Metallo-beta-lactamase Research Articles

Investigating the validity of mCIM and sCIM phenotypic methods in screening Pseudomonas aeruginosa isolates producing IMP, VIM, and NDM metallo-beta-lactamases isolated from burn wounds.

Metallo-beta-lactamase-producing Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen in burn wounds, often exhibiting high levels of antibiotic resistance, which complicates treatment strategies. This study deals with the validity of the modified Carbapenem Inactivation Method (mCIM) and the simplified Carbapenem Inactivation Method (sCIM) phenotypic tests for screening metallo-beta-lactamase (MBL) production by P. aeruginosa isolates from a referral burn center in Iran. Forty isolates were obtained between January and June 2021 and identified using conventional biochemical methods. Antimicrobial susceptibility testing was conducted following Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines. mCIM based on CLSI 2023 guidelines was used to detect carbapenemase production. sCIM was also used based on previously developed protocols. PCR was performed to detect blaIMP, blaVIM, and blaNDM genes. The results were analyzed using SPSS and MedCalc. We observed a 90% resistance rate to imipenem and high resistance to other antibiotics, with multidrug-resistant (MDR) strains constituting 95% of the isolates. The mCIM test demonstrated high sensitivity (87.50%) and high negative predictive value (89.47%) and moderate specificity (70.83%) and moderate positive predictive value (66.67%) for detecting MBLs. In contrast, the sCIM test was unreliable, indicating a need for more standardized testing protocols. This study underscores the importance of accurate and timely detection of carbapenemase production to guide effective treatment.

Phenotypic identification of Metallo-ß- lactamase resistance Gram negative bacteria from a clinical specimen in Sidama, Ethiopia.

Metallo-beta lactamase resistance is one of the carbapenem resistances that worsen the world nowadays. A new variant of carbapenem-resistant has only limited reports from Africa including Ethiopia. This study aimed to determine Metallo -ß- lactamase resistance Gram-negative bacteria in Hawassa University Comprehensive Specialized Hospital January-June 2023. A cross-sectional study was conducted in which consecutive patients infected with Gram-negative bacteria were included in the study. A structured questionnaire was used to collect the data with oriented nurses if the patients/or caregivers gave consent to participate in the study. Clinical specimens are processed based on the standard operating procedure of the Microbiology laboratory and Clinical laboratory standard institute guidelines. Culture and sensitivity testing was used to isolate the bacteria. Gram staining and biochemical tests was used to identify the bacteria to genus and species. Kirby disc diffusion technique was used to determine the susceptibility of antibiotics. Statistical Software for Social Science (SPSS) version 21 is used for data entry and analysis. Descriptive statistics and logistic regression were used to interpret the data. The odds ratio at 95% confidence interval (CI) and p-value < 0.05 were taken as a statistically significant association. Our study included 153 isolates from different specimens, 83 (54.2%) were from male patients and 70 (45.8%) were from females. Klebsiella pneumonia was the predominant 43, followed by Escherichia coli 32, Acinetobacter spp 25, Pseudomonas spp 15, Enterobacter agglomerus 9, Klebsiella ozaenae 6, Enterobacter cloacae 5, Klebsiella oxytoca 4, (Klebsiella rhinoscleromatis, Proteus mirabilis and Morganella morganii) 3, Providencia stuartii 2 and (Citrobacter spp & Proteus vulgaris) 1. The rates of multi, extensive and pan-drug resistance bacteria accounted for 128/153 (83.7%), 77 /153(50.3%), and 26/153 (17.0%), respectively. Carbapenem resistance was 21 (13.7%), of this 7.2% were Enterobacteriaceae, 5.2% were Acetinobacter spp. and 1.3% Pseudomonas spp. Metallo-beta-lactamase was 17 (11.1%), of this, Enterobacteriaceae were 9(5.9%), Acetinobacter spp. 7(4.6%), and Pseudomonas spp. 1(0.7%). There were no variables statistically significantly associated with metallo-beta-lactamase-resistant. Our study revealed that Metallo-beta-lactamase resistance was circulating in the study area. There was a high rate of carbapenem resistance, multi, extensive and pan-drug resistance. Therefore, a measure should be taken to alleviate the emerging threat that leaves the patients without the option of treatment.

