Metallo Β-lactamase Research Articles

PREVALENCE OF ANTIMICROBIAL RESISTANCE AND MOLECULAR CHARACTERIZATION OF CARBAPENEMASE ENCODING GENE IN ESCHERICHIA COLI ISOLATES IN HOSPITAL OF HUE UNIVERSITY OF MEDICINE AND PHARMACY

Background: Currently, the existence of Extended-spectrum beta-lactamases (ESBL) and carbapenemase genes as well as genetic diversity has led to the rapid spread of resistance between E. coli species and the emergence of bacteria with pan-resistance. Objectives: To investigate the prevalence of antimicrobial resistance, prevalence of ESBL-producing E. coli, and carbapenemase-coding gene in E. coli isolates at Hospital of Hue University of Medicine and Pharmacy. Materials and method: The susceptibility to 13 antimicrobials of 246 E. coli isolates was detemined by the Kirby-Bauer method. ESBL-producing potential E. coli isolates were confirmed phenotypic by the Double Disc Synergy Test (DDST) and Combination Disc Test (CDT). Using multiplex PCR to detect genes encoding carbapenemase belonging to class B (Metallo β-lactamase), class D (oxacillinase), and blaKPC gene. Sequencing of carbapenemase genes were used to identify carbapenemase variants. Results: 91.46% of E. coli strains were identified as multidrug-resistant; most of them resistant to penicillins, fluoroquinolone, sulfonamide; high frequency of resistance to 3rd generation cephalosporin, chloramphenicol, gentamycin. The high isolation rate of ESBL-producing E. coli (50%) was detected. The resistance to carbapenems was largely mediated by the expression of acquired carbapenemases: blaKPC-2, blaNDM-4, and blaOXA-48. This study first reported blaNDM-4-harboring E. coli isolates in Vietnam. Conclusion: The high prevalence of multidrug-resistant and ESBL-producing E. coli, as well as the coexistence of ESBLs and carbapenemase genes, could seriously limit options for clinical treatment.

Emergence of Nosocomial Pneumonia Caused by Colistin-Resistant Escherichia coli in Patients Admitted to Chest Intensive Care Unit.

(1) Background: Colistin is a last-resort antibiotic used in treating multidrug-resistant Gram-negative infections. The growing emergence of colistin resistance in Escherichia coli (E. coli) represents a serious health threat, particularly to intensive care unit (ICU) patients. (2) Methods: In this work, we investigated the emergence of colistin resistance in 140 nosocomial E. coli isolated from patients with pneumonia and admitted to the chest ICU over 36 months. Virulence and resistance-related genes and E. coli pathotypes in colistin-resistant and colistin-sensitive isolates were determined. (3) Results: Colistin resistance was observed in 21/140 (15%) of the nosocomial E. coli isolates. The MIC50 of the resistant strains was 4 mg/L, while MIC90 was 16 mg/L. Colistin-resistant isolates were also co-resistant to amoxicillin, amoxicillin/clavulanic, aztreonam, ciprofloxacin, and chloramphenicol. The mechanism of colistin resistance was represented by the presence of mcr-1 in all resistant strains. Respectively, 42.9% and 36.1% of colistin-resistant and colistin-sensitive groups were extended-spectrum β-lactamase (ESBL) producers, while 23.8% and 21% were metallo β-lactamase (MBL) producers. blaTEM-type was the most frequently detected ESBL gene, while blaIMP-type was the most common MBL in both groups. Importantly, most resistant strains showed a significantly high prevalence of astA (76.2%), aggR (76.2%), and pic (52.4%) virulence-related genes. Enteroaggregative E. coli (76%) was the most frequently detected genotype among the colistin-resistant strains. (4) Conclusion: The high colistin resistance rate observed in E. coli strains isolated from patients with nosocomial pneumonia in our university hospital is worrisome. These isolates carry different drug resistance and virulence-related genes. Our results indicate the need for careful monitoring of colistin resistance in our university hospital. Furthermore, infection control policies restricting the unnecessary use of extended-spectrum cephalosporins and carbapenems are necessary.

Uropathogenic Escherichia coli in India-an Overview on Recent Research Advancements and Trends.

