Metallo-beta-lactamase Research Articles

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

Background/Objectives: 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. Methods: 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). Conclusions: 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.

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.

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.

Antibacterial Activity of Some Iranian Herbal Essential Oils as Disinfectant Agents on Surfaces Contaminated with Methicillin -resistant -Staphylococcus Aureus and Carbapenem - resistant -Pseudomonas Aeruginosa

Background & Objectives: Different essential oils (EOs) with antibacterial activities are promising natural sources for providing novel disinfectant agents for hospital surfaces Materials & Methods: The component and antibacterial effects of six EOs, including Cuminum cyminum (CCEO), Artemisia sieberi (ASEO), Laurus nobilis (LNEO), Ferula gummosa (FGEO), Lippia citriodora (LCEO), and Cymbopogon citratus (CIEO) were assessed by GC -MS and 96 -well micro -plates (IC50), against Staphylococcus aureus (S. aureus) ATCC 25923, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 and clinical isolates of methicillin -resistant S. aureus (MRSA) and metallo -beta -lactamase (MBL) -producing P. aeruginosa. Then, the antibacterial effects of FGEO, the most effective EO, were evaluated on the trolley surface in a hospital for 1, 3, 5 and 10 min intervals. Results: CCEO, ASEO, and FGEO exerted the highest antibacterial activity against S. aureus, while CIEO and LNEO inferred the highest activity against P. aeruginosa. In addition, FGEO mitigated the growth of S. aureus and P. aeruginosa on the trolley surface (P<0.05). Conclusion : The studied EOs could be novel encouraging agents to develop further green antimicrobial agents against different infections. In addition, FGEO exhibited considerable antibacterial effects on the surface of the trolley.

Potential involvement of beta-lactamase homologous proteins in resistance to beta-lactam antibiotics in gram-negative bacteria of the ESKAPEE group

BackgroundEnzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens.In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher’s exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems.We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Unmasking Hidden Threats Global Spread of MBL Resistance Exposed

This study aims to establish a routine monitoring system for MBL enzymes to provide timely data to healthcare professionals and policy makers, enabling informed decision making on antibiotic use and resistance management. Using a combination of molecular biology techniques and data analysis, we monitor MBL activity in various institutional settings. The increasing prevalence of multidrug-resistant (MDR) bacteria is a significant threat to public health globally. Metallo-beta-lactamase (MBL), an enzyme that confers resistance to a wide range of beta-lactam antibiotics, is particularly concerning due to its ability to spread rapidly in healthcare and community settings. Despite the importance of this issue, systematic monitoring and understanding of MBL remains inadequate. Our findings reveal a significant, previously unreported presence of MBLs, underscoring the urgent need for targeted antibiotic stewardship programs. The implications of this study emphasize the importance of integrating enzyme monitoring into standard healthcare practices to reduce the spread of MDR bacteria. Highlights: Regular Monitoring: Essential for tracking MBL enzyme prevalence and guiding antibiotic use. Advanced Techniques: Molecular biology methods enhance MBL detection and analysis. Policy Integration: Crucial for implementing enzyme monitoring in healthcare to combat MDR bacteria spread. Keywords: MBL Enzymes, Antibiotic Resistance, Healthcare Monitoring, Molecular Biology, Stewardship Programs

Antimicrobial susceptibility profile of Klebsiella pneumoniae isolated from some dairy products in Libya as a foodborne pathogen.

Klebsiella pneumoniae is one of the most common causes of clinical and asymptomatic mastitis in dairy cattle, as well as in milk and dairy products that affect milk quality. Mastitis caused by K. pneumoniae is even more serious due to its poor response to antibiotic therapy. The aim of this study was to detect and identify the presence of K. pneumoniae in milk and dairy products produced in Libya. A total of 234 samples were randomly collected from various locations in Libya. Samples were examined for the presence of K. pneumoniae using conventional cultural techniques, including cultivation in violet red bile agar plus 4-methylumbelliferyl-ß-D-glucuronide broth and CHROM agar, followed by polymerase chain reaction identification and partial sequencing of 16S rRNA. Of the 234 samples of milk and dairy products collected, 16 (6.8%) isolates revealed mucoid colonies on agar media that were phenotypically suggested to be K. pneumoniae. Identification of isolates was confirmed using molecular techniques (16S rRNA). Among the examined samples, K. pneumoniae was recovered from camel's milk, raw cow's milk, raw fermented milk, Maasora cheese, Ricotta cheese, soft cheese, full cream milk powder, milk powder infant formula, cereal baby food, and growing-up formula. Antibiotic susceptibility testing was performed on 12 of the 16 K. pneumoniae isolates, and the results showed that K. pneumoniae isolates were resistant to more than eight antibiotics; interestingly, two isolates showed metallo-beta-lactamase (MBL) production. K. pneumoniae is considered a risk to human health because many of these products do not comply with the microbiological criteria of international and/or Libyan standards. This study emphasized the relationship between K. pneumoniae and raw milk, cheese, milk powder, and infant milk retailed in Libya. There is a need to take the necessary measures to ensure effective hygiene practices during production in dairy factories, handling, and distribution on the market, in particular at a small local production scale.

