Metallo-beta-lactamase Genes Research Articles

Food chain milieus: Potential reservoirs and sources of metallo-beta-lactamase-producing antibiotic-resistant bacteria in Southeast Nigeria

Concerted efforts are needed to mitigate the development and transmission of bacterial resistance in the general environment. Metallo-beta-lactamase (MBL) is a carbapenemase that allows bacteria to resist the antimicrobial onslaught of carbapenems (e.g., imipenem [IPM]). We investigated the prevalence of MBL-producing Pseudomonas aeruginosa in poultry farms. Samples from poultry birds (n = 65) were studied for the isolation of antibiotic-resistant bacteria according to the Clinical and Laboratory Standards Institute criteria. The modified Hodge test was used to phenotypically detect MBL. Polymerase chain reaction (PCR) was used to confirm MBL production in the test isolates. To confirm the presence of plasmids in the isolates, a plasmid curing experiment was conducted. High levels of reduced susceptibility to cefotaxime (77.8%), IPM (69.4%), gentamicin (63.9%), amikacin (58.3%), fosfomycin (55.5%), ciprofloxacin (66.7%), tetracycline (75%), ertapenem (55.5%), sulfamethoxazole-trimethoprim (66.7%), and cefoxitin (52.8%) were recorded in the P. aeruginosa isolates. A total of 23 isolates of P. aeruginosa produced MBL. The presence of MBL genes in these P. aeruginosa isolates (n = 23) was confirmed by PCR, which detected blaIMP-1 (47.8%) and blaIMP-2 (26.1%). The isolates were multidrug resistant (50%) and carried plasmids, indicating horizontal transmission of resistance genes. Genes for bacterial resistance can be transmitted through mobile genetic elements. Therefore, sustainable interventions are needed to mitigate antibiotic use in poultry practices in Nigeria to curb the development of MBL-producing bacteria arising from selective (antibiotic) pressures in such an environment. These interventions can help to detect and stop infections caused by resistant bacteria before they become clinical cases in our hospitals.

Molecular study of blaVIM and blaIMP genes in Acinetobacter baumannii strains isolated from burn patients in Duhok City, Iraq.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogenic bacterium mainly associated with hospital acquired infections and in immunocompromised individuals who stay in hospitals for a long time. In recent years, it has become increasingly resistant to many different types of antibiotics. The production of the metallo-beta-lactamase (MBL) enzyme is one of the primary causes of this resistance. This study aimed to detect the presence of MBL genes that belong to the verona integrin metallo-β-lactamase (bla-VIM) and imipenemase (bla-IMP) groups in the isolates of Acinetobacter baumannii from burn patients. One hundred and seventeen (117) isolates of A. baumannii were obtained from patient specimens using traditional methods followed by using the VITEK 2 (BioMérieux, Les Pennes-Mirabeau, France) identification system. Metallo β-lactamases were detected in the imipenem-resistant strains by using imipenem disks on Muller-Hinton agar. The polymerase chain reaction (PCR) technique was utilized to examine 117 isolates for the detection of MBLs encoding genes such as bla-VIM, and bla-IMP. Imipenem resistance was detected in 78.6% of the patients. The PCR assays of the isolates identified bla-VIM-1, bla-VIM-2, bla-IMP-1 and bla-IMP-2 genes at the rates of 17%, 40.1%, 29.9% and 4.2%, respectively. The findings suggest that the majority of A. baumannii isolates harbour one or more of the detected genes, signifying that the production of MBLs plays a pivotal role in resistance mechanisms.

Molecular technique for precise antibiotics administration in patients with cancer.