Urinary tract infection (UTI), a prevalent disease in India, also ranks among the most common infections in developing countries. The rapid emergence of antibiotic-resistant uropathogenic Escherichia coli (UPECs), the leading etiologic agent of UTI, in the last few years, led to an upsurge in the health care cost. This caused a considerable economic burden, especially in low-middle income country, India. This review aimed to provide an explicit overview of the recent advancements in E. coli-mediated UTI in India by incorporation of valuable information from the works published in PubMed and Google Scholar in the last six years (2015 to August, 2020). The literature survey demonstrated UPECs as the most predominant uropathogen in India, especially among females, causing both asymptomatic bacteriuria (ABU) and symptomatic UTI. An overall increasing national trend in resistance to penicillins, cephalosporins, aminoglycosides, fluoroquinolones, and sulfonamides was perceived irrespective of ABU and symptomatic UPECs during the aforementioned study period. High incidences of multidrug resistance, extended-spectrum β-lactamases, metallo β-lactamases, and AmpCs in UPECs were reported. Notable information on the pathogenic profiles, phylogroups, pathogenicity islands, and evidence of pathoadaptive FimH mutations was described. Alternative therapeutics and potential drug targets against UPECs were also reconnoitered. Therefore, the nationwide widespread occurrences of highly virulent MDR UPEC together with the limited availability of therapeutics highlighted the urgent need for promotion and invention of alternative therapeutics, search for which had already been started. Moreover, investigation of several mechanisms of UPEC infection and the search for potential drug targets might help to design newer therapeutics.

Faropenem reacts with serine and metallo-β-lactamases to give multiple products.

Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.

The Rising Threat of Drug Resistant Pseudomonas aeruginosa- A Nightmare for Intensive Care Unit Patients

Introduction: Multidrug Resistant (MDR), Extensively Drug Resistant (XDR) and Pan Drug Resistant (PDR) variants manifest a high level of intrinsic resistance to antimicrobial drugs by the help of efflux pump, biofilm formation and aminoglycoside modifying enzymes. The potentiality of Pseudomonas spp. to produce variety of drug resistance mechanism has led to evolution of drug resistant phenotypes this poses a challenge for clinicians in the treatment of severe infection among Intensive Care Unit (ICU) patients. Aim: To determine the phenotypic profiling of b-lactamases and burden of MDR, XDR and PDR Pseudomonas aeruginosa (P. aeruginosa) in ICU patients. Materials and Methods: The present cross-sectional prospective study was carried in the Department of Microbiology, Santosh Medical College and Hospital, Ghaziabad, Uttar Pradesh, India, after permission from Institutional Ethics Committee (IEC). A total of 115 isolates of P. aeruginosa were isolated from 502 human clinical samples from January 2019 to February 2021 and all the clinical samples were non duplicate. Antimicrobial Susceptibility Testing (AST) was performed for all isolates by standard Kirby- Bauer disc diffusion method on Mueller Hinton Agar (MHA). Phenotypic profiling of Extended Spectrum b-Lactamase (ESBL), Metallo b-Lactamase (MBL) and Ampicillinase C (AmpC) was performed by disc potentiation test; Imipenemase (IMP) - Ethylenediamine Tetraacetic Acid (EDTA) combined disc test and Cefoxitin Cloxacillin Double Disc Synergy Test (CC-DDST), respectively. The obtained results were statistically analysed in numbers and percentages using MS Excel 2013 version. Results: Out of 502 total human clinical samples, 115 isolates were P. aeruginosa giving the prevalence rate of 23%. Among 115 Pseudomonas isolates, 60 (52%) were MDR phenotypes, 8 (7%) were XDR phenotypes and there was no PDR phenotypes isolated in present study as all isolates were sensitive to Ticarcillin/Clavulanic acid, Colistin and Polymyxin B. Out of 115 isolates, 59 (51%) were ESBL producers, 26 (23%) were MBL producers, and 6 (5%) were AmpC producers. Conclusion: Strict antibiotic policies and regular surveillance programme of antimicrobial resistance must be tailored to fend off the emergence of drug resistant Pseudomonas aeruginosa.

Evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant Gram-negative organisms.

BackgroundMulti-drug resistant (MDR) Gram-negative bacteria are an emerging threat, both in hospital and community settings. As very few antibiotics are effective against such infections, the need of the hour is a new antibiotic or drug combination which can overcome the effect of extended-spectrum β-lactamases (ESBL) and metallo β-lactamases (MBL). A new antibiotic combination of ceftriaxone, sulbactam and disodium edetate (CSE) has recently been proposed to tackle the MDR organisms.ObjectiveOur study was carried out to assess the susceptibility of ESBL- and MBL-producing Gram-negative organisms to CSE.MethodsThe study was conducted in a tertiary-care hospital in Delhi, India, from February 2017 to June 2017. A total of 179 MDR (85 ESBL + 94 MBL) Gram-negative isolates from various clinical samples, identified by an automated system (Vitek 2) were tested against CSE using the Kirby-Bauer disc diffusion method. Susceptibility to CSE was recorded based on interpretative zone sizes of ceftriaxone as per 2017 Clinical and Laboratory Standards Institute guidelines.ResultsThe most common isolate was Escherichia coli (76/179; 42.4%) followed by Klebsiella pneumoniae (53/179; 29.6%) and Acinetobacter baumanii (27/179; 15.1%). The in vitro susceptibility of ESBL- and MBL-producing Gram-negative isolates to CSE was found to be 58/85 (68.2%) for ESBL and 37/94 (39.4%) for MBL.ConclusionThe in vitro susceptibility results obtained for CSE against ESBL-producing organisms is promising. It has the potential to emerge as a carbapenem-sparing antibiotic, active against ESBL-producing strains. Further clinical studies are required to establish the clinical efficacy of CSE against MDR pathogens.