Molecular characterization of carbapenemase and extended spectrum beta-lactamase producing Acinetobacter baumannii isolates causing surgical site infections in Ethiopia

BackgroundAcinetobacter baumannii is an opportunistic pathogen that can cause a variety of nosocomial infections in humans. This study aimed to molecularly characterize extended-spectrum beta-lactamase (ESBL) producing and carbapenem-resistant Acinetobacter species isolated from surgical site infections (SSI).MethodsA multicentre cross-sectional study was performed among SSI patients at four hospitals located in Northern, Southern, Southwest, and Central parts of Ethiopia. The isolates were identified by microbiological methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was determined using disk diffusion. The presence of phenotypic ESBL and carbapenemase production was detected by employing standard microbiological tests, including combined disk diffusion (CDT). ESBL and carbapenem resistance determinants genes were studied by polymerase chain reaction (PCR) and sequencing.ResultsA total of 8.7% Acinetobacter species were identified from 493 culture-positive isolates out of 752 SSI wounds. The species identified by MALDI-TOF MS were 88.4% A. baumannii, 4.7% Acinetobacter pittii, 4.7% Acinetobacter soli, and 2.3% Acinetobacter lactucae. Of all isolates 93% were positive for ESBL enzymes according to the CDT. Using whole genome sequencing 62.8% of the A. baumannii harbored one or more beta-lactamase genes, and 46.5% harbored one or more carbapenemase producing genes. The distribution of beta-lactamases among Acinetobacter species by hospitals was 53.8%, 64.3%, 75%, and 75% at JUSH, TASH, DTCSH, and HUCSH respectively. Among ESBL genes, blaCTX−M alleles were detected in 21.4% of isolates; of these 83.3% were blaCTX−M−15. The predominant carbapenemase gene of blaOXA type was detected in 24 carbapenem-resistant A. baumannii followed by blaNDM alleles carried in 12 A. baumannii with blaNDM−1 as the most common.ConclusionsThe frequency of Acinetobacter species that produce metallobetalactamases (MBLs) and ESBLs that were found in this study is extremely scary and calls for strict infection prevention and control procedures in health facilities helps to set effective antibiotics stewardship.

Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples

Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.

Bacteriological evaluation, antibiogram, and phenotypic detection of MBL-producing gram-negative bacteria isolated from outdoor patients with Otorrhea attending Ayub Medical Complex

Otitis media is frequently prevalent in developing countries and is primarily caused by bacteria. The current study aimed to study the patients to assess the occurrence rate, susceptibility pattern, and prevalence rate of metallo beta-lactamase (MBL) producing gram-negative bacteria to prevent the prevalent strains and direct the physicians to appropriate antibiotic therapy. The research study was conducted over nine-months, from November 2020 to July 2021, in which a total of 110 samples were collected from the outdoor-patients’ ear, nose, and throat (ENT) wards using sterile swabs and inoculated immediately on suitable culture media. The isolates were identified via conventional biochemical tests, and the susceptibility pattern of each isolate was tested using the Kirby-Bauer disc diffusion method. Out of the 110 total samples, merely 88.18% (97/110) ear samples were detected positive for the various strains of bacteria both in male and female patients with a mean age of 18.21 ± 15.86, contributing 54.64% and 45.36%, respectively. The most prevalent bacterial strain was Staphylococcus aureus (49.48%), followed by Pseudomonas aeruginosa (16.50%), Proteus spp. (13.40%), S. epidermidis (7.22%), E. coli (6.19%), S. saprophyticus (4.12%), and Klebsiella spp. (3.09%). Of the gram-negative isolated strains, only 18.75% (3/16) of the P. aeruginosa strains exhibited MBL production. A high prevalence rate was observed in the age-group 0–4 years (19.58%), followed by the age-groups, 5–9 and 15–19 (16.49% each), and the age-group >39 (10.31%). Levofloxacin, ciprofloxacin, amikacin, and fusidic acid were highly effective against the gram-positive, whereas ciprofloxacin, amikacin, and levofloxacin were potent against the gram-negative (except P. aeruginosa), while carbenicillin, meropenem and imipenem against the P. aeruginosa. The MBL-producers were highly (100%) resistant to IMP and MEM while highly (100%) sensitive to CAZ. In conclusion, the current study showed a high prevalence of S. aureus, P. aeruginosa, and MBL-producing P. aeruginosa (18.75%). Nevertheless, a high degree of resistance to antibiotics was noted, which could not be ignored.