Identification of beta-lactamase genes and molecular genotyping of multidrug-resistant clinical isolates of Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a clinically relevant pathogen that has raised considerable public health concerns. This study aims to determine the presence of beta-lactamase genes and perform molecular genotyping of multidrug-resistant (MDR) K. pneumoniae clinical isolates.MethodsClinical isolates of MDR K. pneumoniae were collected from educational hospitals affiliated with Babol University of Medical Sciences. The isolates of K. pneumoniae were identified through standard microbial and biochemical tests. Antibiotic resistance was assessed using disk diffusion, modified Hodge test (MHT), combined disk, and polymerase chain reaction (PCR) methods. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was performed for molecular typing.ResultsA total of 42 MDR K. pneumoniae isolates were obtained from various clinical specimens. The highest antibiotic resistance was observed for ampicillin (100%), while the lowest resistance was noted for amikacin (19.04%). The MHT indicated that 38.09% of K. pneumoniae isolates produced carbapenemase enzymes. Metallo-beta-lactamase (MBL) production was found in 54.76% of isolates. Molecular detection of beta-lactamase genes revealed the presence of blaNDM (21.42%), blaKPC (42.85%), blaTEM (76.19%), blaSHV (47.16%), and blaCTX−M (80.95%) genes. ERIC-PCR molecular typing identified seven distinct genetic patterns among the isolates.ConclusionsThis investigation demonstrates the high resistance levels of K. pneumoniae strains. The beta-lactamase genes with the highest and lowest frequencies correspond to blaCTX−M and blaNDM genes, respectively. ERIC-PCR dendrograms suggest a common origin for K. pneumoniae clinical isolates and the propagation of similar clones within hospital wards. These findings indicate that K. pneumoniae isolates are highly virulent, necessitating the development of more effective resistance-fighting techniques and gene transfer research.Clinical trial numberNot applicable.

Molecular Investigation of Metallo B-Lactamase genes in Klebsiella Pneumoniae Bacteria from Clinical Isolates

Klebsiella pneumoniae is a significant nosocomial bacteria, that causes a large range of infections e.g. bacteremia, pneumonia, meningitis, and urinary tract infections. The current study intends to detect the existence of specific genes (blaNDM-1, blaNDM-2, blaVIM, blaIMP) associated with metallo-beta-lactamase (MBL) production in multidrug-resistant Klebsiella pneumoniae isolates attained from various clinical isolate. These genes are responsible for conferring resistance to a wide range of antibiotics, making an infection caused by such bacteria difficult to treat. Two hundred and seventy eight samples are collected from patients in Baquba Teaching Hospital during the period between December 2023 and May 2024. The samples included urine, sputums, burn, and wound swabs in addition to blood samples. The isolates are diagnosed microscopically and biochemically and the VITEK-2 compact system was used for diagnosis confirmation. It is found that 39.4%; n=69 is K. pneumoniae with 26%; n=18 of isolates are Multidrug resistant. Also, this study ascertains that multidrug-resistant (MDR) K.pneumoniae has the highest resistance against different types of antibiotics, including: AMP: 100%, AMC73: 36%, PIP: 81.16%, ATM: 72.46%, FEP: 71.01%, CAZ: 62.32%, CRO: 56.5%, IPM: 27.53%, MEM: 26.19%, AK: 47.82%, TOB: 43.47%, GM: 36.23%, LEV: 31.82%, OFX: 28.98%, CIP: 24.63% and SXT: 65.22%. Referring to phenotypic detection of MBL, 11 from 18 of MDR K. pneumoniae (61.11%) indicate a positive result. Also, during the molecular detection of Metallo B-lactamase genes, it was found that (81.81%; n=9) isolates were positive for blaNDM-1, blaNDM-2, and blaVIM gene, while no isolates showed positive results to blaIMP. Objective: Molecular detection of metallo-beta-lactamase genes in the bacteria Klebsiella pneumoniae.

A Rare Report of Carbapenem Resistant Vibrio fluvialis Isolated from a case of Walled off Necrosis: A Sequelae of Acute Necrotising Pancreatitis

Vibrio fluvialis (V. fluvialis) is commonly found in coastal environments and is an emerging pathogen encountered in diarrhoeal outbreaks and sporadic extraintestinal cases. V. fluvialis infections usually manifest as watery bloody diarrhoea, nausea, vomiting, gastroenteritis and peritonitis. In this case report, an unusual presentation of Multidrug Resistant (MDR) V. fluvialis along with Klebsiella pneumoniae co-infection from a case of Walled Off Necrosis (WON)- A complication of Acute Necrotising Pancreatitis (ANP) is discussed. The patient presented with the acute abdomen and had peripancreatic collection for which Ultrasound (USG) guided 12F pigtail drain placed over the right lower quadrant of the abdomen and conservatively managed. Routine cultures were done and the growth on Sheep Blood Agar (SBA) plate and MacConkey (MAC) agar plates were subjected for automated phenotypic identification by Matrix Assisted Laser Desorption Ionisation Time of Flight-Mass Spectrometry (MALDITOF-MS) and Vitek-2 systems and was identified as V. fluvialis. Also, molecular tests like 16S rRNA Polymerase Chain Reaction (PCR) followed by Sanger’s sequencing was done and the identification was further confirmed. Phenotypic RESIST-5 TRURAPID O.K.N.V.I. commercial lateral flow immunochromatographic assay for detection of clinically relevant and common carbapenemase genes like NDM, OXA-48, VIM, IMP and KPC was done which revealed this V. fluvialis isolate as New Delhi Metallo betalactamase (NDM) type of Carbapenemase producer. Patient was managed with levofloxacin and doxycycline and patient condition improved and was discharged.