Characterization of biofilm formation, pyocyanin production, and antibiotic resistance mechanisms in drug-resistant Pseudomonas aeruginosa isolated from children in Egypt

Children can be exposed to more bacterial infections, which has become of importance, especially when bacteria resist many of the antibiotics used today. Twelve Pseudomonas aeruginosa strains were collected and identified. Antibiotic sensitivity testing, biofilm formation, and pyocyanin production were evaluated. A cluster analysis was performed. Phenotypic and genotypic detection of efflux pump activity and Metallo-β Lactamase (MβLs) resistance mechanisms were studied. A marked resistance (100%) resulted toward antibiotics including cefotaxime, cefoxitin, ceftriaxone, and meropenem. Imipenem and amikacin antibiotics showed sensitivity percentages of 41.7 and 33.3, respectively. 66.7% were able to form a biofilm at which they were categorized as moderate (25%) and weak (41.7%). Phenotypically, 41.67% of the isolates were MBL metallo-β-lactamase-positive, but genotypically, blaVIM gene was detected only in one isolate, while blaIMP was not detected in any isolate. MexR and MexZ genes were detected in all isolates (100%). NfxB gene and MexT gene were found in 27.27% and 45.45% of isolates, respectively. All the efflux genes were found collectively in three isolates. This study highlights the occurrence of antibiotic resistance, besides the production of important virulence factors (biofilm formation and pyocyanin production). Also, the gene occurrence of antibiotic resistance mechanisms was reported among our P. aeruginosa. This virulent bacterial behavior is alarming which needs attention to the way we use antibiotics.

Genetic and phenotypic determinants of resistance to antibiotics in Aeromonas spp., strains isolated from pediatric patients.

Intestinal and extraintestinal infections by Aeromonas spp., remain controversial, due to the existence of healthy carriers of Aeromonas spp. In children under five years old, the diarrhea of infectious origin constitutes the second cause of mortality and remains a major concern for public health. The aim of this work was to detect the pheno/genotype of β-lactamases and class 1 integrons in Aeromonas spp., strains isolated from pediatric patients in a tertiary referral hospital in Mexico. Sixty-six strains of Aeromonas spp., were isolated from clinical samples of pediatric origin and were identified by RFLP-PCR 16S rRNA. Resistance phenotype according to CLSI, genetic and phenotypic detection of extended-spectrum β-lactamases (ESBL) and metallo-b-lactamases (MBL) was performed. Finally, characterization of class 1 integrons was performed. Aeromonas spp., strains of diarrheic origin were more predominant. A wide heterogeneity was detected, where A. caviae was the predominant specie. Second-generation cephalosporins, fluoroquinolones, and nitrofurans had best antimicrobial activity; moreover, antibiotics of the β-lactamic and lincosamides families showed lower inhibitory activity. Phenotypically, prevalences of 4.55% and 3.03% were detected for MBL (intestinal origin) and ESBL (extraintestinal origin), respectively. blaIMIS-cphA and blaTEM-1 genes, and nineteen class 1 integrons carrying two variants of cassettes corresponding to adenylyl transferases (aadA), and dihydrofolate reductases (dfrA). Monogenic array with aadA1 cassette was predominantly. ESBL and class 1 integrons, in Aeromonas collected from pediatric patients, determines a major detection challenge for the clinical microbiology laboratory and represents a remarkable epidemiological risk of nosocomial spread of multidrug-resistant determinants.

