Metallo Β-lactamase Research Articles

Novel Antibiotic Resistance Genes Identified by Functional Gene Library Screening in Stenotrophomonas maltophilia and Chryseobacterium spp. Bacteria of Soil Origin

As one of the most diverse habitats of microorganisms, soil has been recognised as a reservoir of both antibiotics and the antibiotic resistance genes (ARGs). Bacteria naturally inhabiting soil or water often possess innate ARGs to counteract the chemical compounds produced by competitors living in the same environment. When such bacteria are able to cause infections in immunocompromised patients, their strong innate antibiotic resistance mechanisms make treatment difficult. We generated functional gene libraries using antibiotic-resistant Stenotrophomonas maltophilia and Chryseobacterium spp. bacteria isolated from agricultural soils in Lithuania to select for the genetic determinants responsible for their resistance. We were able to find novel variants of aminoglycoside and β-lactam resistance genes, with β-lactamases isolated from the Chryseobacterium spp. functional gene library, one of which is a variant of IND-like metallo-β-lactamase (MBL) IND-17 and the other of which is a previously uncharacterised MBL we named CHM (Chryseobacterium metallo β-lactamase). Our results indicate that soil microorganisms possess a diversity of ARG variants, which could potentially be transferred to the clinical setting.

In Vitro and In Vivo Development of a β-Lactam-Metallo-β-Lactamase Inhibitor: Targeting Carbapenem-Resistant Enterobacterales.

β-lactams are the most prescribed class of antibiotics due to their potent, broad-spectrum antimicrobial activities. However, alarming rates of antimicrobial resistance now threaten the clinical relevance of these drugs, especially for the carbapenem-resistant Enterobacterales expressing metallo-β-lactamases (MBLs). Antimicrobial agents that specifically target these enzymes to restore the efficacy of last resort β-lactam drugs, that is, carbapenems, are therefore desperately needed. Herein, we present a cyclic zinc chelator covalently attached to a β-lactam scaffold (cephalosporin), that is, BP1. Observations from in vitro assays (with seven MBL expressing bacteria from different geographies) have indicated that BP1 restored the efficacy of meropenem to ≤ 0.5 mg/L, with sterilizing activity occurring from 8 h postinoculation. Furthermore, BP1 was nontoxic against human hepatocarcinoma cells (IC50 > 1000 mg/L) and exhibited a potency of (Kiapp) 24.8 and 97.4 μM against Verona integron-encoded MBL (VIM-2) and New Delhi metallo β-lactamase (NDM-1), respectively. There was no inhibition observed from BP1 with the human zinc-containing enzyme glyoxylase II up to 500 μM. Preliminary molecular docking of BP1 with NDM-1 and VIM-2 sheds light on BP1's mode of action. In Klebsiella pneumoniae NDM infected mice, BP1 coadministered with meropenem was efficacious in reducing the bacterial load by >3 log10 units' postinfection. The findings herein propose a favorable therapeutic combination strategy that restores the activity of the carbapenem antibiotic class and complements the few MBL inhibitors under development, with the ultimate goal of curbing antimicrobial resistance.

Performance of Phenotypic Tests to Detect β-Lactamases in a Population of β-Lactamase Coproducing Enterobacteriaceae Isolates.