Expression of MBL Genes and Biofilm Genes among Clinical Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa, a versatile Gram-negative bacterium, poses a serious threat due to its adeptness at developing resistance mechanisms. Metallo-beta-lactamases produced by P. aeruginosa are pivotal in conferring resistance to beta-lactam antibiotics, compromising treatment effectiveness. The bacterium’s proficiency in forming biofilms further enhances its persistence, especially in clinical settings. In this study conducted at Krishna Hospital and Medical Research Center, Karad, 205 clinical isolates of Pseudomonas aeruginosa were investigated for their metallo-beta-lactamase (MBL) production and biofilm-forming capabilities. The phenotypic detection of MBL production was carried out using the Imipenem-EDTA combination disc test, while biofilm formation was assessed through the microtiter plate technique. Genotypic detection involved conventional polymerase chain reaction targeting MBL and biofilm genes. Results showed that 28.29% of isolates produced MBL phenotypically, with 16.59% possessing MBL genes, notably the blaNDM-1 gene in 15.12% of isolates. Regarding biofilm production, 85.85% of isolates were biofilm producers, and biofilm-encoding genes were present in 80.49% of isolates. The most frequently encoded genes were algD (79.51%), pelF (58.0%), and pslD (46.34%). Comparing phenotypic and genotypic methods for MBL detection, a statistically significant average agreement was observed. While there was an increasing trend in biofilm genotypic positive patterns with stronger biofilm phenotypic patterns, the correlation was not statistically significant. The study concludes that every resistant clinical isolate should be screened for MBL and biofilm production using simple phenotypic tests, with confirmation by PCR if possible. This comprehensive analysis provides insights into the prevalence and genetic basis of MBL and biofilm in P. aeruginosa clinical isolates, contributing to strategies for combating antibiotic resistance.

Monte Carlo simulation for dosage optimization of the best available therapy for bloodstream infections secondary to carbapenemase-producing Klebsiella pneumoniae in critically ill patients

ObjectiveWe aimed to use Monte Carlo simulation, based on pharmacokinetic/pharmacodynamic targets, to investigate and determine the optimal dosage of the available combination therapies for carbapenem-resistant Klebsiella pneumoniae (CRKP) in critically ill patients. MethodsWe collected CRKP clinical isolates from Phramongkutklao Hospital between October 2020 and June 2022. A molecular study of resistant genes was performed using polymerase chain reaction. Broth microdilution checkerboards were used to evaluate the mono- and synergistic antibiotic activities. Monte Carlo simulation was used to determine the optimal antibiotic regimens, based on the probability of target attainment (PTA) and cumulative fraction of response. ResultsThe 54 CRKP isolates were resistant to tigecycline (100%), colistin (75.9%), amikacin (70.4%), and gentamicin (63.0%). The most common carbapenemase genotype was blaoxacillinases (OXA)-48-like (42.6%), followed by blaNew Delhi metallo beta-lactamase (NDM) (29.6%) and coexistence of blaOXA-48-like and blaNDM (22.2%). Based on the PTA, synergistic and additive activities against CRKP isolates were observed with appropriate dosages of tigecycline–colistin (67.9%), tigecycline–gentamicin (62.2%), and tigecycline–amikacin (51.4%). ConclusionsTigecycline–colistin was the best available combination therapy for critically ill patients with CRKP, especially NDM. When used in combination with tigecycline, a colistin creatinine clearance of <90 mL/min can raise the cumulative fraction of response target and less nephrotoxicity.

FOSFOMYCIN SUSCEPTIBILITY IN MULTIDRUG-RESISTANT UROPATHOGENS: A RETROSPECTIVE STUDY IN THE ERA OF ANTIMICROBIAL RESISTANCE