Molecular detection of the New Delhi metallo-β-lactamase clinical variant with double mutation- V88L and M154L in Escherichia coli isolates from South India

Carbapenem is usually prescribed as a last resort of antibiotic for treating drug resistant Gram-negative bacterial infections. However, the rise in carbapenem resistance due to the emergence of Zn2+ dependent New Delhi metallo-β lactamase (NDM) variants imposes a huge threat to global health. In Indian context, focusing on molecular epidemiology and underpinning the genetic insights of such continuously evolving superbug is perquisite to guide any form of control strategies or therapeutic interventions. Herein, we investigated the occurrence of NDM variants in Escherichia coli (n = 70) isolated from clinical samples. PCR assay targeting blaNDM gene detected 5 E. coli isolates as NDM positive. Sequencing analyses of blaNDM region of the plasmid DNA determined two types of variants viz., NDM-1 (n = 3) and NDM-5 (n = 2). Notably, NDM-5 variants harbored V88L and M154L mutations, which are known to increase the fitness of NDM-5 variant towards host immune system and zinc starvation. On further characterization, three NDM variants showed the ESBL and AmpC β-lactamase genes, along with 3 different plasmid replicon types (L/M, Y, FIC). Virulence profiling showed traT gene as the most predominant gene among the variants. The emergence of NDM variants especially NDM-5 like variants with critical mutations in enterobacteriaceae increases the significant menaces to the global healthcare. The fact that these NDM variants harboring other antibiotic resistance markers, along with plasmid replicons and virulence genes can be transmitted underlines the gravity of this public health issue.

Molecular detection of extended spectrum β-lactamases, metallo β-lactamases, and Amp-Cβ-lactamase genes expressed by multiple drug resistant Pseudomonas aeruginosa isolates collected from patients with burn/wound infections

Objectiveshis study was aimed to identify diverse β-lactamase genes produced by multiple drug-resistant Pseudomonas aeruginosa isolates procured from burn/wound infected patients hospitalized in General Al-thawrah hospital of Hodeidah city, Yemen during (July to December 2018). MethodsFrom 200 burn wound swabs, total 200 single strain per patient was isolated by culturing them on nutrient agar, MacConkey agar and blood agar, of which 98 were identified and confirmed as P. aeruginosa.The isolates were subjected for antibiotic susceptibility test according to the CLSI guidelines; molecular characterization of β-lactamases-encoding genes were carried out with multiplex PCR. ResultsOverall, significantly high resistance was observed for ceftazidime, cefepime, gentamicin, tobramycin, amikacin and ciprofloxacin against P. aeruginosa (N = 98) with percent resistance of77.5% (n = 76), 72.4% (n = 71), 85.7% (n = 54), 74.5% (n = 73), 83.7% (n = 82), and 66.3% (n = 65), respectively. however, carbapenems showed comparatively low resistance as 20–21% while another routinely used anti-pseudomonas combination, piperacillin-tazobactam showed 54% of resistance. Out of 98 Pseudomonas studied, 65 isolates (66.3%) were found to be multi-drug resistant. Also, presence of various β-lactamases which was confirmed by PCR based amplification. Presence of various ESBLs was evident as 43% (n = 28), 30.7% (n = 20), 30.7% (n = 20), 24.6% (n = 16) and12.3% (n = 8) of isolates were identified with blaVEB, blaGES, blaCTX-M, blaOXA-10, blaTEM, and blaSHV genes respectively. Among the MBL-expressing isolates (n = 20, 30.7%) all exhibited blaVIM gene while Amp-C gene was observed in 40isolates (61.5%). ConclusionThe outcome of this study demonstrated significant increase in antibiotic resistance due to expression of diverse β-lactamases owing to increased MDR P. Aeruginosa in Yemen. We suggest antibiotic treatments comprising of combinations of antibiotics in order to preserve the utility of carbapenems and piperacillin-tazobactam in Yemen and to curb acquisition and dissemination of antibiotic resistance genes in P. Aeruginosa in this territory.

English

Eighty-five uropathogen isolates were collected and differentiated on the basis of their Gram stain reaction. Among the collected isolates, 25 (29.4%) were Gram positive cocci and 60 (70.6%) were Gram negative bacilli. Antibiotic susceptibility profile towards 15 different antibiotics concluded that impenime (IMP), amikacin (AK) and Pipperacillin/tazobactam (TZP) were the most effective against Gram negative; while linezolid (LZD), and vancomycin (VA) were the most potent against Gram positive. The highly drug resistant isolates were identified as Pseudomonas aeruginosa and Klebsiella pneumoniae using 16srRNA; the two strains were subjected to different doses of gamma-radiation and the sub lethal dose of both strains was 5.0 kGy. The D10 values were recorded (1.2 and 1.1 kGy) for P. aeruginosa and K. pneumoniae), respectively. In addition, gamma-irradiation technique decreases the resistance of K. pneumoniae towards IMP and the resistance of P. aeruginosa towards more than one antibiotic (TZP, AZM and CIP). The results also, revealed that both of the tested strains had the ability to produce Extended Spectrum β-lactamase (ESBL) before and after gamma-radiation but only P. aeruginosa had the ability to produce Metallo β-lactamase (MBL), that is, ESBL and MBL co-production was detected in P. aeruginosa. So, these findings must be supported by other studies on the level of genes to prove the possibility for using gamma-irradiation technique to overcome microbial drug resistance problem. Key words: Urinary tract infection, antibiotic resistance, β-lactamases, gamma radiation.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of japanese society of chemotherapy, the japanese association for infectious diseases, and the japanese society for clinical microbiology in 2016: General view of the pathogens’ antibacterial susceptibility

