MBL Production Research Articles

Genotypic detection of metallo-Beta-Lactamases among multidrug resistant Klebsiella pneumoniae isolated from urine samples of UTI patients

Metallo-beta-lactamases producing bacteria are of public health concern, owning to the fact that bacteria that harbour genes for their production are notably resistant to several antibiotics. This study was carried out to genotypically investigate the prevalence of MBL-production in multidrug resistant K. pneumoniae isolated from urine samples of UTI patients attending a tertiary teaching hospital in Awka, Anambra State, Nigeria. Two hundred (200) mid-stream urine samples were collected from UTI patients, and were analyzed using standard microbiological procedures. Antibiotic susceptibility testing (AST) of the isolates was carried out using Kirby-Bauer disc diffusion method and combination disc test was employed to phenotypically screen the isolates for MBL production. The genotypic screening was performed by Polymerase chain reaction (PCR), using specific primers. Fifty-one (51) K. pneumoniae isolates were recovered from the urine samples. AST revealed that the isolates exhibited high resistance to Amoxicllin-clavulanic acid (100%), Ampiclox (98.04%), Imipenem (98.04%), Cefotaxime (96.08%), Cefuroxime (96.08%), Nitrofurantoin (70.59%), and were relatively susceptible to Ofloxacin (54.91%), Gentamycin (50.98%) and Levofloxacin (47.06%). A 79.02% of the K. pneumoniae isolates showed resistance to more than two classes of antibiotics, thus termed multidrug resistance strains. Of the 38 (41.76%) isolates of K. pneumoniae that were resistant to imipenem, 19 (20.88%) were confirmed as MBL-producers phenotypically, while 19 (20.88%) were non-MBL producers. Genotypically, blaVIM (21.98%) was the most detected MBL gene, followed by blaOXA-48 (18.68%) and blaIMP (15.38%). This study revealed the prevalence of multidrug resistant and MBLs producing K. pneumoniae in urine samples of the selected UTI patients.

In-vitro activity of the novel β-lactam/β-lactamase inhibitor combinations and cefiderocol against carbapenem-resistant Pseudomonas spp. clinical isolates collected in Switzerland in 2022.

To evaluate the in-vitro activity of the novel commercially-available drugs, including meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam (IPR) as well as cefiderocol (FDC), against carbapenem-resistant Pseudomonas spp. (CRP) isolates. All CRP isolates collected at the Swiss National Reference Laboratory (NARA) over the year 2022 (n = 170) have been included. Most of these isolates (n = 121) were non-carbapenemase producers. Among the 49 carbapenemase producers, 47 isolates produced metallo-β-lactamases (MBL) including NDM-1 (n = 11), VIM-like (n = 28), IMP-like (n = 7), and both NDM-1 and VIM-2 (n = 1) and two isolates produced the class A carbapenemase GES-5. Susceptibility testing was determined by broth microdilution method (BMD), or disk diffusion test, and results interpreted following EUCAST guidelines. The susceptibility rates for MEV, CZA, C/T and IPR were found to be 41%, 45%, 59% and 58%, respectively, for the whole set of isolates tested. Among non-carbapenemase producers, susceptibility rates for these β-lactam/β-lactamase inhibitors (BL/BLI) combinations were higher, determined at 55%, 61%, 83%, and 82%, respectively. The overall susceptibility of carbapenemase-producing Pseudomonas spp. to novel BL/BLI was relatively low, while 80% of these isolates demonstrated susceptibility to FDC, with a similar proportion (79%) observed among MBL producers. A total of 10 MBL-producing isolates (6%), mainly NDM-1, were found to exhibit resistance to all drugs tested, with the exception of colistin. FDC exhibited an excellent in-vitro activity against this collection of CRP recovered from Switzerland in 2022, including MBL producers. The new BL/BLI combinations displayed significant activity against non-carbapenemase CRP, with IPR and C/T showing the highest susceptibility rates.