Objective: Urinary tract infection (UTI) is one of the most prevalent clinical entities affecting people worldwide. The accelerating rate of Antimicrobial resistance due to the unimpeded and rampant use of antimicrobials with over-the-counter availability of drugs has limited the therapeutic options for the treatment of UTIs. Fosfomycin, an old broad-spectrum antimicrobial with good pharmacokinetics has regained importance for the treatment of multidrug-resistant (MDR) isolates. The purpose of this study was to determine the in vitro Fosfomycin susceptibility of common uropathogens and to study the resistance pattern of these organisms against commonly prescribed antimicrobial agents. Methods: A retrospective cross-sectional study was conducted in the Bacteriology section of the Microbiology laboratory at Adesh Institute of Medical Sciences and Research, Bathinda, Punjab for duration of 6 months from December 2022 to May 2023 from urine samples received from all clinically suspected cases of UTI. Samples were processed immediately as per standard microbiological techniques, followed by culture by a semi-quantitative method. Kass criteria was followed for interpretation of significant bacteriuria according to which significant growth was considered if the colony count was more than 105 colony forming units (CFU)/mL. Culture positives were analyzed by Gram staining and on the basis of colony characteristics, Gram staining, final identification, and Antimicrobial Susceptibility were done through Vitek 2 compact system. Results: A total of 2292 urine samples received in the Microbiology laboratory were processed and cultured during the study period, which yielded 509 significant bacterial isolates i.e.509/2292 (22.2%) culture positivity. Among 509 culture-positive samples, Escherichia coli 235/509 (46.1%) was the most common uropathogens isolated followed by Klebsiella pneumoniae 107/509 (21.1%), Enterococcus species 40/509 (7.8%). Fosfomycin depicted good in vitro susceptibility of a minimum of 94% in both Gram-negative and Gram-positive uropathogens as compared to Nitrofurantoin, which showed sensitivity of 74% and 85%, respectively. Maximum resistance was observed toward Cephalosporins i.e., Ceftriaxone in E. coli (60%) and K. pneumoniae (64%), respectively, followed by 50% in Acinetobacter baumannii. Maximum resistance to Ciprofloxacin (62%) was seen in case of A. baumannii. 172/405(42.4%) isolates of Enterobacteriaceae family were extended-spectrum β-lactamase (ESBL) producers with an average Fosfomycin susceptibility of 95.9%. Among the total isolated uropathogens, 135/509 (26.5%) were MDR, out of which 116/135 (85.9%) depicted Fosfomycin susceptibility. Metallo-beta-lactamase (MBL) production was seen in 14.3% of the isolated Gram-negative uropathogens. 63/73(86.3%) of the MBL producers were found susceptible to Fosfomycin. Conclusion: Fosfomycin has emerged as an effective alternative for the treatment of common uropathogens including the MDRs, ESBL producers, and the MBLs in the era of increasing antimicrobial resistance. It has the potential to act as a promising oral agent for the treatment of UTI in both community and healthcare setups.

Molecular Characterization of Multidrug-Resistant and Hypervirulent New Delhi Metallo-Beta-Lactamase Klebsiella pneumoniae in Lazio, Italy: A Five-Year Retrospective Study.

Antimicrobial resistance represents a challenge to public health systems because of the array of resistance and virulence mechanisms that lead to treatment failure and increased mortality rates. Although for years the main driver of carbapenem resistance in Italy has been the Klebsiella pneumoniae KPC carbapenemase, recent years have seen an increase in VIM and NDM metallo-beta-lactamases (MBLs). We conducted a five-year survey of New Delhi Metallo-beta-Lactamase (NDM)-producing Klebsiella pneumoniae (NDM-Kpn) clinical isolates from the Lazio region, Italy; the study aimed to elucidate the molecular mechanisms underpinning their resistant and virulent phenotype. Antimicrobial susceptibility was evaluated by automated systems and broth microdilution. In silico analysis of acquired resistance and virulence genes was performed using whole-genome sequencing (WGS), molecular typing through MLST, and core genome multi-locus sequence typing (cgMLST). A total of 126 clinical NDM-Kpn isolates were collected from 19 distinct hospitals in the Lazio region. Molecular analysis highlighted the existence of NDM-1 (108/126) and NDM-5 (18/126) variants, 18 Sequence Types (STs), and 15 Cluster Types (CTs). Notably, 31/126 isolates displayed a virulence score of 4, carrying ybt, ICEKp, iuc, and rmp genes. This study identified a variety of NDM-Kpn STs, mainly carrying the blaNDM-1 gene, with a significant number linked to high-risk clones. Of these isolates, 24.6% showed high-level resistance and virulence, emphasizing the risk of the spread of strains that combine multi-drug-resistance (MDR) and virulence. Proactive surveillance and international collaborations are needed to prevent the spread of high-risk clones, as well as further research into new antimicrobial agents to fight antibiotic resistance.

Detection of Carbapenemase-Producing Enterobacterales: A Comparative Analysis of Available Phenotypic Methods