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2016.The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between February 2016 and August 2016 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute.Susceptibility testing was evaluated in 1062 strains (143 Staphylococcus aureus, 210 Streptococcus pneumoniae, 17 Streptococcus pyogenes, 248 Haemophilus influenzae, 151 Moraxella catarrhalis, 134 Klebsiella pneumoniae, and 159 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 48.3%, and those of penicillin-susceptible S. pneumoniae was 99.5%. Among H. influenzae, 14.1% of them were found to be β-lactamase-producing ampicillin-resistant strains, and 41.1% to be β-lactamase-non-producing ampicillin-resistant strains. Extended spectrum β-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo β-lactamase were 4.5% and 0.6%, respectively.

New Delhi metallo-β-lactamase-1 inhibitors for combating antibiotic drug resistance: recent developments

The most common mechanism of the resistance to β-lactam antibiotics is the expIdentification of cisplatin and palladiuression of β-lactamases, which can hydrolyze the β-lactam moiety and deactivate these antimicrobials. The worldwide prevalence of New Delhi metallo-β-lactamase-1 (NDM-1), also known as carbapenemase has created distress among clinicians. NDM-1 is a type of metallo β-lactamase (type of β-lactamases which require metal ions for their catalytic action) produced in pathogens carrying blaNDM-1 gene. These NDM-1 producing pathogens are resistant to all β-lactam antibiotics including carbapenems and are greatest threat to public health as they can easily extend via horizontal gene transfer. Various NDM-1 variants have been evolved over time and it may further evolve in the future due to several factors such as bacterial gene mutation and overuse of antibiotics. In the past 10 years, various NDM-1 inhibitors have been reported showing diverse chemical structure. In spite of a great development in terms of structural and mechanistic information, the design of a potent inhibitor of NDM-1 to be approved clinically remains challenging, this could be due to structural complexity of the enzyme that limits the development of clinically useful NDM-1 inhibitors. Along with designing of novel and effective NDM-1 inhibitors, measures should be taken for controlling multidrug resistance. Currently, there is non-availability of NDM-1 inhibitors clinically therefore clinicians are facing a huge challenge in treating infections due to multidrug-resistant bacteria. This review article has been planned to discuss about antibiotic resistance, NDM-1, mechanism of antibiotic resistance by NDM-1, and recent updates in the development of NDM-1 inhibitors as well as mechanism of action of NDM-1 inhibitors.

In vitro activity of tigecycline against local clinical isolates of some Enterobacteriaceae

BackgroundInfectious diseases that Enterobacteriaceae cause are spreading on a wide scale. Examples of these infections are gastrointestinal tract infections, meningitis, pneumonia, septicemia, urinary tract infections, and wound infections. Antibiotic resistance among Enterobacteriaceae is a critical problem that makes treatment difficult. Tigecycline is a broad spectrum antibiotic that is effective against multi-drug resistant organisms (MDR) and beneficial in the therapy of infections caused by Enterobacteriaceae.ObjectivesThis study aims to evaluate the in vitro activity of tigecycline against clinical isolates of Enterobacteriaceae species and detect the possible resistance mechanisms of them against β-lactams.MethodsThe sensitivity of different isolates to antibiotics was determined by standard disc diffusion method. Phenotypic detection of resistance mechanisms such as extended spectrum β-lactamase (ESBL), AmpC, ESBL& AmpC co-producers and metallo β-lactamase (MBL) β-lactamases enzymes producer isolates was investigated.ResultsA total of eighty three Enterobacteriaceae clinical isolates were collected. The common bacteria isolated were Escherichia coli and Klebsiella pneumoniae. Multidrug resistance was found in 59.04% of tested isolates. The isolates were resistant to sulphamethaxzole-trimethoprim, ciprofloxacin, tetracycline and impenem. The highest resistance was found to sulphamethaxazole-trimethoprim followed by ciprofloxacin, tetracycline and imipenem. Phenotypic detection of resistance mechanisms revealed that 69.4% of clinical isolates were ESBL producers, 12.2% were AmpC producers, 8.2% were ESBL and AmpC co-producers, while 10.2% were MBL producers. There was no resistance found to tigecycline among all Enterobacteriaceae isolates tested.ConclusionThis study showed that tigecycline has potent in vitro activity against ESBL, AmpC, combined ESBL and AMPC and MBL β-lactamases producing Enterobacteriaceae.