MOLECULAR CHARACTERIZATION OF MBL IN UROPATHOGENIC E. COLI ISOLATED FROM PATIENTS OF TERTIARY CARE HOSPITAL

Background: Antibiotic resistance is on an increasing trend, particularly in gram-negative bacteria. The production of metallo β-lactamase (MBL) puts the health sector at great risk as it further limits the treatment option for MDR strain. MBL is mostly reported in Enterobacteriaceae, which poses resistance to almost all β-lactam antibiotics except monobactam. The current study aims to determine the prevalence, antibacterial sensitivity pattern, and molecular characterization of MBL in Uropathogenic E. coli from clinical samples of hospitalized patients in Khyber Pakhtunkhwa. Methods: From tertiary care hospitals in Peshawar, 250 Urine samples were collected from indoor hospitalized patients. Gold standard microbiological methods were used to identify UPEC from these clinical samples.For that,urine samples was inoculated onto Cysteine Lactose Electrolyte. Deficient (CLED) agar plate, and MacConkey Agar.Positive growth of E.coli identified through Gram staining, colony morphology, Biochemical Tests and E.coli 16srRNA gene amplification .Antibiotic sensitivity was determined by the disc diffusion method on Muller Hinton agar. For the detection of MBL production double disc synergy, and a combination disc test of the antibiotics were used. Furthermore, multiplex PCR was used for the molecular characterization of the MBL (blaIMP, blaVIM, and blaNDM) genes. Results: Of the 250 samples, only 110 samples were confirmed as Uropathogenic E. coli based on colonial morphology, biochemical testing, and molecular level by targeting the 16SrDNA. Female was found more susceptible to UTI compared to male. High prevalence was found in the age group 45-65 years. UPEC was found highly resistance to Ciprofloxacin (90%), followed by Cefotaxime and Ceftriaxone (86%), Ceftazidime and Augmentin (81%), Tazobactam (61%). while the lowest resistance was reported against Meropenum (20%) Imipenem (18%) and Amikacin (37%). PCR-based confirmed prevalence of MBL encoding genes was blaNDM (42%), blaVIM (32%), and blaIMP (26%). Conclusion: The study proposed a higher prevalence of urinary tract infections (UTIs) in females aged group 54-65 years compared to males. An analysis of antibiotic sensitivity revealed Imipenem and Meropenem to be the most effective antimicrobial agents, while Ciprofloxacin, Cefotaxime and Amoxicillin were found to be the less effective. UPEC were found highly resistance to Ciprofloxacin 91%, and ceftazidime 86%, while comparatively less resistance to meropenem, and imipenem,20% and 18% respectively. Genotype BlaNDM of the MBL is highly prevalent (42%) among UPEC.Furthermore, the presence of MBL genes was detected in over 19% of UPEC, and in different combinations.The upraise of the MBLs resistance in uropathogenic E. coli is an alarming sign for clinicians to decide on treatment options for complicated UTIs.

Antimicrobial susceptibility of enterobacterales causing bloodstream infection in United States medical centres: comparison of aztreonam-avibactam with beta-lactams active against carbapenem-resistant enterobacterales

BackgroundBloodstream infection (BSI) is associated with poor outcomes especially when effective antimicrobial therapy and control of infection source are delayed. As the frequency of Enterobacterales producing metallo-β-lactamases (MBL) and/or OXA-48–like carbapenemases is increasing in some United States (US) medical centres, effective antimicrobials to treat the infections caused by these organisms are urgently needed. Aztreonam-avibactam is under clinical development for treatment of infections caused by Gram-negative bacteria, including MBL producers.ObjectivesTo evaluate the antimicrobial susceptibility of Enterobacterales causing BSI in US medical centres and compare the activity of aztreonam-avibactam with ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, cefiderocol, and other antimicrobials used to treat BSI.Methods4,802 Enterobacterales were consecutively collected (1/patient) from 72 US medical centres in 2020–2022 and susceptibility tested by broth microdilution. Aztreonam-avibactam was tested with avibactam at a fixed concentration of 4 mg/L. A pharmacokinetic/pharmacodynamic susceptible breakpoint of ≤ 8 mg/L was applied for aztreonam-avibactam for comparison. Carbapenem-resistant Enterobacterales (CRE) isolates were tested for β-lactamase–encoding genes using Next-generation sequencing.ResultsAztreonam-avibactam was highly active against Enterobacterales; only 2 isolates showed aztreonam-avibactam MICs > 8 mg/L: 1 meropenem-susceptible E. coli and 1 K. aerogenes (CRE). All carbapenemase producers and 98.0% of CRE were inhibited at an aztreonam-avibactam MIC of ≤ 8 mg/L. CRE susceptibility rates were 81.6% for ceftazidime-avibactam, 65.3% for meropenem-vaborbactam, 61.2% for imipenem-relebactam, and 87.8% for cefiderocol. Aztreonam-avibactam retained activity (MIC, ≤ 8 mg/L) against all (100.0%) meropenem-vaborbactam nonsusceptible (n = 17), 99.5% of imipenem-relebactam nonsusceptible (n = 206), and 90.0% of ceftazidime-avibactam nonsusceptible (n = 10) isolates. The most common carbapenemases were KPC-2/3 (57.1% of CREs), OXA-48–like (16.3%), and NDM (14.3%). A carbapenemase gene was not observed in 12.3% of CREs. Ceftazidime-avibactam and meropenem-vaborbactam were active against 100.0% of KPC producers, but ceftazidime-avibactam showed limited activity against MBL producers and meropenem-vaborbactam showed limited activity against OXA-48–like and MBL producers. The most active non–β-lactam comparators against CRE were gentamicin (49.0% susceptible) and amikacin (44.9% susceptible).ConclusionsAztreonam-avibactam demonstrated potent activity against a large collection of Enterobacterales isolated from patients with BSI in US hospitals, including CRE, MBL producers, and isolates resistant to recently approved β-lactamase inhibitor combinations.