Background: Carbapenems serve as a critical last-line defence against infections from multidrug-resistant Enterobacterales. The increasing prevalence of CRE (carbapenem-resistant Enterobacterales) poses significant challenges in clinical settings. This greatly complicates the handling of gram-negative bacilli (GNB) infections and represents a significant global health threat. Aim: This study evaluates and contrasts different phenotypic techniques used to identify carbapenemase-producing Enterobacterales isolated from various clinical samples. Objectives: To compare the accuracy and reliability of Combined Disc Test (CDT) and Modified Carbapenem Inactivation Method/EDTA - Carbapenem Inactivation Method (mCIM/eCIM) in identifying Metallo-beta-lactamase (MBL)-producing Enterobacterales. Methods: All clinical specimens submitted for culture and sensitivity testing at the Department of Microbiology were analysed to isolate and identify Enterobacterales. Carbapenemase production was assessed through using the phenotypic CDT Confirmation was carried out using the mCIM and eCIM in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: During a two-year period, 254 strains of Enterobacterales were isolated. Among those, 80/254 (31.49%) isolates exhibited resistance to the Imipenem (IMP) disc. The CDT identified 30/80 (37.5%) isolates as serine carbapenemase producers and 50/80 (62.5%) isolates as MBL producers. In comparison, the mCIM/eCIM methods detected 26/80 (32.5%) isolates as serine carbapenemase producers, 48/80 (60.0%) isolates as MBL producers, and 6/80 (7.5%) isolates were tested negative for carbapenemase production. Conclusion: This study evaluates the precision and effectiveness of the mCIM/eCIM and CDT methods for identifying CRE. The combination of the mCIM and eCIM tests may be a more reliable phenotypic method for detecting carbapenemase compared to the CDT.

Antibacterial activity and inhibitory effects of allicin, sultamicillin tosylate and diallyl trisulfide of garlic on metallo beta-lactamases produced from Pseudomonas aeruginosa

Antibacterial activity and inhibitory effects of allicin, sultamicillin tosylate and diallyl trisulfide of garlic on metallo beta-lactamases produced from Pseudomonas aeruginosa

Evaluation of the CARBA PAcE test, a colorimetric imipenem hydrolysis test for rapid detection of carbapenemase activity.

There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we evaluated a colorimetric imipenem hydrolysis test, called the CARBA PAcE test, to detect carbapenemase-producing Gram-negative bacteria (GNB). We tested a collection of 270 GNB isolates with a characterized carbapenemase content. Our testing set included 205 carbapenemase-producing, carbapenemase-resistant Enterobacterales (CP CRE) with 40 Klebsiella pneumoniae carbapenemase (KPC), 49 New Delhi metallo beta lactamase (NDM), 49 OXA-48-like, 15 Verona integron-mediated metallo-β-lactamase (VIM), three IMP, 43 IMI, six isolates producing more than one carbapenemase, and 65 non-carbapenemase-producing Enterobacterales (20 ESBL producers, 35 non-carbapenemase-producing, carbapenem-resistant [non-CP CRE], and 10 carbapenem-susceptible Enterobacterales [non-CP, non-CSE, third-generation cephalosporin and carbapenem susceptible]). We compared the performances of the CARBA PAcE test, the qualitative colorimetric β-CARBA test, and the modified CarbaNP test to a gold standard of carbapenemase gene detection by PCR. Specificities of all tests were high: 95.4% (62/65) for CARBA PAcE test, 98.5% (64/65) for β-CARBA test, and 100% (65/65) for the modified CarbaNP test. Sensitivity varied by carbapenemase: all three tests had a sensitivity of 100% for NDM, VIM, and IMP and 97.5% for KPC. Sensitivity to detect IMI was 0% for the CARBA PAcE and β-CARBA tests and 11.6% for the modified CarbaNP test. Sensitivity to detect OXA-48-like was 89.7% for the CARBA PAcE test, 87.7% for the β-CARBA test, and 14.2% for the modified CarbaNP test. Reading the results of the CARBA PAcE assay was difficult. The CARBA PAcE assay is highly sensitive for detecting NDM, VIM, IMP, and KPC, but slightly less sensitive for OXA-48-like. It does not detect IMI. It is highly specific, and its overall diagnostic accuracy is similar to that of β-CARBA. Its operational advantages are rapid turnaround time, ease of use, and long shelf life, but reading of results is subjective.IMPORTANCEWe evaluated the ability of the CARBA PAcE test to detect carbapenemases in 274 Gram-negative isolates with a known carbapenemase content. Specificity was high for all carbapenemases tested (96.9%). Sensitivity was high for KPC, NDM, VIM, and IMP (97.5-100%); but lower for OXA-48-like (89.7%). Activity of IMI could not be detected. Taken together, our results indicate that CARBA PAcE is a useful alternative in regions where NDM and KPC are predominant. The limitations of the test are difficulty in reading results and incompatibility with mSuperCARBA.

A Comprehensive Study of Bacterial Etiological Agents in Sterile Body Fluids and Antimicrobial Susceptibility Patterns Among Hospitalized Patients at an Academic Medical Center in India.