Objectives This study aimed to evaluate the performance of routinely used phenotypic tests to detect β-lactamase production in isolates coproducing multiple β-lactamase types. Methods Commonly used phenotypic tests for the detection of extended spectrum β-lactamases (ESBL), AmpC β-lactamase, and carbapenemases were compared with detection and sequencing of β-lactamase genes (as the reference test) in 176 uropathogenic Enterobacteriaceae coproducing multiple β-lactamases from two hospitals in the Western Province of Sri Lanka. Results Majority of the isolates (147/176, 83.5%) carried β-lactamase genes with (90/147, 61%) harboring multiple genes. The Clinical and Laboratory Standards Institute screening method using cefotaxime (sensitivity [Se], 97; specificity [Sp], 93; accuracy [Ac], 94) and ceftriaxone (Se, 97; Sp, 91; Ac, 93) was the most effective to detect ESBLs. The modified double disc synergy test (Se, 98; Sp, 98; Ac, 97) and combined disc test (Se, 94; Sp, 98; Ac, 96) showed good specificity for confirmation of ESBLs. Cefoxitin resistance (Se, 97; Sp, 73; Ac, 85) and the AmpC disc test (Se, 96; Sp, 82; Ac, 86) were sensitive to detect AmpC β-lactamase producers coproducing other β-lactamases but showed low specificity, probably due to coproduction of carbapenemases. Meropenem was useful to screen for New Delhi metallo β-lactamases and OXA-48-like carbapenemases (Se, 97; Sp, 96; Ac, 96). The modified carbapenem inactivation method showed excellent performance (Se, 97; Sp, 98; Ac, 97) in identifying production of both types of carbapenemases and was able to distinguish this from carbapenem resistance due to potential mutations in the porin gene. Conclusion Microbiology laboratories that are still depend on phenotypic tests should utilize tests that are compatible with the types of β-lactamase prevalent in the region and those that are least affected by coexisting resistance mechanisms.

Meropenem Incorporated ZnO Nanoflakes as Nano Antibiotics: Efficient Antimicrobial Activity against Metallo β-lactamase Producing Clinical Isolates

The number of fatalities caused by multidrug-resistant (MDR) bacteria is over 700,000 annually due to widespread antibiotic usage. So, there is a need of new antibiotics, materials that work like antibiotics, or combinations of antibiotics with nanomaterials that could help in treating the infections which is caused by MDR bacteria. The present study describes the synthesis of ZnO nanoflakes using a co-precipitation method. The ZnO nanoflakes and ZnO nanoflakes combinations with carbapenem antibiotics were tested against carbapenem-resistant (CR) clinical isolates. The SEM analysis showed surface morphology of the synthesized nanoflakes-like structure of ZnO. All 67 CR isolates were tested and showed inhibitory action at varying concentrations of ZnO nanoflakes. ZnO nanoflakes were found to have an inhibitory effect against Escherichia coli and Klebsiella pneumoniae at lowest concentration of 1.25 mg.ml-1 of ZnO NPs with average zone size (mean ±SD) 1.91±2.94 mm and 2.00±4.14 mm and the average zone size of ZnO nanoflakes against Acinetobacter baumanni and Pseudomonas aeruginosa was 9.89±0.76 mm and 10.17±0.39 mm at 2.5 mg.ml-1 concentration. The combined action of ZnO nanoflakes with Meropenem 10 mcg demonstrated synergetic activity against CR pathogens, with an average zone of inhibition measuring 15.2 mm in diameter. ZnO nanoflakes illustrated considerable antibacterial activity against MBL-producing gram-negative clinical isolates at the lowest concentration. Chemically synthesized ZnO nanoflakes may offer a superior future expectation as a nano-antibiotic to treat the infection caused by CRE bacteria.

Synthesis and biological evaluation of novel β-lactam-metallo β-lactamase inhibitors.

β-lactamases are enzymes that deactivate β-lactam antibiotics through a hydrolysis mechanism. There are two known types of β-lactamases: serine β-lactamases (SBLs) and metallo β-lactamases (MBLs). The two existing strategies to overcome β-lactamase-mediated resistance are (a) to develop novel β-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop β-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (≤2 mg L-1) against New Delhi metallo-β-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available β-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (Ki = 0.85 and 1.87, respectively). This study has revealed a novel series of β-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs.