The Challenge of Treating Infections Caused by Metallo-β-Lactamase-Producing Gram-Negative Bacteria: A Narrative Review.

Gram-negative multidrug-resistant (MDR) bacteria, including Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa, pose a significant challenge in clinical practice. Infections caused by metallo-β-lactamase (MBL)-producing Gram-negative organisms, in particular, require careful consideration due to their complexity and varied prevalence, given that the microbiological diagnosis of these pathogens is intricate and compounded by challenges in assessing the efficacy of anti-MBL antimicrobials. We discuss both established and new approaches in the treatment of MBL-producing Gram-negative infections, focusing on 3 strategies: colistin; the recently approved combination of aztreonam with avibactam (or with ceftazidime/avibactam); and cefiderocol. Despite its significant activity against various Gram-negative pathogens, the efficacy of colistin is limited by resistance mechanisms, while nephrotoxicity and acute renal injury call for careful dosing and monitoring in clinical practice. Aztreonam combined with avibactam (or with avibactam/ceftazidime if aztreonam plus avibactam is not available) exhibits potent activity against MBL-producing Gram-negative pathogens. Cefiderocol in monotherapy is effective against a wide range of multidrug-resistant organisms, including MBL producers, and favorable clinical outcomes have been observed in various clinical trials and case series. After examining scientific evidence in the management of infections caused by MBL-producing Gram-negative bacteria, we have developed a comprehensive clinical algorithm to guide therapeutic decision making. We recommend reserving colistin as a last-resort option for MDR Gram-negative infections. Cefiderocol and aztreonam/avibactam represent favorable options against MBL-producing pathogens. In the case of P. aeruginosa with MBL-producing enzymes and with difficult-to-treat resistance, cefiderocol is the preferred option. Further research is needed to optimize treatment strategies and minimize resistance.

Identification Of Metallo?-Lactamses and Integron Genes In Pseudomonas Aeruginosa Isolated From Burn Injury Patients: Phenotype And Genotype

Pseudomonas aeruginosa, which generates metallo-?-lactamase (MBL), is the cause of infections linked to burn patients, and this is a growing global problem. The objectives of the recent study were to determine antibiotic susceptibility of P. aeruginosa isolates, the presence of MBLs genes, and Integron gene class-1 . One hundred burn patients at the Al-Hussein Teaching Hospital in Al Smawah, Iraq, were isolated clinically. Twenty (20%) of these were determined to be P. aeruginosa by 16sRNA and biochemical testing. To further identify the antibiotic sensitivity for each isolate was employed by Disc Diffusion Method . The phenotypic MBL production For detected isolates was evaluated by Combined Disc Diffusion Method (CDD). Integron gene class -1 (Int-1) and Genes encoding MBLs were identified by Polymerase Chain Reaction (PCR). P. aeruginosa was shown to be totally resistant to ceftriaxone, ampicillin, piperacillin, gentamicin, and ciprofloxacin in this investigation. 90% of the isolates were determined to be multidrug resistant, and different levels of moderate to lowest resistance were observed in Amikacin, Meropenem, Aztreonam, Cefixime, and Levofloxacin. While 90% of the isolates possessed the Int-1 gene, our recorded the absence of All MBLs genes (Bla IMP, Bla VIM, Bla GIM ) From All isolates under investigation. Illustrating for the first time how crucial it is to put in place appropriate infection control measures in order to give patients the best care possible and stop the development of these resistant microbes among burn patients.

Evaluation of gradient strip diffusion for susceptibility testing of aztreonam-avibactam in metallo-β-lactamase-producing Enterobacterales.