Background Sterile body fluids are devoid of any microbial presence, including commensal bacteria. However, bacterial invasion of these fluids can result in life-threatening infections, often leading to significant morbidity and mortality. Timely detection and precise identification of pathogens, along with antimicrobial susceptibility testing, are critical for optimizing therapeuticinterventions and improving patient outcomes. Objective To study the prevalence of bacterial infections in various body fluids in hospitalized patients and to determine the antimicrobial susceptibility patternand the phenotypic detection of extended-spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC beta-lactamase producers within bacterial isolates. Materials and methods Sterile body fluid samples, excluding blood and urine, were collected and cultured at the Department of Microbiology, Krishna Institute of Medical Sciences, Western Maharashtra, India, from November 2022 to 2023. The microorganisms isolated from these fluids were identified using standard biochemical tests. Antibiotic sensitivity was assessed through the disc diffusion assay (zone of inhibition) and phenotypic identification of beta-lactamaseenzymes was performed using the combined disc diffusion method. Results During the study period, 180 sterile fluid specimens were collectedrepresenting 48 cerebrospinal fluid (CSF), 53 pleural fluid, 23 peritoneal fluid, and other sterile body fluid samples. Out of these, (n=32, 17.77%) samples were culture-positive. Gram-negative bacteria were oftentimes isolated at 84% (27/32), while gram-positive were 16% (5/32). Escherichia coli was frequently isolated and (n=9, 28.12%) exhibited maximum sensitivity to gentamicin and fosfomycin (n=7, 77.78%) and maximum resistance to cefoperazone-sulbactam (n=8, 88.88%). Pseudomonas aeruginosawas isolated as the second most common organism and showed maximum susceptibility to fosfomycin (n=5, 83.34%) and maximum resistance to gentamicin, cefotaxime, cefoxitin, etc. (n=5, 83.34%). Among gram-positive isolates, coagulase-positive Staphylococcus was high in prevalence rate and (n=3, 9.37%) presented 100% sensitivity to vancomycin and maximum sensitivity to tetracycline(n=2, 66.67%) and 100% resistance to ciprofloxacin, cefoxitin, erythromycin, and other antibiotics. Among gram-negative isolates, MBLproducers were 48.15%, ESBLproducers were 40.74%, and 18.51% were AmpCbeta-lactamase producerswith a multidrug-resistant (MDR) occurrence rate of 93.75%. Conclusion Infections affecting sterile body fluids are critical due to their high mortality and morbidity rates. Timely identification of the causative organisms and their antibiotic susceptibility is essential. The prompt initiation of appropriate antibiotic therapy can decreasethe duration of hospitalization and mitigate the emergence of drug resistance. The presence of MDR organisms in sterile body fluids constitutes considerable challenges in the management of critically ill patients.

Genotypic Characterisation of Carbapenemases in Clinical Isolates of Acinetobacter baumannii in a Tertiary Care Teaching Hospital of Kerala

Background: The multidrug-resistant and carbapenem-resistant Acinetobacter baumannii strains are an increasing global concern. The primary cause of carbapenem resistance in A. baumannii is production of various beta-lactamases with versatile hydrolytic capacities. The present study investigates the prevalence of different carbapenemases among clinical isolates of carbapenem-resistant A. baumannii from a tertiary care teaching hospital in Kerala, India. Methods: Non-duplicate isolates from sputum, endotracheal aspirate, pus, urine, and blood samples were included in the study. Isolate identification and antibiotic susceptibility testing of the isolates were done using vitek 2 system. A conventional polymerase chain reaction was used to detect the carbapenemase-encoding genes in the isolates. Results: A total of the 126 isolates studied, among these 105 (83.3%) isolates were from intensive care unit patients and 21(16.6%) were from non-ICU inpatients. Most of the isolates were obtained from respiratory specimens-78(61.9%) followed by pus-33(26.2%), urine-12(9.5%) and blood- 3(2.4%). The prevalence of carbapenemase genes were as follows:blaOXA-51(n=126, 100%) blaOXA-23 (n=98, 77.7%), blaOXA-58 (n=7, 5.6%), blaNDM (n=66, 52.4%), blaIMP (n=26, 20.6%), and blaVIM (n=20, 15.9%). Among the total isolates 78 (61.9%) isolates harbored metallo-beta-lactamase genes, 47(37.3%) isolates harboured a single carbapenemase gene, while 69(54.8%) isolates harbored two or more genes. Conclusion: This study reveals a higher prevalence of metallo-beta lactamases and the co-occurrence of multiple carbapenemase genes in Carbapenem resistant A. baumannii isolates.

Impact of AbaI mutation on virulence, biofilm development, and antibiotic susceptibility in Acinetobacter baumannii

The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen.

Comparing the activity of broad-spectrum beta-lactams in combination with aminoglycosides against VIM-producing Enterobacteriaceae.