e18749 Background: Infectious diseases are the second most common cause of mortality in oncology patients. Multiple hospital admissions and previous exposure to antibiotics are risk factors for infection with microorganisms having antibiotic resistance gene in these patients.Molecular technique helps in targeted antibiotic administration at the earliest. Methods: This was a retrospective observational study over a period of one year from November 2021 to October 2022 in Tata Memorial Hospital, Mumbai, on oncology patients with blood stream infection (BSI) whose blood sample underwent film array assay. The BioFire Blood culture Identification 2 Panel (BCID2) which identifies Imipenemases (IMP), Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo beta-lactamases (NDM), oxacillinase-48 like (OXA-48), Verona integron-encoded metallo beta-lactamase (VIM), CTX M and methicillin resistance gene like mec A/C was used. BSI was categorised according to the resistance gene and recommended antibiotics of choice as per the Infectious Disease Society of America (IDSA). CTX-M - Carbapenem  OXA-48 like/ KPC - Ceftazidime-avibactam, Polymixin  Metallo beta-lactamase (MBL) i.e VIM, IMP, NDM - Ceftazidime-avibactam plus aztreonam, polymyxin, Cefiderocol  The impact of appropriate antibiotic administration on 30-day mortality was seen. Results: A total of 79 blood samples were submitted for film array assay during the study period. The mean age of the study population was 17± 4 years. 41(51.9%) patients had acute myeloid leukemia (AML), 29 (36.7%) had acute lymphocytic leukemia (ALL), 4 (5%) patients had lymphoma and 5 (6.3%) had other malignancies. 54(68.3%) BSI had microorganism with antibiotic resistance gene, 16 (20.3%) did not carry resistance gene and in 9 (11.4%), microorganism was not identified in film array assay, though conventional culture showed contaminants. Of the 54 BSI with microorganisms having antibiotic resistance gene, 37(68.5%) had MBL gene either alone or along with CTX-M or OXA-48 like genes, 12 (22.2%) had CTX-M and 5 (9.2%) had both CTX-M and OXA -48 like genes. Empirical antibiotic did not match the antibiotic resistance pattern in 40 (74.1%), antibiotic matched the resistance pattern in 14(22.9%). Antibiotic adjustment was done in 32 (86%) of MBL, 4 (80%) of OXA-48 like with CTX-M and 9 (75%) CTX-M. The 30-day mortality was significantly less (28% vs 80%, p = 0.042) in patients who received appropriate antibiotic versus those who continued inappropriate antibiotics in MBL BSI. Conclusions: Majority of the study population had BSI with antibiotic resistance gene and underwent a change in the antibiotic after the assay. There was lesser 30-day mortality in patients who received antibiotics appropriate for the identified resistance gene. Molecular technique is helpful for precise antibiotic administration in cancer patients who have a higher risk of infection with organisms having resistance gene.

Assessment of bacteria exposure in vitro activity to 1st, 2nd, 3rd and 4th generation-cephalosporins and Comparison effects

The emergence and spread of resistance to cephalosporin generations are threateningto create species resistant to all currently available agents. Recently, we have seen thedevelopment and spread of bacteria carrying metallo-betalactamase genes that areresistant to cephalosporins (and all beta-lactams). This study was designed tocomparison the effects of first, second, third and fourth- generation-cephalosporin ondifferent bacterial species, which include: Escherichia coli, Enterobacter cloacae,proteus mirabilis, Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcuspneumonia and Staphylococcus aureus. 11 cephalpsporins antibiotics were used inthis study, which include: cefalexin, cefazolin, cephalothin, cefuroxime, cefoxitin,cefaclor, ceftibuten, cefotexime, ceftazidime, cefpirome and cefepime. kirby bauermethod was used to detect the activity of these antibiotics in vitro. Results showedthat cefpirome and cefepime antibiotics belonging to 4th generation cephalosporin,exhibit antibacterial spectrum effective, except Str. pneumonia and K. pneumonia.Some cases, 1st generation cephalosporin exhibit more antibacterial spectrum effectivethan other cephalosporin generation. In conclusion. This study indicated thatinsignificant influence among four cephalosporins generation on different bacterialspecies. Although, cephalosporins antibiotics have variant activity against differentbacterial species, but the resistant development among bacteria become the publicproblem during the past 2 decades.

Detection of NDM Variants (bla NDM-1, bla NDM-2, bla NDM-3) from Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae: First Report from Nepal.