The emergence of metallo-β-lactamase (MBL)-producing Enterobacterales presents unique clinical treatment challenges. Recently developed β-lactam/ β-lactamase inhibitor combination agents, while effective against other carbapenemase-producing organisms, are notably ineffective against MBL producers. While MBLs do not hydrolyze monobactams (aztreonam), many MBL-producing organisms are resistant to aztreonam through alternate mechanisms, leaving cefiderocol as the sole monotherapy treatment option recommended for MBL producers. Recent guidelines for the treatment of MBL-harboring organisms have added combination therapy with aztreonam and ceftazidime-avibactam, using ceftazidime-avibactam as a source of the β-lactamase inhibitor avibactam. Current laboratory testing options for the combination of aztreonam-avibactam are limited to broth microdilution (BMD) and broth disk elution (BDE) methods, which are not practical in most clinical laboratories. In this study, we evaluated the performance of aztreonam/avibactam gradient strips on 103 MBL-producing Enterobacterales patient isolates as well as an additional 31 isolates from the CDC AR Bank. All MBL Enterobacterales patient isolates included in this study harbored a New Delhi metallo-β-lactamase (blaNDM) gene. Essential agreement of gradient strip minimal inhibitory concentrations (MICs) for patient isolates compared to BMD was 93.2%. While there are no established breakpoints for aztreonam-avibactam, category agreement (CA) for patient isolates was 97.1% when using the CLSI aztreonam breakpoints. There were no major or very major errors observed. There were three minor errors. Precision for aztreonam-avibactam gradient strip diffusion was 100%. These data demonstrate that the use of gradient strip diffusion for aztreonam-avibactam MIC determination in MBL-producing Enterobacterales is a viable option for clinical laboratories.

Extended spectrum and metalo beta lactamase producing gram negative bacterial pathogens from cockroaches collected at hospital, Southern Ethiopia

BackgroundCockroaches can pose a significant health risk in hospital environments because they may serve as reservoirs and vectors for nosocomial pathogens. Cockroaches harbor epidemiologically significant extended spectrum and metalo beta lactamase producing Gram negative bacterial pathogens, which complicate nosocomial infections.ObjectivesThe main aim of this study is to determine aetiology and phenotypic extended spectrum and metalo beta lactamase producing Gram negative bacteria pathogens from cockroaches collected in hospitals.MethodsA cross-sectional study was employed from February to May 2022 to determine the antibiotic resistance producing bacterial isolates from cockroaches by giving special emphasis to metalo beta lactamase and extended spectrum beta lactamase production from different wards of WSUCSH. Cockroaches were collected with hands wearing sterile gloves. External homogenate was prepared and incubated microbiologically by using different culture media and differentiated biochemically. Antimicrobial susceptibility testing was performed by disk diffusion method. ESBL production was conducted using double disc synergy method and double disk method was used to detect MBL enzyme detection. Descriptive statistics was used to determine prevalence and percentage.ResultOut of 245 cockroaches, 108 Gram negative bacteria were isolated. K. pneumoniae 29(26.9%) was the most predominant bacteria and Enetrobacter spp. 8(7.4%), was the least. All, K. pneumoniae, P. mirabilis, and Enterobacter isolates were pan-resistant to Ampicillin. P.aeruginosa and P.mirabilis antibiotics showed ≥ 80% resistant for amoxicillin/clavulanic acid antibiotics. Cefotaxime, ceftazidime, ceftriaxone and imipenem showed relative efficacy compared with other antibiotics. Out of 78 amoxicillin-clavulanic acid resistant isolates, 42(34.7%) were ESBL producers. ESBL production is more depicted by P. aeruginosa, A. baumannii, K. pneumoniae and E. coli. The overall prevalence of MBL production is 29(23.1%). K. pneumoniae P. aeruginosa, E.coli, A. baumannii, Enterobacter spp and K.oxytoca revealed MBL production.ConclusionThe overall prevalence of ESBL and MBL producing nosocomial agents from hospital cockroaches was 34.7% and 23.1% respectively. P.aeruginosa, A.baumannii, K.pneumoniae and E.coli showed pronounced ESBL production. All bacterial isolates except P. mirabilis and C. freundii showed MBL production. The needed to evaluate our antibiotic stewardship program and antibiotic resistance detection for treatment is mandatory. The impact of cockroach as a source of AMR should be sought.

Antibiotic Resistance Profiles of Extended-Spectrum β-Lactamase (ESBL)- and Metallo-β-Lactamase (MBL)-Producing Klebsiella pneumoniae Isolates From Diabetic Foot Ulcers: Implications for Treatment Strategies.