Metallo-beta-lactamase (MBL)-producing carbapenem-resistant Enterobacteriaceae (CRE) infections continue to pose a serious threat to healthcare. Due to their unique active site, MBLs evade the activity of many novel beta-lactam/beta-lactamase inhibitor combinations, which have been specifically targeted toward those carbapenemases with serine active sites. Furthermore, resistance to most, if not all, other clinically relevant antimicrobial classes leaves few reliable therapeutic options. Combination therapy has thus played a vital role in the treatment of MBL-producing CRE infections. In this study, we utilized the static time-kill assay to investigate clinically relevant concentrations of cefepime, piperacillin-tazobactam, and meropenem alone and in combination with either amikacin or the novel plazomicin to determine if combinations of routinely used beta-lactam therapy with an aminoglycoside would achieve bactericidal activity against eight clinically isolated Verona integron-encoded MBL (VIM)-producing CRE. Furthermore, we compared this activity to the combination of aztreonam/avibactam, which has shown potent activity against MBL-producing CRE. Both aztreonam/avibactam and meropenem with either aminoglycoside were rapidly bactericidal within 4 hours and remained bactericidal through 24 hours against all isolates with few exceptions. Combinations including cefepime and piperacillin-tazobactam were also rapidly bactericidal, but activity after 24 hours was inconsistent depending upon the partner aminoglycoside and isolate. Further investigation is warranted to elucidate optimal antibiotic exposures against MBL-producing CRE, including novel agents in the pipeline.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are one of the most pressing antimicrobial-resistant threats at present. In addition to exhibiting resistance to many, if not all, commonly used antimicrobial agents, CRE achieves these resistant phenotypes through a variety of mechanisms, each of which can uniquely affect available treatment options. The present study is an in vitro investigation of several Verona integron-encoded metallo-beta-lactamase (VIM)-producing CRE isolated from patients at our academic medical center. Because metallo-beta-lactamases (MBLs) are inherently resistant to many of the novel treatments designed to treat CRE due to their different active site composition, we tested several antimicrobial combinations containing routinely utilized broad-spectrum beta-lactams and aminoglycosides. Our results further our understanding of combination therapy options against VIM-producing CRE, including with non-carbapenem-beta-lactams cefepime and piperacillin. By optimizing combinations of existing antimicrobial agents, we hope to expand the available armamentarium against these resistant pathogens.

Characterisation of carbapenemase-producing Gram-negative bacilli among clinical isolates in a tertiary care centre in Kerala, South India

Background: Infections caused by carbapenem-resistant Gram-negative bacteria are a cause for concern due to the limited choice of antibiotics available for their treatment. Aims, Settings and Design: This study screened multidrug-resistant (MDR) Gram-negative bacilli isolated from clinical samples over a period of one year, for carbapenem resistance and characterised them using phenotypic methods such as combined disc diffusion test (CDDT), modified Hodge test (MHT), E-test for metallo-beta-lactamase (MBL) and molecular method, PCR. Materials and Methods: Two hundred and ten MDR Gram-negative bacilli were screened for carbapenem resistance using Imipenem and Meropenem disc diffusion. These were further checked for carbapenemase production by CDDT, MHT and E-test for MBL. Those positive by E-test were subjected to PCR. Uniplex PCR for New Delhi metallo-beta-lactamase-1 was used for Escherichia coli and Klebsiella pneumoniae , and multiplex PCR for Imipenemase and Verona imipenemase was used for Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Results: Twenty-three (11%) isolates were found to be carbapenem-resistant and included E. coli (six) K. pneumoniae (three), P. aeruginosa (five) and A. baumannii (nine). Seventeen (74%) isolates were positive by phenotypic methods and were subjected to PCR. Out of eight Enterobacteriaceae isolates subjected to PCR, all were positive for bla NDM gene. All were negative for bla KPC gene. All five A. baumannii isolates subjected to PCR were found to contain bla VIM gene. Two out of four P. aeruginosa isolates were positive for bla IMP , one was positive for bla VIM gene. One P. aeruginosa isolate was positive for both bla IMP and bla VIM gene. Conclusions: In view of the increasing resistance of Gram-negative bacilli to carbapenems, rational use of antibiotics needs to be emphasised.

Occurrence and Antibiotic Resistance Profiles of Metallo Beta Lactamase Producing Pseudomonas aeruginosa among Clinical Isolates in Enugu Metropolis, Nigeria