Antibiotska terapija kod kritično obolelih pacijenata sa sepsom - od koncepta FK/FD do novih lekova

Early detection of sepsis and its severity is critical for initiating proper therapy, including antibiotics, as soon as possible to maximize survival chances. Understanding the pathophysiological changes in septic shock that impact antimicrobial pharmacokinetics and pharmacodynamics (PK/PD), understanding the basics of PK/PD, and knowing PK-PD strategies in septic shock patients are all critical for appropriate therapy. By definition multi-drug resistant (MDR) microorganisms are those with acquired non-susceptibility to at least one agent in three or more antimicrobial categories. In the race between bacteria and novel antibiotics development, unfortunately, the number of new antibiotics/indications is not keeping pace with resistance and needs, especially for MDR Gram-negative microorganisms. When we fast forward to the approved antibiotics in the last five years, the list is rather short. Lefamulin is a novel pleuromutilin antibiotic which manifests activity against most Gram-positive pathogens. Cefiderocol is an injectable siderophore cephalosporin. Like other b-lactam antibiotics, it inhibits Gram-negative bacterial cell wall formation by binding to penicillin-binding proteins. Imipenem/cilastatin/relebactam is a new b-lactam/b-lactamase inhibitor combination with activity against MDR Gram-negative bacteria, including many CRE but excluding Metallo-b-lactamase (MBL)-producing Enterobacterales and CRAB. Those three antibiotics were FDA approved in 2019 and European Medicines Agency (EMA) approved in 2020. Plazomicin is a new semisynthetic aminoglycoside with activity against several MDR Gram-negative organisms, including CRE (FDA-approved in 2018). Meropenem-vaborbactam is a fixed-dose combination product of a carbapenem and a cyclic boronic acid b-lactamase inhibitor with potent activity against resistant Gram-negative bacteria like Klebsiella pneumoniae carbapenemase (KPC)-producing CRE; it is inactive against CRAB (FDA-approved in 2017; EMA-approved in 2018). Eravacycline is a new completely synthesized fluorocycline. It has a high level of effectiveness against Grampositive and Gram-negative bacterial strains that have developed tetracycline-specific resistance mechanisms; it is inactive against Pseudomonas aeruginosa (FDAand EMA-approved in 2018). Concluding this list of recently approved antibiotics is omadacycline, novel aminomethylcycline and a derivative of minocycline, with a chemical structure similar to tigecycline. Like other tetracyclines, omadacycline inhibits bacterial protein synthesis and possesses broad-spectrum antibacterial activity against Gram-positive and Gram-negative aerobic, anaerobic, and atypical bacteria including CRAB. It is inactive against Pseudomonas aeruginosa (FDA-approved in 2018). There are three novel carbapenems sulopenem, tebipenem pivoxil hydrobromide and benapenem. There is a group of antibiotics in phase III clinical trials: the combination aztreonam/ avibactam, sulbactam/durlobactam, cefepime/enmetazobactam, cefepime/zidebactam, cefepime/taniborbactam. An interesting approach to antimicrobial treatment is antimicrobial drug repurposing. Drug combinations could be a strategy to extend the life of antibiotics in the XXI century due to the multi-targeting mechanisms of agents. New active substances are urgently required to stop the spread of antibiotic-resistant bacteria. However, it could be a rather cumbersome and expensive procedure. Antimicrobial misuse and/or overuse contribute greatly to this very important global healthcare problem.

NDM-producing Enterobacterales prevalence associated to COVID-19 in a tertiary hospital

Colonizations/Infections caused by carbapenem-resistant Enterobacterales are of great clinical and epidemiological importance due to their rapid dissemination and high mortality rates. In this scenario, the use of antibiotics intensified by the COVID-19 pandemic has brought about a great warning on the real impact that this pandemic could have on antimicrobial management programs and long-term antimicrobial resistance rates. The objective of this study was to evaluate the increase of New Delhi Metallo β-Lactamase (NDM)-producing Enterobacterales cases in COVID-19 units of a complex Brazilian tertiary hospital. This retrospective observational study included all patients admitted to the hospital identified as colonized or infected by NDM-producing Gram negative bacilli (GNB), from January 2017 to April 2021. Forty-two NDM-producing Enterobacterales were identified in 39 patients. The rate of NDM cases per total surveillance cultures increased progressively between 2017 and 2021 (chi-2 for trend, p < 0.0001) and was associated with a higher occurrence specifically in COVID units (Fisher exact, p < 0.0001). The molecular investigation of the NDM-producing Klebsiella pneumoniae strains revealed the emergence of diverse clones during the COVID-19 period, also with possible evidence of horizontal transmission among patients within COVID units. NDM-producing Enterobacterales with multiple and different clonalities in the COVID-19 units also raised questions about the importance of other factors besides horizontal clonal transfer, including the increase of antimicrobial consumption by these patients.