BackgroundIncreasing burden of carbapenem resistance among Enterobacterales is attributable to their ability to produce carbapenemase enzymes like metallo-beta-lactamase (MBL), Klebsiella pneumoniae carbapenemase (KPC), and OXA-type. This study aimed to determine the prevalence of carbapenemases and MBL genes ((blaNDM-1, blaNDM-1 and blaNDM-3) among E. coli and K. pneumoniae isolates.MethodsA total of 2474 urine samples collected during the study period (July–December 2017) were processed at the microbiology laboratory of Kathmandu Model Hospital, Kathmandu. Isolates of E. coli and K. pneumoniae were processed for antimicrobial susceptibility testing (AST) by disc diffusion method. Carbapenem-resistant isolates were subjected to Modified Hodge Test (MHT) for phenotypic confirmation, and inhibitor-based combined disc tests for the differentiation of carbapenemase (MBL and KPC). MBL-producing isolates were screened for NDM genes by polymerase chain reaction (PCR).ResultsOf the total urine samples processed, 19.5% (483/2474) showed the bacterial growth. E. coli (72.6%; 351/483) was the predominant isolate followed by K. pneumoniae (12.6%; 61/483). In AST, 4.4% (18/412) isolates of E. coli (15/351) and K. pneumonia (3/61) showed resistance towards carbapenems, while 1.7% (7/412) of the isolates was confirmed as carbapenem-resistant in MHT. In this study, all (3/3) the isolates of K. pneumoniae were KPC-producers, whereas 66.7% (10/15), 20% (3/15) and 13.3% (2/15) of the E. coli isolates were MBL, KPC and MBL/KPC (both)-producers, respectively. In PCR assay, 80% (8/10), 90% (9/10) and 100% (10/10) of the isolates were positive for blaNDM-1, blaNDM-2 and blaNDM-3, respectively.ConclusionPresence of NDM genes among carbapenemase-producing isolates is indicative of potential spread of drug-resistant variants. This study recommends the implementation of molecular diagnostic facilities in clinical settings for proper infection control, which can optimize the treatment therapies, and curb the emergence and spread of drug-resistant pathogens.

Study of the relationship between antibiotic resistance markers and virulence markers in NDM-positive Klebsiella pneumoniae strains circulating in various waters and human loci

Introduction. The propagation of multi-resistance to antibiotics among hospital isolates of Klebsiella pneumoniae (K. pneumoniae) is a subject of growing concern worldwide. At present, growing data of association between resistance and hypervirulence in clinical isolates of K. pneumoniae emerges. However, the occurrence of these pathogens in the environment remains an open question. The aim of this study was to evaluate and compare antibiotic resistance determinants occurrence in Klebsiella pneumoniae isolates from water sources (environmental and sewage), human sources (practically healthy people and patients with inflaammatory bowel disease (IBD), and extraintestinal infections (ExII)). Materials and methods. The PCR assay of carbapenemase genes IMP, NDM, VIM, KPC, OXA-48 was performed with the commercial “Amplisense” kits according to the manufacturer's instructions. The assay was used to evaluate the occurrence of antibiotic-resistance genes in 223 isolates of Klebsiella pneumoniae from various sources: 42 isolates from sewage, 19 isolates from surface water sources, 30 isolates from biological material (blood, urine, surgical wounds, bronchoalveolar lavage) of patients with extraintestinal infections (ExII), 69 isolates from patients with inflammatory bowel diseases (IBD), and 63 isolates from faeces of practically healthy people. Results. The ExII group revealed various antibiotic resistance genes. The most prevalent gene was OXA (30% had this gene only, other 26,6% had also KPC or NDM). NDM as the only resistance gene was observed in 23,3% of ExII isolates. KPC gene was observed in 3,3% of ExII group. Two isolates from IBD group contained NDM gene along with VIM gene. Only NDM gene was found in all the other groups of Klebsiella pneumoniae isolates (13-28% isolates in every group, no statistical difference). NDM was shown to be associated with virulence genes iutA and rmpA that are responsible for iron consumption and hypermucoid phenotype. Conclusion. The most abundant resistance genes in the studied Klebsiella pneumoniae isolates were NDM (13.5%) and OXA (8%). At the same time, NDM was the only gene found in all groups (11-28%). NDM metallobeta-lactamase gene was associated with rmpA and iutA genes, giving an example of the connection between virulence and resistance properties. A significant amount of resistant isolates from healthy donors and surface waters indicates the need for additional study of the role of NDM positive isolates in pathogenicity of Klebsiella pneumoniae.