Introduction: Antibiotic resistance is rising worldwide, posing a challenge for contemporary medicine. Metallo-beta-lactamases (MBLs) confer resistance to beta-lactam antibiotics other than monbactams. It threatens public health because of its vast scope of action and quick spread. The study was undertaken to assess the occurrence of MBL in clinical isolates of Pseudomonas aeruginosa in the Enugu metropolis and to determine their resistance profile. Materials and Methods: The work was conducted from October 2020 to July 2021 in the microbiology laboratory of the University of Nigeria Teaching Hospital, Ituku-Ozalla. A total of 127 non-duplicate bacterial isolates recovered from clinical samples including wounds, urine, sputum, ear discharge, and catheter tip processed in the microbiology laboratory of four referral hospitals within the Enugu metropolis was used for the study. Isolates were identified and characterized using standard microbiology protocols. Antimicrobial susceptibility was done using the Kirby-Bauer disc diffusion method. Phenotypic detection of Metallo-beta-lactamase production was done using the Combined Disk diffusion Test (CDDT). Results: Of the 127 isolates, 68 (53.5%) were resistant to imipenem. Among these, 35 strains were positive for MBL production while 33 isolates were non-MBL producers. The highest percentage occurrence of MBL producers was recorded from catheter tips 33% followed by urine 30.6%. Both MBL producers and non-MBL producers displayed high resistance to most of the antibiotics used except Aztreonam. Conclusion: The overall occurrence of MBL among Pseudomonas aeruginosa in our study was found to be 27.6%. MBL-producing strains showed higher resistance than the non-MBL-producing strains. Aztreonam was the most potent antibiotic. The most effective approaches to combating this organism include early detection, stringent antibiotic regimens, and adherence to infection control measures.

Prevalence of metallo-beta-lactamase in clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis in Benin City, Nigeria

Carbapenems are the prime choice of treatment for severe cases of infections caused by Multi-Drug-Resistant Pseudomonas aeruginosa and Proteus mirabilis. Nevertheless, Metallo-Beta-Lactamase (MBL) production by these organisms has led to carbapenem resistance which is a global threat. This study aimed to determine the prevalence and antimicrobial susceptibility profile of MBL in clinical isolates of Pseudomonas aeruginosa and Proteus mirabilis in Benin City, Nigeria. 354 non monotonous clinical isolates of Pseudomonas aeruginosa (282) and Proteus mirabilis (72) used were obtained from various clinical samples from tertiary hospitals in Benin City. Identification of these isolates were done using standard microbiological techniques. Antimicrobial susceptibility test was performed using Kirby-Bauer disk diffusion method. MBL production was detected using Imipenem Ethylene-Diamine-Tetra-Acetic Acid combined disc test method. Of the total 354 clinical isolates tested, 115 (32.48%) were MBL and the prevalence of this resistant isolates was significantly higher in Pseudomonas aeruginosa (46.8%) compared to Proteus mirabilis (P=0.0001). Among the metallo-beta-lactamase producing isolates of Pseudomonas aeruginosa higher prevalence were reported from Pleural fluid and cerebrospinal fluid samples with 6 (100%) and 3 (100%) respectively. This difference was statistically significant (P=0.0001). Susceptibility testing showed that isolates that produced beta-lactamase demonstrated poorly against cephalosporin, amoxicillin-clavulanate, gentamicin and floroquinones than non-beta- lactamase producers. A prevalence of 46.8% was reported for MBL producing Pseudomonas aeruginosa and 12.5% was reported for MBL producing Proteus mirabilis. Isolates that produced the MBL enzyme were more resistant to antibacterial agents. Measures to control and curb the spread of MBL producing clinical isolates are highly advocated

PREVALENCE OF ASYMPTOMATIC BACTERIURIA IN ANTENATAL WOMEN ATTENDING TERTIARY CARE HOSPITAL-A CROSS-SECTIONAL STUDY

Objective: Urinary tract infection (UTI) is one of the most common bacterial infections during pregnancy due to anatomical changes and physiological adaptations during pregnancy. Asymptomatic bacteriuria is the significant presence of bacteria in the urine of an individual without symptoms. Untreated asymptomatic bacteriuria (ASB) in pregnancy predisposes to symptomatic UTI in 25% of infected women. Screening of antenatal women help in early diagnosis and treatment of ASB and thus to prevent maternal and fetal morbidity and mortality. The present study was carried out to determine the prevalence of UTI in pregnant women and to study the bacteriological profile and antimicrobial sensitivity patterns of uropathogens. Methods: A Cross-sectional study was conducted for a period of six months and midstream urine specimens were collected from 480 pregnant females and were processed by standard protocols. All subjects were clinically identified to have no signs and symptoms of UTI. Antibiotic susceptibility testing was done as per CLSI guidelines. Results: Prevalence rate of asymptomatic bacteriuria was seen 10% in pregnant women. Majority of the culture-positive patients belonged to the age group of 26-30 y (31.25%). 70.84% were Gram-negative isolates and 29.16% were Gram-positive organisms. The commonest pathogen isolated was Escherichia coli (33.33%). In the present study, Extended Spectrum Beta-Lactamase (ESBL) production was seen in (20.58%) isolates, and Metallo Beta-Lactamase (MBL) production was seen in (17.64%) isolates. Conclusion: This study reveals the importance of screening of pregnant women for UTI. Emerging multi-drug resistance seen in uropathogens emphasizes the need to rationalize use of antibiotics, which eventually prevent development of resistant strains.