Phenotypic Detection of Carbapenem Resistance in Clinical Isolate of Klebsiella Pneumoniae in Tertiary Care Hospital

The emergence and rapid spread of Multidrug resistant isolates of Klebsiella pneumoniae causing nosocomial infections, which require the great afford for the treating the patients by the clinician. The emergence of Metallo β-lactamase (MBL) producing isolates poses a challenge to routine microbiology laboratories. The purpose of this study was to assess the accuracy of various phenotypic methods for detecting MBL production among K. pneumoniae isolate. The current study may provide the necessary data to formulate a hospital antibiotic policy and to prevent the spread of multidrug resistance in the community. Total 240 K. pneumoniae isolates were included in our study which was isolated from various clinical samples from patients visiting a tertiary care hospital in central India. A total of 148 (61.7%) of K. pneumoniae were isolated from male patients whereas 92 (38.3%) isolates were from female patients. Out of 240 K. pneumoniae, 102 (42.5%) was detected Imipenem resistance by conventional (Kirby-Bauer) disc diffusion method which was confirmed by minimum inhibitory concentration (MIC) test (Epsilometer test). By the MIC Carbapenem resistance K. pneumoniae were confirmed for MBL producer by using phenotypic test. Combined disc synergy test (CDST) (Imipenem 10 mcg and Imipenem 10mcg + 0.5 m EDTA) and Modified Hodge test (MHT).The MHT test found most sensitive approach for detecting MBL in K. pneumoniae. This procedure is simple and clear and it may use in routine clinical labs for detection of MBL because carbapenems are last resort for treating bacterial infections. In recent years, carbapenem resistance has rampantly increased across the globe.

144. Gram Negative Bacteria with Increased Minimal Inhibitory Concentration for Cefiderocol among Pediatric Patients in a Northern Italy Hospital.

Abstract Background Cefiderocol is a siderophore cephalosporin active against Gram negative (GN) carbapenem resistant bacteria, approved in Italy in September 2020 for adult patients. In pediatric population off label use is allowed when severe infections from multidrug resistance (MDR) GN bacteria occur. Recently, some authors reported the emergence, among strains of Enterobacterales resistant to ceftazidime avibactam, of cross-resistance with cefiderocol. The aim of this study is to describe GN strains with increased Minimal Inhibitory Concentration (MIC) for cefiderocol isolated in our hospital. Methods This is a retrospective, single-center study conducted in Istituto Giannina Gaslini Pediatric Hospital (Genoa, Italy) from 1st January 2020 to 31st April 2022. We collected all strains of MDR GN bacteria whit increased MIC for cefiderocol isolated in our hospital. Cefiderocol susceptibility testing was performed by disk diffusion assay with the disk of 30 μg (Kirby-Bauer method, KB) according to EUCAST’s recommendations and KB breakpoints. We collected demographical data, previous colonization (if note), carbapenemases production, clinical outcomes (infections from MDR; death at 7 and 30 days; ICU admission). Results Overall, 10 GN strains with increased MIC for cefiderocol were collected. 9/10 were Enterobacterales (4 Escherichia coli, 2 Enterobacter cloacae complex, 3 Klebsiella pneumoniae), one was Acinetobacter baumannii. 8/10 patients were admitted to other hospital in the six months before admission in our hospital, data about previous colonization were unknown. All MDR isolated produced metallo-β-lactamase [MBL] (5/10 New Delhi metallo-β-lactamase [NDM], 5/10 Verona integron-encoded metallo β-lactamase [VIM]). Four patients developed infection due to MDR (2 urinary tract infections, 1 bloodstream infection, 1 surgical wound), 2/4 patients died during the hospitalization. Conclusion Our study confirms that MDR producing MBL carbapenemases may have reduce susceptibility to cefiderocol, even if never exposed to this siderophore cephalosporin. Due to the possibility of cross resistance with MDR resistant to ceftazidime/avibactam, cefiderocol should be carefully prescribe in empirical treatment of MBL producers strains. Disclosures All Authors: No reported disclosures.