Background Diabetic foot ulcers (DFUs) are prevalent complications of diabetes mellitus, often leading to severe infections and adverse clinical outcomes. Klebsiella pneumoniae, a gram-negative bacterium, has emerged as a significant causative agent in DFU infections, raising concerns due to its increasing antibiotic resistance, particularly in extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) production. Aim This study aimed to comprehensively assess the prevalence, antibiotic resistance profiles, and clinical correlates of ESBL- and MBL-producing K. pneumoniae isolates specifically derived from DFUs. Methods A cross-sectional observational study was conducted at Krishna Vishwa Vidyapeeth from January 2023 to June 2023, involving 126 patients diagnosed with DFUs. Clinical and demographic data were collected, and wound swabs underwent microbiological analysis. Phenotypic detection methods were employed to identify ESBL and MBL production, followed by standardized antibiotic susceptibility testing. Results Among the 126 isolates tested, 36 (28.6%) were identified as ESBL-producing and 21 (16.7%) as MBL-producing strains. ESBL-producing isolates exhibited high resistance rates to antibiotics such as ampicillin (92.3%), amoxicillin-acid (84.6%), and cephalosporins, including ceftriaxone (76.9%), and cefepime (73.8%). MBL-producing isolates demonstrated even broader resistance profiles, including resistance to fluoroquinolones (ciprofloxacin, 60.0%; levofloxacin, 57.1%), aminoglycosides (gentamicin, 42.9%), and carbapenems (meropenem, 38.1%; imipenem, 35.7%). Conclusion This study identifies a significant prevalence of ESBL- and MBL-producing K. pneumoniae in DFUs, showcasing high antibiotic resistance rates. Comorbidities correlate significantly with the presence of resistant isolates, necessitating treatment strategies for effective management.

Ventilator Associated Pneumonia in Tertiary Care Hospital, Maharajgunj, Kathmandu, Nepal

Introduction: Ventilator Associated Pneumonia (VAP) is the most common nosocomial infection among intensive care unit (ICU) patients and lack of much information in Nepal. So, the aim of this study was to determine prevalence and bacteriological profile of VAP with special reference to multi-drug resistant (MDR), Methicillin-resistant Staphylococcus aureus(MRSA), Metallo-β-Lactamase(MBL), Extended-Spectrum β-Lactamase(ESBL)-producing bacterial strains. Methods: A total 150 tracheal specimens were studied during June 2011 to May 2012 at Department of Microbiology, TUTH as described by American Society for Microbiology (ASM). Combination disk method was done for the detection of ESBL and MBL producing isolates. Results: Prevalence of VAP was found to be 34%. Acinetobactereal coaccticusbaumannii complex (44%) was the commonest isolate, followed by Klebsiellapneumoniae (22%), Pseudomonas aeruginosa (16%) and Staphylococcus aureus (12%). Among MDR Gram negative bacteria (GNB), 39% were MBL and 33% were ESBL-producers. All GNB (61) were sensitive to Polymyxin B and Colistinsulphate, whereas, 48% were found resistant to Carbapenems. Prevalence of MRSA was 75%, which were all sensitive to Vancomycin. Conclusion: High prevalence of VAP, MDR along with MRSA or ESBL or MBL producing strains was found in the study. Thus, suitable control measures must be adopted to cope up this alarming situation with genetic characterization.

Assessment of in vitro antimicrobial activity of ceftazidime-avibactam and phenotypic synergy testing with aztreonam against carbapenem resistant Gram-negative bacilli in a tertiary care hospital

Objectives: Ceftazidime avibactam (CZA) is a drug used against carbapenemase producing Gram-negative bacterial infections. Avibactam (AVI) is a non-beta-lactam-beta-lactamase inhibitor which has no action against metallo-β-lactamase (MBL) enzymes. This inadequacy is counteracted by combining CZA with aztreonam (ATM). The present study aims to denote the in vitro susceptibility pattern of the CZA and CZA-ATM combination in our area. Materials and Methods: In this study conducted prospectively from January to June 2023, the samples growing Enterobacterales and Pseudomonas aeruginosa were proceeded for carbapenemase detection by phenotypic testing for EDTA carbapenem inactivation method and modified carbapenem inactivation method. The minimum inhibitory concentration MIC of CZA was determined by E-strip and interpreted as per clinical and laboratory standard institute (CLSI) guidelines, while synergy testing of CZA and ATM was performed using ATM discs. Statistical Analysis: All data were entered in Microsoft Excel and analyzed for basic statistics. Results: The study included 150 carbapenem resistant organisms (131 Enterobactarales and 19 P. aeruginosa). Among these Enterobacterale strains, 72 (54.9%) were MBL producers. CZA resistance was detected in 69.3% of Klebsiella spp., 61.53% of Escherichia coli, and 50% of Serratia spp. Among Klebsiella spp. and E. coli, 88.9% and 65.2% of MBL isolates showed in vitro synergy to CZA-ATM. Conclusions: The study highlights a good in vitro sensitivity pattern of the CZA and ATM combination. However, we also highlight a growing percentage of non-synergistic interactions that need further genetic evaluation.