Virulence characterization and clonal analysis of uropathogenic Escherichia coli metallo-beta-lactamase-producing isolates

BackgroundUropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL.MethodsA cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR.ResultsAmong 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments.ConclusionMBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Genetic diversity and molecular analysis of metallo beta lactamases among imipenem resistant clinical isolates of Pseudomonas aeruginosa from Peshawar, Pakistan.

Objectives:Pseudomonas aeruginosa is an opportunistic pathogen with remarkable adaptation ability to thrive in diverse environmental conditions. This study aimed at phenotypic and molecular analysis of metallo beta lactamases (blaIMP, blaVIM, blaNDM-1 and blaSPM-1) and genetic diversity analysis among imipenem resistant clinical isolates of Pseudomonas aeruginosa.Methods:This study was conducted from May 2017 to June 2018. The study included 187 Pseudomonas aeruginosa isolates collected from different clinical specimens from Peshawar, Pakistan. The isolates were analyzed for resistance to imipenem. Combined disc test (CDT) was then performed for phenotypic detection of metallo beta lactamases among imipenem resistant isolates of Pseudomonas aeruginosa. Molecular detection of metallo beta lactamases genes i.e. blaIMP, blaVIM, blaNDM-1 and blaSPM-1 was analyzed through polymerase chain reaction. Genetic diversity was determined through RAPD-PCR.Results:MBL production was observed in 76% (n=19) isolates. The occurrence of MBL genes blaIMP, blaNDM-1 and blaVIM was 68% (n=17), 48% (n=12), and 4% (n=1) respectively. The blaSPM-1 gene was not detected. High genetic diversity was observed in current study. Out of 182 isolates 171 isolates showed different RAPD profiles (93.95% polymorphism); 160 were unique RAPD strains and based on similarity coefficient ≥ 80%, 22 isolates were clustered into 11 distinct clones.Conclusion:A high prevalence of blaIMP and blaNDM-1 among imipenem resistant isolates of Pseudomonas aeruginosa is alarming that calls for proper control and prevention strategies. RAPD technique was found to be a good genotyping technique when limited resources are available.

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.

Karbapenemlere Dirençli Pseudomonas aeruginosa Kökenlerinde Metallo-Beta-Laktamaz Enzimlerinin Fenotipik ve Moleküler Yöntemlerle Araştırılması

Introduction: Carbapenem resistant Pseudomonas aeruginosa strains cause serious problems in treatment. A large number of identified metallo-beta-lactamase (MBL) enzymes produced by P. aeruginosa are one of the most important mechanisms in resistance to carbapenems. MBL genes are located on the chromosome or plasmid, and they can easily spread between different bacterial strains. The activities of these enzymes are zinc-dependent, and they are inhibited by ethylenediaminetetraacetic acid (EDTA). Therefore, this advantage is used in MBL identification tests. In this study, it was aimed to determine MBL among P. aeruginosa strains. Materials and Methods: MBL existence was investigated in 35 P. aeruginosa strains accepted to be mildly susceptible/resistant to any of the carbapenem group of antibiotics through phenotypic and genotypic methods. Phenotypic tests were performed as double disk synergy test (DDST), combined disk diffusion tests (CDDT) by using 0.1 M and 0.5 M EDTA, MBL E-test, and modified Hodge test (MHT). blaIMP, blaVIM, blaGIM, blaSIM, blaSPM genes and blaNDM gene were investigated by multiplex polimerase chain reaction (PCR) and PCR, respectively. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 standard bacteria were used in tests. VIM-1, VIM-2, IMP-13, SPM-1, NDM-1 type MBL-producing P. aeruginosa strains were used as positive controls. Results: Among the carbapenems resistant P. aeruginosa isolates, positivity of MBL was found as 54.2% by MBL E-test, 42.8% by DDST, 94.2% and 37.1% by CDDT method using 0.5 M and 0.1 M EDTA, respectively. Modified Hodge test and genotypic method did not detect MBL. Conclusion: In order to correctly evaluate the results of the phenotypic method, the investigation of resistance genes by molecular methods is also required. The most common metallo-beta-lactamase enzymes responsible for resistance to carbapenem in Pseudomonas were not observed. It was thought that different mechanisms might be responsible for the identified carbapenem resistance.