Multidrug-Resistant Acinetobacter: Detection of ESBL, MBL, bla NDM-1 Genotype, and Biofilm Formation at a Tertiary Care Hospital in Eastern Nepal.

The Acinetobacter species is an important hospital-acquired pathogen. The rapid development of resistance to multiple drugs and the ability to form biofilm make these bacteria more adaptable to survive in healthcare facilities, thus posing a challenge to their effective management. This study aimed to characterize clinical isolates of Acinetobacter spp and to study their antimicrobial susceptibility patterns and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended-spectrum β-lactamases (ESBLs), metallo β-lactamases (MBLs), carbapenemase production, and presence of blaNDM-1 gene. A total of 324 Acinetobacter species were isolated from various clinical specimens which were submitted to the Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal, and were studied for antibiotic susceptibility testing, detection of ESBL and MBL production, and formerly biofilm formation was performed by standard microbiological methods. PCR was carried out to determine the presence of the blaNDM-1 gene. The predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). Among those, all A. species 128 (40%) were multidrug resistant (MDR). In which 13 (4.0%) were ESBL producers, 70 (61.9%) were MBL, and 12 (10.6%) were carbapenemases producers. The blaNDM1 gene was present in 33 isolates. Thirty-seven percent (121/324) of isolates formed biofilm. The majority of A. species were resistant to cefotaxime 73.8% (239) and cefepime 74.4% (241). A significant proportion of biofilm producers were MDR (p < 0.001). Drug-resistant Acinetobacter formed a substantial proportion of this hospital's samples with a large presence of the bla NDM-1 gene. A matter of great concern is the association of multidrug-resistant phenotype with biofilm formation. This situation warranted stringent surveillance and adherence to infection prevention and control practices.

Identification and characterization of a novel metallo β-lactamase, SZM-1, in Shenzhen Bay, South China.

Metallo β-Lactamases (MBLs) degrade most clinical β-lactam antibiotics, especially Carbapenem, posing a huge threat to global health. Studies on environmental MBLs are important for risk assessment of the MBLs transmission among connected habitats, and between environment and human. Here, we described a novel metallo β-Lactamases, named SZM-1 (Shenzhen metallo-β-lactamase), from an Arenimonas metagenome-assembled genome recovered from the river sediment in the Shenzhen Bay area, south China. Phylogenetic analysis, primary sequence comparison, structural modeling suggested that the SZM-1 belongs to B1 MBL family, likely harboring a typical di-zinc catalytic center. Furthermore, the gene encoding the MBLs was cloned into Escherichia coli TOP10 for Carba NP test and antimicrobial susceptibility test. The results indicated that the SZM-1 had carbapenemase activity, and conferred the carrier to increased resistance toward carbapenems. Taken together, our results raise alarms about the emergence and spread of the SZM-1, and suggest further surveillance, especially in hospital settings and clinical isolates, to determine whether blaSZM–1 is a mobilizable antibiotic resistance.