1177 QUANTITATIVE QPCR-BASED BIOBURDEN TEST FOR THE DETECTION OF CARBAPENEM-RESISTANT ENTEROBACTERIACEAE (CRE): NOVEL TEST FOR SURVEILLANCE AND RISK-STRATIFICATION

Current strategies to combat duodenoscope-associated carbapenem-resistant Enterobacteriaceae (CRE) transmission, a low-prevalence but high-risk clinical problem, are either capital intensive (ethylene oxide sterilization) or time- and resource-intensive (reference laboratory PCR or culture and sequester strategy). PCR tests do not discriminate live vs. dead organisms and quantify them. Our aim was to develop and perform initial analytical verification and clinical sample testing of a bioburden qPCR test that quantifies the number of live CRE present on duodenoscopes or biofluids within 3 hours of sampling. We designed TaqMan assays against each intended target gene sequences (16S rRNA, Klebsiella pneumonia carbapenemase (KPC) and New Delhi metallo β-lactamase (NDM)) that can detect and quantify genomic DNA (gDNA) across 5-9 log concentrations with >95% qPCR amplification efficiency. We developed an optimum gDNA extraction method to extract gDNA with maximum efficiency. Propidium monoazide (PMA)/ enhancer concentrations and incubation/light exposure time was optimized to yield maximum cycle threshold (Ct) difference in qPCR quantification between live and dead CRE (Figure. 1). We successfully tested and quantified live CRE from serial dilutions of intentionally spiked (with different CRE concentrations) patient-derived bio-fluids (bile and gastrointestinal secretions) acquired during ERCPs (n=15). Duodenoscope samples were collected by flushing 10 ml of sterile saline through the accessory channel and dipping and agitating the end of the duodenoscope and performing ten up and down movements of the elevator in a container of sterile saline Propidium monoazide (PMA)/enhancer can detect, differentiate and quantitate 10% of spiked-in live bacteria among the dead. Our DNA extraction method outperforms commercially available DNA extraction kits that we tested, in time, cost and efficiency. Assay reproducibly detects >100 intentionally spiked NDM1+ and KPC1+ CFUs either within patient derived biofluids or endoscope wash fluids (Saline) (figure 2). There is no detectable PCR inhibition from patient derived abdominal fluids, and biliary specimens. We report on the successful development and initial testing of a rapid-turnaround quantitative PCR bioburden test that can detect and quantify small numbers of live CRE on duodenoscopes and biofluids. Experiments are underway to test the accuracy, precision, and the analytical sensitivity of the assay. Future studies will include test validation in a multi-center duodenoscope and other medical device-derived biofluid testing and creation of a digital PCR interface.Figure 2Amplification plot demonstrating detection of live CRE among intentionally spiked biofluids.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Occurrence of blaTEM and blaCTXM Genes and Biofilm-Forming Ability among Clinical Isolates of Pseudomonas aeruginosa and Acinetobacter baumannii in Yaoundé, Cameroon.

Background: Pseudomonas aeruginosa (PSA) and Acinetobacter baumannii (ACB) are non-fermentative bacteria mostly associated with nosocomial infections in humans. Objective: This study aimed to determine the antimicrobial resistance profiles and virulence gene of PSA and ACB previously isolated from humans in selected health facilities in Yaoundé, Cameroon. Methods: A total of 77 and 27 presumptive PSA and ACB isolates, respectively, were collected from the Yaoundé teaching hospital. These isolates were previously isolated from various samples including pus, blood and broncho-alveolar lavage. The identities of the isolates were determined through polymerase chain reaction (PCR) amplification of PSA and ACB specific sequences. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disc diffusion method. Phenotypical expression of AmpC β-lactamases (AmpC), extended spectrum β-lactamases (ESBLs) and metallo β-Lactamases (MBLs) were determined using the combined disc method. Bacterial genomes were screened for the presence of β-lactamases blaTEM and blaCTXM genes using specific PCR. The pathogenicity of PSA and ACB was assessed through amplification of the lasB, exoA, pslA and exoS as well as OmpA and csuE virulence genes, respectively. Results: Of the 77 presumptive PSA isolates, a large proportion (75 to 97.4%) were positively identified. All (100%) of the presumptive 27 ACB harbored the ACB-specific ITS gene fragment by PCR. Twenty five percent of the PSA isolates produced ESBLs phenotypically while more than 90% of these isolates were positive for the lasB, exoA, pslA and exoS genes. A large proportion (88%) of the ACB isolates harboured the OmpA and csuE genes. blaTEM and blaCTXM were detected in 17 and 4% of PSA, respectively, while a much higher proportion (70 and 29%) of the ACB isolates possessed these resistance determinants respectively. Conclusion: Our findings reveal the occurrence of both virulence and drug-resistant determinants in clinical PSA and ACB isolates from patients in health care settings in Yaoundé, Cameroon, thus suggesting their role in the pathological conditions in patients.