Genetic Characterization of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-Beta-Lactamase (MBL) Producing Klebsiella pneumoniae from Diabetic Foot Ulcer (DFU)

ABSTRACT Background: Antibiotic resistance in common pathogenic bacteria is linked with the genetic makeup. The genetic basis of antibiotic resistance may vary in different species or pathophysiological conditions. Objectives: We studied the antibiotic resistance in Klebsiella pneumonia isolates from DFU in the western Indian population. We also studied the presence of ESBL and MBL mechanisms of antibiotic resistance along with the prevalence of the genes involved in ESBL (TEM ESBL , SHV ESBL , and CTX-M ESBL ) and MBL (NDM-1 bla , KPC bla , OXA-48 bla , and VIM bla ) production. Results: A total of 161 K. pneumoniae isolates were analyzed; among which 50.93% were positive for ESBL and 45.96% were positive for MBL production. Most of the isolates were resistant to antibiotics used in the present study and partially resistant to Imipenem and Amikacin. There was no relation between the antibiotic resistance of the isolates and the production of ESBL or MBL mechanism of antibiotic resistance. Further, TEM ESBL was the most prevalent gene in K. pneumoniae isolates followed by CTX-M ESBL , NDM-1 bla , SHV ESBL , and KPC bla . VIM bla was the least prevalent gene found in K. pneumoniae isolates. There was no difference in the prevalence of the genes with respect to the presence or absence of ESBL and MBL mechanism of resistance. Further, there was no relation between the prevalence of the genes and antibiotic resistance in K. pneumoniae isolates. Conclusion: These results along with the literature review suggest that the prevalence of the genes involved in antibiotic resistance mechanisms are widespread in India and their distribution varies in different studies.

Phenotypic and molecular characterization of extended spectrum- and metallo- beta lactamase producing Pseudomonas aeruginosa clinical isolates from Egypt

BackgroundAntimicrobial resistance among Pseudomonas aeruginosa (P. aeruginosa), a leading cause of nosocomial infections worldwide, is escalating. This study investigated the prevalence of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) among 104 P. aeruginosa clinical isolates from Alexandria Main University Hospital, Alexandria, Egypt.MethodsAntimicrobial susceptibility testing was performed using agar dilution technique, or broth microdilution method in case of colistin. ESBL and MBL prevalence was assessed phenotypically and genotypically using polymerase chain reaction (PCR). The role of plasmids in mediating resistance to extended-spectrum β-lactams was studied via transformation technique using plasmids isolated from ceftazidime-resistant isolates.ResultsAntimicrobial susceptibility testing revealed alarming resistance rates to carbapenems, cephalosporins, and fluoroquinolones. Using PCR as the gold standard, phenotypic methods underestimated ESBL production while overestimating MBL production. Eighty-five isolates (81.7%) possessed only ESBL encoding genes, among which 69 isolates harbored a single ESBL gene [blaOXA-10 (n = 67) and blaPER (n = 2)]. Four ESBL-genotype combinations were detected: blaPER + blaOXA-10 (n = 8), blaVEB-1 + blaOXA-10 (n = 6), blaPSE + blaOXA-10 (n = 1), and blaPER + blaVEB-1 + blaOXA-10 (n = 1). Three isolates (2.9%) possessed only the MBL encoding gene blaVIM. Three ESBL + MBL- genotype combinations: blaOXA-10 + blaAIM, blaOXA-10 + blaVIM, and blaPER + blaOXA-10 + blaAIM were detected in 2, 1 and 1 isolate(s), respectively. Five plasmid preparations harboring blaVEB-1 and blaOXA-10 were successfully transformed into chemically competent Escherichia coli DH5α with transformation efficiencies ranging between 6.8 × 10 3 and 3.7 × 10 4 CFU/μg DNA plasmid. Selected tested transformants were ceftazidime-resistant and harbored plasmids carrying blaOXA-10.ConclusionsThe study highlights the importance of the expeditious characterization of ESBLs and MBLs using genotypic methods among P. aeruginosa clinical isolates to hinder the development and dissemination of multidrug resistant strains.

Clonal Distribution and Its Association With the Carbapenem Resistance Mechanisms of Carbapenem-Non-Susceptible Pseudomonas aeruginosa Isolates From Korean Hospitals.

Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC β-lactamase. Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.

