > ciprofloxacin > levofloxacin). Resistance rates were high (58-89%) for all of the tested beta-lactams. Among the tested strains, 5 were ESBL producers (4 Aeromonas spp. and 1 Escherichia sp.), 2 were MBL producers (1 Stenotrophomonas sp. and 1 Citrobacter sp.) and 3 were AmpC producers (2 Pseudomonas spp. and 1 Morganella sp.). The ARGs qnrS2 and blaTEM were detected in Aeromonas spp. and Escherichia sp. The results highlighted the role of Aeromonas as a vector. The study reports bacteria of multidrug resistance nature in the wastewater environment of Pakistan, which harbor ARGs of clinical relevance and could present a public health concern.

Incidence of Beta-Lactamase Enzymes among Klebsiella pneumonia Isolates Causing Urinary Tract Infections in Aliabad, North-East Iran

AimsIn the past decade, drug resistance in Gram negative bacilli has become a serious problem. The production of extended spectrum beta-lactamase (ESBL), AmpC beta-lactamase, and metallo beta-lactamase (MBL) enzymes in Klebsiella pneumoniae strains is the mechanism of drug resistance among these commonly isolated Gram negative bacteria from clinical specimens. The aim of this study was to assess the frequency of β-lactamase enzymes, including extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and AmpC beta-lactamases, in K. pneumonia strains isolated from urine samples referred to medical laboratories in Aliabad.Materials & MethodsA total of 780 urine samples were collected from patients suspected of having UTI from March to June 2017. In positive urine samples, K. pneumonia isolates were identified by biochemical tests. Antibiotic resistance pattern was determined by disk diffusion method, and phenotypic confirmatory test was performed for detecting ESBLs, MBLs, and AmpC BLs producers.FindingsOut of 378 positive samples for UTI, 97 K. pneumonia strains were isolated. Most of the isolates (more than 90%) were resistant to ampicillin and amoxicillin; however, imipenem and amikacin were effective antibiotics against the isolates. The frequency of ESBLs, MBLs, and AmpC BLs producers was determined as 33.3, 21.3, and 5.1%, respectively.ConclusionsIn this study, 14 isolates were simultaneously positive for ESBL and AmpC BL production, and 2 isolates were co-producer of ESBL and MBL. This finding could have a great impact on the management and treatment of UTI cases. Therefore, detection of beta‑lactamases is of great importance for controlling and reducing the spread of ESBL, AmpC BL, and MBL producing strains.

Phenotypic synergy testing of ceftazidime–avibactam with aztreonam in a university hospital having high number of metallobetalactamase producing bacteria

Background Ceftazidime–avibactam combination with aztreonam and role of rapid synergy reporting has not been widely evaluated. Also the synergy correlation with various betalactamases has not been widely studied. Methods We studied phenotypic synergy testings and molecular detection of betalactamases in our university hospital where we have large number of mellatobetalactmase producing bacteria. We tested two phenotypic synergy methods for ceftazidime-avibactam with aztreonam (Disc-E strip method, E strip-Agar method) for rapid reporting to clinicians (153 isolates). The treatment (colistin, ceftazidime-avibactam, ceftazidime-avibactam with aztreonam) was guided as indicated in the synergy testings. The resistance genes in bacteria were identified by polymerase chain reaction (PCR) and correlated with synergy results. Results The highest synergy was seen in Klebsiella pneumoniae by Disc-E strip and E strip-Agar method (86% and 84% respectively). About 70% of Pseudomonas aeruginosa and 29% of Escherichia coli showed synergy. Molecular methods revealed multiple resistance gene combinations and bla NDM (96%) was predominant gene in isolates showing synergy. Among isolates that were sensitive to ceftazidime–avibactam, the predominant genes were bla OXA-48 and bla IMP. Rapid laboratory reporting led to proper utilization of antibiotic combinations. Conclusions Ceftazidime–avibactam and aztreonam rapid synergy testing will be highly beneficial in treatment of infections by metallobetalactamase producing resistant bacteria, especially K. pneumoniae and P. aeruginosa.

Phenotypic and Molecular Detection of the Metallo-Beta-Lactamases in Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Clinical Samples

Background: Pseudomonas aeruginosa is an opportunistic pathogen that causing high morbidity and mortality, and responsible for a high proportion of hospital infections. Recently, P. aeruginosa infections having carbapenem group antibiotic-resistant have been increasing and serious problems are being experienced in treatment. Carbapenem resistance mechanisms are the loss of OprD porin protein, excretion of drug with the efflux system and production of metallo-beta-lactamase (MBL). Objectives: The aim of this study was to investigate the production of MBL in carbapenem resistant P. aeruginosa isolates phenotypically and genotypically. Study design: In this study, 58 P. aeruginosa isolates were included from various clinical samples between May 2014 and March 2015 at the Microbiology Laboratory of Tertiary Care Hospital of Mersin University, Faculty of Medicine. Methods: The strains were identified by automated system (Vitek 2, BioMerieux, France); and antibiotic susceptibilities were performed by using the disc diffusion method and the automated system (Vitek 2). Gradient test was performed for imipenem (IPM) and meropenem (MEM) to confirm the carbapenem resistance. Combined disc test (CDT) and MBL E-test were used for the detection of MLB phenotypically. The most common MBL genes, blaIMP-1, blaIMP-2, blaVIM1, blaVIM-2, blaSPM, blaGIM-1 were investigated with polymerase chain reaction (PCR). Direct DNA sequence analysis was performed to PCR positive isolates. Results: Of the 58 isolates, 57 (99%) were found resistant to IPM, 43 (74%) to MEM, one was intermediate susceptible to IPM and 15 (25%) to MEM by automated system. Of the 58 isolates, 57 were found to be resistant and one was intermediate susceptible to IPM, 34 isolate were found to be resistant, 17 were intermediate susceptible and 7 were susceptible to MEM by gradient test. MBL positivity was detected on 37 (63.7%) isolates by CDT and 16 (27%) by E-test. MBL gene region was detected on 8 isolates (13.7%) by PCR and 6 isolates were found to be VIM-1 gene region positive and 2 isolates were GIM-1 gene region positive. This study is the first study showing the GIM-1 gene in P. aeruginosa in Turkey. DNA sequence analysis data was compared with the reference NCBI sequence data in the PubMed-BLAST program to confirm gene regions. Conclusions: Phenotypic tests can be used as screening test for the detection of MBL activity in carbapenem resistant P. aeruginosa isolates. However, confirmation of the results by molecular tests and determination of the gene regions responsible for resistance are very important epidemiologically.