Disruption of Metal Homeostasis and its Effect on Bacterial Growth and Antibiotic Resistance

As effective as antibiotics have been since they were first discovered, antibiotic resistance proves to be a worldwide threat, endangering their efficacy. Over the years, there has been a significant decline in development of novel class antibiotics. These two factors have made once easily treated infections difficult to manage. Carbapenem‐resistant Enterobacteriaceae (CRE) are an emerging threat due to their high levels of antibiotic resistance. CRE have become resistant to almost all antibiotics, and cause 23,000 infections per year in the United States. Metals are a vital nutrient in all living organisms including bacteria and, have thus been targeted by antimicrobial agents. Bacteria, like all living organisms, must regulate their metal homeostasis in order to survive and replicate. Therefore, mechanisms able to diminish the availability of the metals to bacterial pathogens can be used to develop new therapeutic drugs.An example of a mechanism of bacterial resistance to antibiotics is the production of inactivating enzymes, such as carbapenemases, an example of which is seen in Klebsiella pneumoniae. Carbapenemases play an important role against b‐lactam antibiotics such as imipenem in Enterobacteriaceae. Carbapenemases are categorized based on their functional and molecular properties. Class B carbapenemases are metallo‐b‐lactamases (MBL) which require zinc in their active site, and are therefore considered metalloenzymes. MBL can hydrolyze all b‐lactams and are not inactivated by b‐lactamase inhibitors.In this study we analyzed antimicrobial susceptibility of K. pneumoniae isolates, KPC 2146, KPC 1705 and KPC 1706 which contain class B, class A and no carbapenemase, respectively. Metal homeostasis of isolates was disrupted by inducing zinc deficiency using TPEN, a metal chelator with high affinity for zinc, and preparing a zinc deficient minimal media. Carbapenemase activity was assessed using CARBA‐NP test and LC‐MS. Bacterial susceptibility of zinc‐deficient isolates to carbapenems was analyzed using spot plating, growth curve, minimum inhibitory concentration and ring of inhibition.Our findings demonstrate zinc dependency of class B carbapenemase in the KPC 2146 isolate, while class A carbapenemase in the KPC 1705 isolate was not affected. Spot plating and growth curve analyses demonstrated that zinc deficiency lowers bacterial growth in KPC 2146 in the presence of imipenem. Zinc deficiency via TPEN addition lowered imipenem, doripenem, ertapenem and meropenem MIC in KPC 2146. Ring of inhibition of KPC 2146 with imipenem was greatly affected by zinc chelation. In conclusion, we demonstrate that disrupting zinc homeostasis affects class B carbapenemase activity, which in turn increases bacterial susceptibility to carbapenems.Support or Funding InformationNika Padyab was supported by funds from the Biomedical Sciences Program at MWU. Drs. Veltri and Hernandez were supported by MWU intramural funds.

Antibiotic resistance pattern of Salmonella species isolated from Typhoid patients in Jigawa state, Nigeria

This study was designed to determine the antibiotic resistance pattern of Salmonella spp. isolated from clinical samples of symptomatic and asymptomatic typhoid patients, selected from some hospitals in Jigawa State, Nigeria. Four hundred bacterial isolates were recovered from these samples, were identified as; Salmonella typhimurium, S. enteritidis, S. typhi, S. paratyphi A, and S. paratyphi B using microgen biochemical system, according to the standard techniques. The antibiogram of all these Salmonella spp. were carried out using standard oxoid disc of different antibiotics. Double disc diffusion technique was employed to determine the isolates’ capacity to produce β-lactamase. Results showed 5(1.25%), 0(0.0%), 0(0.0%) of the samples collected were positive for extended spectrum β-lactamases (ESBL), AmpC β-lactamase, and Metallo β-lactamases (MBL), respectively. The antibiogram profile of the isolates showed that 100% of these isolates are susceptible to Imipenem, only 3% exhibited resistance to ceftriaxone, whereas 7% demonstrated resistance to ciprofloxacin. To the best of our knowledge, this the first study of this kind in the Jigawa state, especially on these Salmonella spp. The study concluded that β-lactamases with the capacity of conferring multidrug resistance were recorded in Salmonella spp. isolated from patients in several hospitals of Jigawa State, Nigeria.