Prevalence of Extended-spectrum Beta-lactamase (ESBL), Metallo Beta-lactamase (MBL) and Carbapenemase Producing Acinetobacter Species Isolated from Various Clinical Samples in Tertiary Care Hospital

Acinetobacter is an important nosocomial pathogen causing health care associated infections. It is highly antibiotic resistant gram-negative bacilli. The study was done to determine the prevalence of Acinetobacter species isolated from various clinical samples with their antibiotic susceptibility pattern. To determine the antimicrobial susceptibility pattern of the isolated Acinetobacter species and also the multidrug resistant mechanisms by phenotypic characterization. The retrospective study was carried out in patients diagnosed with Acinetobacter infection in the Microbiology Department, Krishna Institute of Medical Sciences, Krishna Vishwa Vidyapeeth, Deemed To Be University, Karad, a tertiary care hospital, including the clinical departments, during the period of two years from November 2020 till November 2022. Organism identification, biochemical test, antibiotic resistance pattern and phenotypic tests such as ESBL, MBL and Carbapenemase production were performed as per the Clinical and Laboratory Standards Institute (CLSI). The Modified Hodge test (MHT) was performed for Carbapenemase production detection in Acinetobacter species. A total of 150 Acinetobacter species were isolated from clinical samples. Acinetobacter baumannii was the most prevalent species 138 (92%), followed by Acinetobacter lwoffii 10 (7%) and Acinetobacter hemolyticus 2 (1%). The isolates showed highest resistance to Ampicillin 130 (87%) and sensitive to Colistin 113 (75.3%). Most of the isolates of of Acinetobacter baumannii showed maximum ESBL 21(14%) and MBL 75 (93%) production. Modified Hodge test showed positive results in Acinetobacter baumannii, only 11 (7%). The study showed that Acinetobacter baumannii was the most prevalent species showing drug resistance by phenotypic detection methods. At present Acinetobacter is a frequent pathogen in hospital acquired infections in critically ill patients admitted to ICU. The isolates of Acinetobacter species in our study showed resistant to most commonly used antibiotics. The study showed that ESBL production in Acinetobacter was 22 (15%) and MBL 80 (53%). Most Acinetobacter isolates were Multi Drug Resistant (MDR). MDR Acinetobacter is widely increasing due to inappropriate use of antibiotics in healthcare hospital. In our study, detection of carbapenemase by Modified Hodge test was positive in 11 (7%) isolates.

Reduce susceptibility to cefiderocol in gram negative bacteria in children: Is hope already lost before it’s even arrived?

BackgroundIn last years the diffusion of carbapenem resistance in Gram-negative bacteria (CR-GNB) is increasing worldwide, mainly due to the expression of carbapenemases. Cefiderocol has molecular characteristics that ideally confers activity against all CR-GNB, but resistant strains have already been identified. We describe cefiderocol susceptibility profile among multi-drug resistant Gram-negative isolated from pediatric patients. MethodsProspective, single pediatric center study, 1st January 2020–15th June 2023. All GNB carbapenemases producers or phenotypically carbapenem-resistant isolated in the study period were tested for cefiderocol susceptibility. Clinical and microbiological data were collected. A descriptive analysis was performed, comparing the groups of cefiderocol-resistant vs. cefiderocol-susceptible Enterobacterales and non-fermenting Gram-negative bacteria (NF-GNB). ResultsForty-seven GNB were tested for cefiderocol susceptibility; 38% were cefiderocol-resistant: 16/30 (52%) among Enterobacterales and 2/17 (12%) among NF-GNB. None of the patients were previously exposed to cefiderocol. Looking at Enterobacterales, resistance to ceftazidime/avibactam was higher among cefiderocol-resistant vs. cefiderocol-susceptible strains (62% vs 36%, respectively), as MBL expression (67% vs. 36%, respectively). Too few NF-GNB were cefiderocol-resistance to draw any conclusion. No difference in ICU admission and mortality was identified comparing cefiderocol-resistant vs. susceptible strains. Patients colonized/infected by cefiderocol-resistant strains had been previously hospitalized more frequently. ConclusionIn our cohort cefiderocol resistance was mostly registered among Enterobacterales, and especially among MBL producers’ strains (that were alongside resistant to ceftazidime/avibactam). This could be explained by the known possible cross resistance mechanism among ceftazidime/avibactam and cefiderocol. Also, correlation of cefiderocol-resistance with previous hospitalization could be associated with horizontal resistance transmission. Looking at our data, we believe that cefiderocol should be use cautiously, especially empirically and in monotherapy, due to the high resistance rate.