Molecular Characterisation of Carbapenemase-Producing Acinetobacter baumannii isolates from Hospitalised Patients in Iraq

Carbapenem-resistant Acinetobacter baumannii has been considered one of the major threats to patients worldwide. To evaluate carbapenemase in several clinical isolates using phenotypic and genotypic approaches. A total of 49 A. baumannii isolates were tested against imipenem and meropenem discs on Muller Hinton agar, then screened phenotypically through the modified Hodge test (MHT), combined disc test (CDT) and modified carbapenem inactivation method (mCIM). The tested isolates have been subjected to polymerase chain reaction (PCR) detection to identify some carbapenemase-encoding genes and one insertion sequence. The carbapenem resistance profile showed 96% and 94% resistance to imipenem and meropenem, respectively. MHT and mCIM were able to produce carbapenemase in 94% and 98% of isolates, respectively, while CDT was able to produce metallo-B-lactamase (MBL) only in 59.2% of isolates. The PCR amplification of blaOXA-51 has been observed in all isolates. We found blaOXA-23 in 98% of isolates. Insertion sequence ISAba1 was present in all positive blaOXA-23 strains (98%). A blaVIM gene encoding MBL was present in 71% of isolates, but none of the isolates has been positive for blaKPC and blaNDM. The high rate of carbapenem resistance in A. baumannii became a serious threat worldwide. Concerning phenotypic tests, mCIM was the most sensitive compared to MHT and CDT. This study established that blaOXA-23 and blaOXA-51 have been the most prevalent among class D carbapenemase, and blaVIM among class B carbapenemase. The present study suggests that there might be silent carbapenemase genes in carbapenem-sensitive strains.

Prevalence of Waterborne blaNDM-1 Gene Producing Carbapenem-resistant Klebsiella pneumoniae from Al-Hillah River Water, Babylon Province, Iraq

The current work suggested the occurrence of blaNDM-1 gene among Klebsiella pneumoniae recovered from surface waters of the Al-Hillah River. Between January and April 2015, water samples (101) were taken from seven different area of the Al-Hillah River, Babylon province, Iraq. K.pneumoniae was reported in percentage of 35 (34.6%). The antibiotics susceptibility profile of K.pneumoniae was determined with disk diffusion assay. The most common resistance was detected for penicillins agents (ampicillin and cloxacillin) with 20(57.14%) and 17(48.57%) resistance rate, respectively. Two isolates of K.pneumoniae were carbapenem-resistant. Phenotypic screening of metallo β-lactamase detection was carried out using imipenem–EDTA double disk synergy test for carbapenem resistant isolates, 2(100%) isolates with positive result. Conventional Polymerase Chain Reaction (PCR) test was used for detection NDM-1 beta-lactamase, 1 (50%) K.pneumoniae isolate harboring this gene.

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

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

Prevalence and Molecular Characterization of Metallo β-Lactamase Producing Gram-Negative Pathogens Causing Eye Infections.

Purpose:Metallo β-lactamases (MβL) production is a worldwide problem, particularly in gram-negative bacteria. As scanty data is available on the prevalence of MBL, the present study is being undertaken to determine the prevalence, antibacterial sensitivity patterns, and molecular characterization of MβL associated resistant genes in gram-negative bacteria isolated from ocular infections.Material and MethodsAt a tertiary eye care center in south India, 359 gram-negative pathogens, 200 isolates from eye infections, and 159 isolates from normal flora of the eye were studied. A gold standard microbiology method was used to identify the isolates. An antibiotic double disc synergy test and a combination disc test were used to detect MβL production. Multiplex PCR was used to investigate the molecular characteristics of the MβL encoding genes blaVIM, blaIMP, and blaNDM.ResultsOf the 359 gram-negative bacterial pathogens, Pseudomonas aeruginosa 108 (30.1%) and Enterobacter agglomerans 46 (12.8%) were commonly isolated. High prevalence of P. aeruginosa 81% (17 strains) was detected as an MβL producer and it shows 100% resistance to 2nd and 3rd generation cephalosporins and meropenem. Multiplex PCR detected only the blaVIM gene in 56 (28%) of various eye infections and 27 (17%) of normal flora of the gram-negative bacteria (GNB). The blaVIM gene is detected predominantly in 51.8% of keratitis and 21.4% of postoperative endophthalmitis. High prevalence of the gene was detected in P. aeruginosa 42.9% (24 of 56) and Alcaligens denitrificans 10.7% (6 of 56) from eye infections. Whereas, in the control group, P. aeruginosa and E. coli each had 14.8% (4 of 27) that were shown positive.ConclusionThe emerging MβLs mediated resistance among P. aeruginosa is a challenging task for ophthalmologists, especially in patients with endophthalmitis and bacterial keratitis. This local knowledge will aid in advising appropriate antibiotic use and avoiding unnecessary antibiotic prescriptions, which are highly warranted.