Prevalence of bla<sub>VIM</sub>, bla<sub>IMP</sub> and bla<sub>NDM</sub> Genes in Carbapenem Resistant Pseudomonas Aeruginosa Isolated from Different Clinical Infections in Diyala, Iraq

The study aimed to determine the prevalence of bla-genes (bla VIM , bla IMP and bla NDM ) encoding MBLs among the isolates of P. aeruginosa, which were recovered from various clinical samples from hospitalized patients in Diyala hospitals. This study was carried out during the period from February 2018 to August 2018. Out of 326 specimens, 81 isolates of P. aeruginosa were recovered. All isolates were tested toward the different class of clinically important antipseudomonal agents(18) by using agar diffusion method. The results of resistance were as following: piperacillin 74.07, ticarcillin 85.18%, Amoxicillin-Clavulanic acid 93.82%, Ticarcillin/clavulanic acid 71.60%, ceftriaxone 87.65%, cefotaxime 85.18% and ceftazidime 75.30%, cefepime 80.24%, streptomycin 90.12%, gentamicin 85.18%, tobramycin 65.43%, amikacin 56.79%, ciprofloxacin 67.90%, levofloxacin 72.83%, oflaxacin 69.13%, Aztreonam 50.61%, Imipenem 11.11% and Meropenem 23.45%.In this investigation, antibiotic susceptibility testing of the P. aeruginosa isolates showed that 20(24.69%), 25(30.8%), 27(33.33%), and 9(11.11%) of the isolates were MDS, MDR, XDR and PDR, respectively. Based on the results from susceptibility testing, 19 (23.45%) of P. aeruginosa isolates were found to be resistant to at least one of carbapenems. Out of 19 of carbapenem resistant P. aeruginosa isolates, 16 (84.21%) were found to produce MBL. Among 16 phenotypic Metallo β-lactamase isolates the results achieved by using PCR revealed that 9 (56.25%) isolates have bla VIM genes, while 4 (25%) isolates carried bla NDM genes. No bla IMP was detected among carbapenem resistant strains in this study.

Carbapenem-Resistant Klebsiella pneumoniae Strains: Susceptibility to Novel Antibiotics and Molecular Detection of the Resistance Mechanisms - A Study from Two Greek Tertiary Teaching Hospitals

Objectives: We evaluated the carbapenem resistance mechanisms of Klebsiella pneumoniae strains isolated in two Greek tertiary teaching hospitals and their susceptibility to currently used and novel antimicrobial agents. Materials and methods: Forty-seven carbapenem resistant K. pneumoniae strains were collected in G. Papanikolaou and Ippokrateio hospital of Thessaloniki from 1/11/2016 to 5/1/2018 and 26/1/2017 to 19/4/2017 respectively. Strain identification and antimicrobial susceptibility was conducted by Vitek 2 system (Biomerieux France). Susceptibility against new antimicrobial agents was examined by disk diffusion method. Polymerase chain reaction (PCR) was used for the detection of blaKPC, blaVIM, blaNDM and blaOXA-48 genes. Results: The EDTA-boronic acid disk synergy test performed on the 24 K. pneumoniae strains from G. Papanikolaou hospital demonstrated that 8 (33.3%) yielded positive for metallo-b-lactamases (MBL) and 16 (66.6%) for K. pneumoniae carbapenemases (KPC) production. Gentamycin demonstrated the highest in vitro activity (82.6%) among the 23 K. pneumoniae strains from Ippokrateio hospital followed by colistin (73.9%) and tigecycline (69.5%). All strains from G. Papanikolaou hospital were sensitive to colistin whereas the 70.8% of them displayed susceptibility to gentamycin. Ceftazidime/ avibactam showed the highest sensitivity (76.6%) in all strains followed by eravacyclin (66.6%). The blaKPC gene was present in 30 strains (63.8%), the blaNDM in 11 (23.4%) and the blaVIM in 6 (12.8%). The blaOXA-48 gene was not detected. Conclusions: Well established antimicrobial agents such as colistin, gentamycin and tigecycline and novel antibiotics like ceftazidime/avibactam and eravacycline may be reliable options for the treatment of invasive infections caused by KPC-producing pathogens.