Evaluation of antimicrobial susceptibility tests for Acinetobacter and Pseudomonas species using disks containing a high dose of meropenem

The emergence and dissemination of carbapenem-resistant species of Acinetobacter and Pseudomonas have become a serious health concern. Routine antimicrobial disk susceptibility tests in clinical laboratories cannot distinguish between isolates that are highly carbapenem-resistant and those that are moderately carbapenem-resistant. The present study describes antimicrobial susceptibility tests using disks containing high doses (1000 μg) of meropenem. The diameters of inhibition zones were significantly negatively correlated with the MICs of Pseudomonas and Acinetobacter species for meropenem (R2: 0.93 and 0.91, respectively) and imipenem (R2: 0.75 and 0.84, respectively). Double disk synergy tests using clavulanic acid or sodium mercaptoacetate can detect ESBL or MBL producers. Susceptibility tests using disks containing high doses of meropenem can easily detect highly carbapenem-resistant isolates in a quantitative manner. These disks may be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.

Роль генетически детерминированного дефицита лектинового пути активации комплемента при хронической инфекции Helicobacter pylori у детей

Aim. To study the association of six polymorphic regions of MBL2 (rs11003125, rs7096206, rs7095891, rs1800450, rs1800451, rs5030737), two regions of FCN2 (rs7851696, rs17549193) and one region of MASP2 (rs72550870) with Helicobacter pylori infection and the severity of neutrophilic inflammation in gastric mucosa among children with recurrent abdominal pain. Design. Genetic association study of single nucleotide polymorphisms, case — control type. Materials and methods. 96 adolescents aged 12–17 years with recurrent abdominal pain were examined. Medical history, general clinical methods and fibrogastroscopy with a biopsy of the gastric mucosa were included in the medical testing. Biopsy samples were recorded and processed to assess the neutrophil infiltration and the presence of H. pylori. The results were interpreted according to the Sydney System of Gastritis Classification. Genotyping of allelic gene variants was carried out using real-time polymerase chain reaction (MBL2, MASP2), as well as the restriction analysis of amplification products of specific genome regions (MBL2, FCN2). Results. A high rate of H. pylori colonization was associated with a high frequency of the L allele of the rs11003125 gene polymorphism of the MBL2 gene and with a decrease in the proportion of high MBL — expressing genotypes. Carriage of the Q allele of the rs7095891 gene polymorphism of the MBL2 gene was associated with less pronounced neutrophilic infiltration, and a high frequency of the T allele of the rs7851696 gene polymorphism of the FCN2 gene was associated with severe neutrophilic inflammation in gastric mucosa. No differences in the distribution of MASP2 genotypes were found. Conclusion. The results obtained suggest that genetic defects in the production of MBL and ficolin-2 may cause chronic helicobacteriosis in children and more severe inflammation of gastric mucosa. Further study of these proteins seems to be promising for new approaches to the treatment and prevention of H. pylori complications in children to be identified. Keywords: Helicobacter pylori, mannose-binding lectin, ficolin, MASP, gene polymorphism.

Screening of antimicrobial activity of Ilex paraguariensis St. Hil. leaf extracts against carbapenemase-producing bacteria.

I. paraguariensis St. Hil. is a south American species of agronomic interest with studies supporting its medicinal properties. As the investigation of active ingredients with antimicrobial effect from medicinal plants is a suitable approach to the current antibacterial resistance problem, the aim of the present study was to determine the antibacterial activity of yerba mate ethanolic extracts against carbapenemase-producing gram-negative bacteria (reference strains and clinical isolates). Extracts showed antibacterial activity against Klebsiella pneumoniae ATCC® BAA-2342™ (KPC producing), Providencia rettgeri (NDM producing), Pseudomonas aeruginosa (MBL producing) and P. aeruginosa (VIM producing) at the concentrations tested. The Minimal-Inhibitory-Concentration and Minimal-Bactericidal-Concentration values ranged between 1 and 32 mg.ml-1 for the reference strains, and between 0.125 and 1 mg.ml-1 for the clinical isolates. The MBC/MIC index characterized the extracts as bactericidal. The combinations of commercial antibiotics and extracts showed a synergistic action on the reference strains studied. The lethal concentration 50 obtained using the Artemia salina toxicity assay were higher than 1 mg.ml-1 for all the extracts, indicating a low toxicity. The in vitro activity and low toxicity suggest that ethanolic I. paraguariensis leaf extracts constitute an outstanding source for new antibacterial compounds, and further studies should be carried out to understand their mechanism of action.

Phenotypic detection of ESBL and MBL producing Klebsiella pneumoniae in patients with nosocomial pneumonia

Phenotypic detection of ESBL and MBL producing Klebsiella pneumoniae in patients with nosocomial pneumonia