Genotypic to Phenotypic Resistance Discrepancies Identified Involving β-Lactamase Genes, blaKPC, blaIMP, blaNDM-1, and blaVIM in Uropathogenic Klebsiella pneumoniae.

IntroductionKlebsiella pneumoniae carbapenemase (KPC) belongs to the Group-A β-lactamases that incorporate serine at their active site and hydrolyze various penicillins, cephalosporins, and carbapenems. Metallo-beta-lactamases (MBLs) are group-B enzymes that contain one or two essential zinc ions in the active sites and hydrolyze almost all clinically available β-lactam antibiotics. Klebsiella pneumoniae remains the pathogen with the most antimicrobial resistance to KPC and MBLs.MethodsThis research investigated the blaKPC, and MBL genes, namely, blaIMP, blaVIM, and blaNDM-1 and their phenotypic resistance to K. pneumoniae isolated from urinary tract infections (UTI) in Bangladesh. Isolated UTI K. pneumoniae were identified by API-20E and 16s rDNA gene analysis. Their phenotypic antimicrobial resistance was examined by the Kirby-Bauer disc diffusion method, followed by minimal inhibitory concentration (MIC) determination. blaKPC, blaIMP, blaNDM-1, and blaVIM genes were evaluated by polymerase chain reactions (PCR) and confirmed by sequencing.ResultsFifty-eight K. pneumoniae were identified from 142 acute UTI cases. Their phenotypic resistance to amoxycillin-clavulanic acid, cephalexin, cefuroxime, ceftriaxone, and imipenem were 98.3%, 100%, 96.5%, 91.4%, 75.1%, respectively. Over half (31/58) of the isolates contained either blaKPC or one of the MBL genes. Individual prevalence of blaKPC, blaIMP, blaNDM-1, and blaVIM were 15.5% (9), 10.3% (6), 22.4% (13), and 19% (11), respectively. Of these, eight isolates (25.8%, 8/31) were found to have two genes in four different combinations. The co-existence of the ESBL genes generated more resistance than each one individually. Some isolates appeared phenotypically susceptible to imipenem in the presence of blaKPC, blaIMP, blaVIM, and blaNDM-1 genes, singly or in combination.ConclusionThe discrepancy of genotype and phenotype resistance has significant consequences for clinical bacteriology, precision in diagnosis, the prudent selection of antimicrobials, and rational prescribing. Heterogeneous phenotypes of antimicrobial susceptibility testing should be taken seriously to avoid inappropriate diagnostic and therapeutic decisions.

Emerge of NDM-1-Producing Multidrug-Resistant Pseudomonas aeruginosa and Co-harboring of Carbapenemase Genes in South of Iran

New Delhi metallo-beta-lactamase-1 (NDM-1) is one of the most important emerging antibiotic resistance. Co-harboring three or four carbapenemases is rare and only a few reports exist in the literature. We described the characteristics of the large epidemic outbreaks and reports co-producing blaNDM-1 with the other carbapenemase genes in P. aeruginosa isolates. This present cross-sectional research was conducted on 369 P. aeruginosa isolates obtained from burn and general hospitals within years 2013 to 2016. Beta-lactamase classes A, B and D genes were identified by PCR method. Modified hodge test (MHT), double-disk potentiation tests (DDPT) and double disk synergy test (DDST) were performed for detection carbapenemase and metallo beta-lactamase (MBL) production of blaNDM-1 positive P. aeruginos isolates. From 236 carbapenem-resistant P. aeruginosa (CRPA), 116 isolates have had MBL genes and twenty-nine isolates were found positive for blaNDM-1 . In CRPA isolates, blaIMP-1 , blaVIM-2 and blaOXA-10 were identified in 27.5%, 21.1% and 32.2% of isolates respectively, while co-producing blaNDM-1 , blaIMP-1 , blaOXA-10 , co-producing blaNDM-1 , blaVIM-2 , blaOXA-10 and co-producing blaIMP-1 , blaVIM-2 were determined in 11 (4.6%), 8 (3.4%) and 27 (11.4%) of isolates respectively. The finding of this co-existence of multiple carbapenemase resistance genes is threating for public health. Dipicolinic acid is a superior MBL inhibitor in DDPT antique than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P. aeruginosa.