The Rapid Emergence of Ceftazidime-Avibactam Resistance Mediated by KPC Variants in Carbapenem-Resistant Klebsiella pneumoniae in Zhejiang Province, China.

Ceftazidime-avibactam (CAV) is a new treatment option against carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, the rapid emergence of CAV resistance mediated by KPC variants has posed a severe threat to healthcare after its clinical application. The characteristics of CAV resistance in CRKP strains needs to be determined in China. A total of 477 CRKP isolates were collected from 46 hospitals in Zhejiang Province from 2018 to 2021. The results demonstrated that CAV had a potent activity against 94.5% of all CRKP (451/477, 95% CI: 93.0–96.1%) and 86.0% of CRKP strains carrying blaKPC genes (410/477, 95% CI: 83.5–88.4%). A total of 26 CAV-resistant strains were found. Among these strains, sixteen harbored metallo-β lactamases, and two carried KPC-2 carbapenemase and mutated ompK35 and ompK36. Eight CRKP strains encoded KPC-33 or KPC-93, belonging to ST11, among which seven strains were detected in patients hospitalized in 2021 after exposure to CAV and one strain was associated with intra-hospital spread. CAV is a potent agent in vitro against CRKP strains. The rapid development of CAV resistance mediated by various KPC variants after a short period of CAV treatment has increased and brought difficulties in treating infections caused by CRKP strains, especially those belonging to ST11. The surveillance of bacterial resistance against CAV is highly recommended due to the steep development of CAV resistance and rapid evolution of KPC enzymes.

Phenotypic Detection of ESBL, AmpC, MBL, and Their Co-occurrence among MDR Enterobacteriaceae Isolates.

Background Emergence of extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and metallo-β lactamases (MBL), and their co-existence among members of Enterobacteriaceae pose newer diagnostic and therapeutic challenges. The present study examines the ESBL, AmpC, and MBL production by various phenotypic methods and their co-occurrence among the multidrug-resistant (MDR) Enterobacteriaceae clinical isolates. Materials and Methods Four hundred non-repetitive Enterobacteriaceae clinical isolates were collected from the Central Referral Hospital, Sikkim. The isolates were used for identification and their antibiotic susceptibility tests were performed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL was detected by double-disc synergy test (DDST) and phenotypic confirmatory disc-diffusion test (PCDDT), AmpC detection by AmpC E-test, and boronic acid disc diffusion (BD) test. MBL was detected using the imipenem–imipenem/EDTA disc and carba-NP tests. Results Around 76% were considered MDR. ESBL was seen in 58% and 50.4% based on DDST and phenotypic confirmation disc-diffusion test (PCDDT), respectively. AmpC was detected in 11.8% and 13.1% using a commercial E-test and boronic acid test, respectively. MBL were identified in 12.8% and 14.8% based on MBL imipenem-EDTA and carba-NP tests, respectively. Co-occurrence of ESBL and AmpC, ESBL and MBL, AmpC and MBL was seen in 5.2%, 11.5%, 1.3%, respectively, whereas a combination of these three β-lactamases was observed in only 0.3% of 304 MDR isolates. Conclusion The findings highlight a high prevalence of β-lactamases and their co-production among the Enterobacteriaceae , mainly in Klebsiella pneumoniae and Escherichia coli isolates. The study further highlights the necessity to identify the MDR β-lactamases stains for effective therapy in severe as well as mild bacterial infections, thereby enabling to reduce the risk of MDR in hospital and community settings.