Rapid evolutionary change in trait correlations of single proteins

Many organismal traits are genetically determined and covary in evolving populations. The resulting trait correlations can either help or hinder evolvability – the ability to bring forth new and adaptive phenotypes. The evolution of evolvability requires that trait correlations themselves must be able to evolve, but we know little about this ability. To learn more about it, we here study two evolvable systems, a yellow fluorescent protein and the antibiotic resistance protein VIM-2 metallo beta-lactamase. We consider two traits in the fluorescent protein, namely the ability to emit yellow and green light, and three traits in our enzyme, namely the resistance against ampicillin, cefotaxime, and meropenem. We show that correlations between these traits can evolve rapidly through both mutation and selection on short evolutionary time scales. In addition, we show that these correlations are driven by a protein’s ability to fold, because single mutations that alter foldability can dramatically change trait correlations. Since foldability is important for most proteins and their traits, mutations affecting protein folding may alter trait correlations mediated by many other proteins. Thus, mutations that affect protein foldability may also help shape the correlations of complex traits that are affected by hundreds of proteins.

Detection of metallo-beta-lactamase-producing genes blaSPM and blaNDM in Pseudomonas aeruginosa isolated from wastewater in Southern Brazil.

Pseudomonas aeruginosa is commonly associated with the ability to acquire antimicrobial resistance. The surveillance of resistance genes in various environmental matrices has gained prominence in recent years, being seen as a potential threat to public health. The objective of this study was to investigate genes encoding metallo-beta-lactamases (MBLs), which confer resistance to carbapenems, in wastewater. Fifteen isolates of P. aeruginosa were collected for five months from samples obtained from a municipal wastewater treatment plant in Rio Grande do Sul. These isolates were subjected to disk diffusion testing using 10 different antimicrobials. Phenotypic enzymatic tests for MBLs were conducted, and positive isolates underwent DNA extraction and gene detection using the polymerase chain reaction. The resistance rate to ceftazidime was 100%, cefepime 73.3%, piperacillin-tazobactam 66.67%, imipenem 53.30%, levofloxacin 46.67%, tobramycin 40%, and ciprofloxacin and amikacin 13.33%. Both meropenem and aztreonam resistances were rare accounting for 6.60% of the tested isolates. Among these isolates, 20% were classified as multidrug-resistant and were found to carry the blaNDM and blaSPM genes. The results suggest that evaluating resistance genes in bacteria from urban raw sewage can provide data that assist in surveillance, as this environment can stimulate increased bacterial resistance.

Use of Aztreonam Plus Ceftazidime/Avibactam Combination for In vitro Susceptibility Testing of CRO Isolates in a Tertiary Care Hospital – Our experience

Abstract Background: Infections caused by metallo-beta-lactamases (MBLs) producing Gram-negative bacteria (mainly Enterobacterales) pose a great challenge for clinicians in treating these multidrug-resistant infections. In recent times, combination antibiotic therapy with ceftazidime–avibactam (CAZ-AVI) with aztreonam (ATM) has been researched by a number of laboratories worldwide and has gained much clinical attention. This study evaluated a practical laboratory method of testing for clinically significant in vitro synergy between CAZ/AVI + ATM in New Delhi MBLs (NDM) producing Gram-negative organisms. Materials and Methods: A total of 100 isolates of carbapenem-resistant Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae) were tested for synergy between CAZ/AVI + ATM using E-strip of CAZ/AVI and disc of ATM. The minimum inhibitory concentration value of CAZ/AVI was measured and the zone of inhibition was observed when ATM was placed parallel to the E-strip on a lawn culture of an isolate. Simultaneously, all isolates were tested for NDM and OXA-48 by Xpert Carba-R. Results: Of 100 isolates, 63/100 (63%) were harboring OXA-48 beta-lactamase, 54/100 (54%) were harboring NDM beta-lactamase and 18/100 (18%) were harboring both (OXA-48 and NDM). In vitro synergy between CAZ/AVI + ATM was noted in 99 isolates (99%). Conclusion: A significant synergy was demonstrated in vitro with CAZ/AVI + ATM combination therapy and seems to be a promising treatment strategy for infections caused by Enterobacterales harboring NDM and OXA-48 beta-lactamases.