Whole genome sequencing and antibiotic diffusion assays, provide new insight on drug resistance in the genus Pedobacter.

ABSTRACTA total of four strains of the ‘environmental superbug’ Pedobacter isolated from sludge produced at Norwegian drinking water treatment plants, were characterized by whole genome sequencing and antibiotic susceptibility assays. As with previous studies on members of this genus, we found that the isolates were multi-drug resistant, and that this resistance included clinically important beta-lactams, aminoglycosides and the fluoroquinolone ciprofloxacin. Using the minION sequencing platform (Oxford Nanopore Technologies) combined with HiSeq PE150 Illumina sequencing data, the four isolates were assembled into genomes of single contigs. Analysis of the genomes revealed potential genetic factors possibly underlying some of the specific resistances observed. Metallo-beta-lactamase activity was detected in one isolate, and the same isolate contained a putative metallo-betalactamase gene resembling pedo-2. Furthermore, several genes related to multidrug efflux systems were found using the resistance database CARD. Additionally, the present study extends our knowledge on the phylogeny of this genus, adding four new genomes to the existing 50.

Carbapenem Resistance in Non-Fermenters: An Overview

This study was conducted with interest in increasing carbapenem resistance in non-fermenters: an important causative agent of nosocomial infection and to standardize the methods for interpretation of their resistance. The aim of this study is to perform disk diffusion testing and minimal inhibitory concentration technique for the identification of carbapenem resistance for imipenem and meropenem. The isolates found resistant to carbapenems were confirmed with the modified Hodge test. The genes responsible for carbapenem resistance were identified by both phenotypic and genotypic methods. Out of 240 non-fermenters isolated 20% showed resistance to carbapenem by disk diffusion. Only 7% showed resistance by the micro broth dilution technique of minimum inhibitory concentration. 3% were panning drug-resistant. Out of 16 carbapenem-resistant isolates, 5 were found to have KPC (Klebsiella pneumonia carbapenem) genes, 9 had MBL (Metallo beta-lactamase) genes and 2 had KPC+MBL genes and none were found to have Amp C and OXA-48 genes phenotypically. Genotypically all the KPC strains had KPC genes and out of 9 MBL strains, 6 had VIM and the remaining 3 strains were negative for both IMP and VIM gene. In conclusion, the interpretation of susceptibility for carbapenems should not be made only with disk diffusion testing. Always check for Minimal inhibitory concentration methods and determination of genes responsible for carbapenem resistance, a double-disc synergy test goes in hand with genotypic detection.

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.

Phenotypic and genotypic detection of metallo-B-lactamases producing Pseudomonas aeruginosa: A study done in two university hospitals

<br><b>Background</b><br>Metallo-beta-lactamase (MBL) producing <i>Pseudomonas aeruginosa </i>has emerged as a threat to hospital-infection control due to its multidrug resistance, but the laboratory detection of these strains is not well defined.<br><b>Objectives</b><br>Screening for MBL production from <i>P. aeruginosa </i>isolates from different clinical specimens using different phenotypic and genotyping techniques. In addition, we aim to apply a PCR test for genetic confirmation of MBL producers.<br><b>Patients and methods</b><br>We used EDTA-disk screen test and molecular diagnostic assay for the detection of MBL-producing <i>P. aeruginosa </i>isolated from different clinical specimens collected in Kasr Alainy and Fayoum University Hospitals. Using Clinical and Laboratory Standards Institute-disk methodology, inhibition-zone diameters were determined in tests with imipenem (IPM) and meropenem disks alone and in combination with 750 μg of EDTA. This test was compared with the MBL E-test. Detection for MBL-production genes (<i>blaIMP</i> and <i>blaVIM</i>) was done using PCR.<br><b>Results</b><br>Of the 50 clinical strains of IPM nonsusceptible <i>P. aeruginosa</i>, 32/50 (64.0%) were MBL positive using disk-diffusion methods, 26/50 (52.0%) were positive for MBL by E-test, while 17/50 (34.0%) were positive for MBL genes: 17/50 (34.0%) for <i>blaVIM</i> and 0/50 (0%) for <i>blaIMP</i>. The EDTA disk-screen test using IPM showed 100% sensitivity and 54.5% specificity for detecting MBLs in clinical strains. While E-test showed 100% sensitivity and 69.7% specificity.<br><b>Conclusion</b><br>The EDTA disk-screen test was simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM nonsusceptible <i>P. aeruginosa</i> isolates be routinely screened for MBL production using the EDTA disk-screen test and PCR confirmation be performed at a regional laboratory.<br>