Isolation and characterisation of carbapenem-resistant Pseudomonas aeruginosa from hospital environments in tertiary care hospitals in Dhaka, Bangladesh

Increasing evidence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) infection in healthcare facilities poses an alarming threat to public health. There is little evidence on the occurrence of this organism in Bangladeshi hospitals. We collected 117 environmental swab samples from two tertiary care hospitals in Dhaka, Bangladesh and tested for Pseudomonas species by nonselective enrichment of swabs followed by plating on Cetrimide agar. We confirmed the isolates as P. aeruginosa by API 20NE test and polymerase chain reaction Polymerase Chain Reaction (PCR) for 16S rRNA gene. We analysed P. aeruginosa isolates for susceptibility against 15 clinically important antibiotics and tested the carbapenem-resistant isolates for metallo β-lactamase (MBL). All CRPA isolates were characterised for carbapenem-resistant genes, virulence genes and biofilm formation genes. Of 117 swab samples, 82 (70%) were tested positive for P. aeruginosa. All P. aeruginosa isolates were multidrug-resistant, and 39% (n=32) of isolates were CRPA. Around 56% (n=18) of CRPA were MBL-producing; 22% (n=7) of isolates were positive for carbapenemase gene blaNDM followed by 16% (n=5) for blaVIM and 13% (n=4) for blaIMP. Sequencing identified these genes as blaNDM-1, blaIMP-13, blaVIM-2 variants. Based on optical density values, 94% (n=30) of CRPA isolates were capable of producing biofilms. All CRPA isolates (n=32) were positive for at least 1 of 6 biofilm-associated genes and 4 of 12 virulence genes tested in the study. Hospital environments in Bangladesh are contaminated with highly virulent CRPA, which might be a potential source of hospital-acquired infections, accentuating the need for strengthening hospital infection control programs.

Carba M Test for Rapid Detection and Simultaneous Differentiation of Carbapenemases among Clinical Isolates of Gram Negative Bacteria

Introduction: Carbapenemase production is the most common mechanism of carbapenem resistance. The carbapenemases belongs to class A, class B and class D of Ambler's molecular classification. Various tests have been designed for screening and confirmation of these enzymes. The acidimetric based Carba NP (Nordmann Poirel) test is simple and detects carbapenemases within two hours. The test could not differentiate between the serine carbapenemases from Metallo β-Lactamase (MBL). Differentiation of these two classes of enzymes will help in choosing the newer carbapenemase inhibitors like avibactam which selectively act on serine carbapenemases. Aim: To design a test that can simultaneously confirm and differentiate Amblers class A and D from class B carbapenemase enzymes among clinical strains of the Gram-Negative Bacteria (GNB). Materials and Methods: An experimental study was conducted between January-December 2018 on 195 strains of carbapenem resistant and 40 strains of carbapenem sensitive GNB. The carbapenemase genes were detected among all the bacteria by multiplex Polymerase Chain Reaction (PCR). The Carba M test was designed and evaluated for the detection and differentiation of class A and D from class B carbapenemases among the study isolates. Results: The Carba M test had 100% sensitivity and specificity for identification of New Delhi Metallo-β-lactamase (NDM) and Verona Integron-encoded Metallo-β-lactamase (VIM) enzymes. The strains which co-produced two MBL enzymes were detected with 100% sensitivity. The test had 42.85% sensitivity for the detection of Oxacillinase (OXA)-48-like enzymes. Conclusion: The Carba M test is useful in detection and simultaneous differentiation of carbapenemase content of the GNB and will help to choose appropriate carbapenemase inhibitors judiciously

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

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