MOLECULAR EPIDEMIOLOGY OF METALLO BETA LACTAMASES IN ACINETOBACTER BAUMANNII AT A TERTIARY CARE HOSPITAL

Background: Carbapenem resistance mediated by metallo beta lactamases (MBL) in Acinetobacter baumannii is a global challenge due to its rapid spread and limited therapeutic options.&#x0D; Objective: To determine the prevalence of MBL in A. baumannii isolates in hospitalized patients by both phenotypic and genotypic methods.&#x0D; Materials 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). Antibiotics that were not tested by VITEK-2 were tested manually by Kirby-Bauer disk diffusion method according to CLSI 2017 and EUCAST 2016 guidelines. The isolates which were resistant to carbapenem (imipenem and/ or meropenem) were tested by phenotypic (imipenem-EDTA combined disk method) and genotypic method for presence of common metallo beta lactamases genes (blaIMP, blaNDM, blaGIM, blaVIM, blaSPM and blaSIM).&#x0D; Results: 84 non duplicate A.baumannii were isolated out of 947 pathogenic gram negative isolates. Majority (47.6%) of isolates were obtained from tracheostomy/endotracheal/bronchoalveolar lavage (TT/ET/BAL) followed by sputum (21.4%). None of the isolates were found to be resistant to colistin and tigecycline. 73 (86.9%) isolates were found to be carbapenem resistant, among these 60 (82.2%) were found to be MBL positive by phenotypic and 32 (43.2%) by genotypic method. MBL genes detected were blaNDM (39.7%), blaGIM (2.7%) and blaVIM (1.4%). None of the isolates were positive for blaIMP, blaSPM and blaSIM.&#x0D; Conclusion: The prevalence of MBL in carbapenem resistant isolates of A.baumannii was 87.7%. blaNDM was the most common gene detected. No significant difference was found in the ability of phenotypic and genotypic methods for MBL detection. The resistance rate of the A.baumannii is high for most antibiotics except for polymyxins (E&amp;B) and tigecycline.&#x0D; Key words: Metallo beta Lactamases, Acinetobacter baumannii, Carbapenem.

Sewage effluent from an Indian hospital harbors novel carbapenemases and integron-borne antibiotic resistance genes

BackgroundHospital wastewaters contain fecal material from a large number of individuals, of which many are undergoing antibiotic therapy. It is, thus, plausible that hospital wastewaters could provide opportunities to find novel carbapenemases and other resistance genes not yet described in clinical strains. Our aim was therefore to investigate the microbiota and antibiotic resistome of hospital effluent collected from the city of Mumbai, India, with a special focus on identifying novel carbapenemases.ResultsShotgun metagenomics revealed a total of 112 different mobile antibiotic resistance gene types, conferring resistance against almost all classes of antibiotics. Beta-lactamase genes, including encoding clinically important carbapenemases, such as NDM, VIM, IMP, KPC, and OXA-48, were abundant. NDM (0.9% relative abundance to 16S rRNA genes) was the most common carbapenemase gene, followed by OXA-58 (0.84% relative abundance to 16S rRNA genes). Among the investigated mobile genetic elements, class 1 integrons (11% relative abundance to 16S rRNA genes) were the most abundant. The genus Acinetobacter accounted for as many as 30% of the total 16S rRNA reads, with A. baumannii accounting for an estimated 2.5%. High throughput sequencing of amplified integron gene cassettes identified a novel functional variant of an IMP-type (proposed IMP-81) carbapenemase gene (eight aa substitutions) along with recently described novel resistance genes like sul4 and blaRSA1. Using a computational hidden Markov model, we detected 27 unique metallo-beta-lactamase (MBL) genes in the shotgun data, of which nine were novel subclass B1 genes, one novel subclass B2, and 10 novel subclass B3 genes. Six of the seven novel MBL genes were functional when expressed in Escherichia coli.ConclusionBy exploring hospital wastewater from India, our understanding of the diversity of carbapenemases has been extended. The study also demonstrates that the microbiota of hospital wastewater can serve as a reservoir of novel resistance genes, including previously uncharacterized carbapenemases with the potential to spread further.

Bio-surfactin stabilised silver nanoparticles exert inhibitory effect over New-Delhi metallo-beta-lactamases (NDMs) and the cells harbouring NDMs.

Silver nanoparticles (AgNPs) are used as an antimicrobial agent since the ages. However, it is unknown whether AgNPs exert inhibitory effects over the bacterial cells carrying metallo-beta-lactamases (MBLs). Here, using bio-surfactin stabilised AgNPs having a size range from 5 to 25 nm we established its antimicrobial effects against NDMs harbouring cells. Antimicrobial effectiveness of AgNPs is assessed on the E. coli cells carrying New Delhi MBL (NDM) genes, which shows that the cells expressing NDM becomes susceptible to AgNPs and when combined with various groups of beta-lactam a synergistic increase in sensitivity is observed. Purified NDMs are also inhibited by AgNPs as revealed by the hydrolysis of nitrocefin (a chromogenic cephalosporin), though the expression NDM genes remain unchanged. Further, the results obtained from biochemical analysis attribute that the Ag+ ions possibly bind to sulfhydryl (SH) group of cystine in NDMs to inactivate these enzymes. Nonetheless, these AgNPs has the ability to exert antimicrobial activity without affecting the host cell viability when used at a moderate concentration. Overall, we conclude that bio-surfactin-stabilised AgNP is a good candidate to serve as an inhibitor of NDMs, either individually or in combination with beta-lactams.

First prevalence of metallo beta-lactamases producing Enterobacteriacea in Iranian cancer patients.

Hospital-associated infections, recently renamed Healthcare-associated infections, are among the most common life-threatening complications of hospitalized patients, especially the immunocompromised patients. Regarding the significant role of Enterobacteriaceae in nosocomial infections and also the increasing trends of carbapenem-resistant strains, the present study aimed to evaluate the antibiotic resistance pattern and the occurrence of metallo-beta-lactamases (MBLs) in Enterobacteriaceae strains from Iranian cancer patients. This hospital-based cross-sectional study was conducted in teaching hospitals of two cities in the central parts of Iran during the 6 months period from December 2015 to May 2016. The Enterobacteriaceae isolates were obtained from different clinical specimens and were identified using standard microbiological methods. Antimicrobial susceptibility pattern for the bacterial isolates was determined using the disk diffusion method. The presence of antibiotic resistance genes was determined by PCR method. The distribution of Enterobacteriaceae isolates were 74 (71.8%) E. coli, 23 (22.3%) Klebsiella spp., 3 (2.9%) Proteus spp., 2 (1.9%) Salmonella spp., and 1 (1%) Shigella spp. The results of antibiotic susceptibility revealed that all of the isolates were multiple-drug resistant (MDR) and 60% of them were (excluded Salmonella and Shigella) carbapenem-resistant. Of all the carbapenem-resistant isolates, 31.7% were MBL-positive. Meanwhile, fosfomycin and minocycline were the most effective antibiotics against MBL-positive bacteria. Moreover, none of the investigated carbapenemases genes were found in MBL-positive isolates. This study highlights the importance of MBLs producing Enterobacteriaceae in causing nosocomial infections in cancer patients. However, carbapenem resistance was not associated with the presence of MBL genes such as IMP, VIM, and SPM. Vatus haeque crent Catilium ausatem nendactui scerem clere forum dicaur hili consceri plin ternul ut audam que factus, que ad ponis. Go vicaet L. Legilici pos.