Β-lactamase Enzymes Research Articles

Identification of Arg32Ser, His92Tyr and Leu147Phe novel mutations in chromosomally mediated β-lactamase SHV and in silico characterization to understand their substrate activity imparting resistance

The emergence of β-lactam resistance is yearning for clinical significance in Enterobacteriaceae, which are categorized under global priority pathogen lists by the World Health Organization. Likewise, the prevalence of numerous β-lactamase enzymes, mutational propensity in such bacteria, and their role in accelerating resistance is still a major concern. Thus, the present work intends to characterize the β-lactamase producing bacteria isolated from acute diarrheal patients to understand their chromosomally acquired resistance pattern through molecular characterization and in silico approaches. The current study highlights the first identified Escherichia fergusonii and Escherichia marmotae species and their β-lactamase encoding genes, blaOKP-A, blaNDM and blaOXA from the unexplored Enterobacteriaceae family from North East India. First-ever reported point mutations such as Arg32Ser, His92Tyr, and Leu147Phe were observed in BlaSHV protein of two Klebsiella pneumoniae isolates S-35 and S-46. In molecular docking, non-catalytic site H-bond interactions of Arg 218, Ala 223, Asn 128, Ser 126, Gln 95, Asp 100, Tyr 101, Ser 102, Ala 274 with a low binding affinity towards BlaSHV was found. This correlates with the high imipenem, ceftazidime, cefuroxime, ceftriaxone, and cefpodoxime resistance in Klebsiella pneumoniae S-35 with the complementary effect of mutations Arg32Ser and Leu147Phe. Besides, the role of His92Tyr mutation in controlling the resistance in Klebsiella pneumoniae S-46 is also illustrated. Thus, our study highlights the novel mutations of β-lactamase and its clinical importance with altered resistance profiles. This could be useful to design better therapeutics and to readjust antibiotic treatment regimes against them and control to grow more resistance under selective pressure. Communicated by Ramaswamy H. Sarma

The Next-Generation β-Lactamase Inhibitor Taniborbactam Restores the Morphological Effects of Cefepime in KPC-Producing Escherichia coli.

ABSTRACTGram-negative bacteria producing carbapenemases are resistant to a variety of β-lactam antibiotics and pose a significant health risk. Given the dearth of new antibiotics, combinations of new broad-spectrum β-lactamase inhibitors (BLIs) with approved β-lactams have provided treatment options for resistant bacterial infections. Taniborbactam (formerly VNRX-5133) is an investigational BLI that is effective against both serine- and metallo-β-lactamases, including the serine carbapenemase KPC. In the current study, we assessed the effectiveness of taniborbactam to restore antibacterial activity of cefepime against KPC-3-producing Escherichia coli by inhibiting the KPC-3-dependent hydrolysis of cefepime. Time-lapse microscopy revealed that cells treated with greater than 1× MIC of cefepime (128 μg/ml) and cefepime-taniborbactam (4 μg/ml cefepime and 4 μg/ml taniborbactam) exhibited significant elongation, whereas cells treated with taniborbactam alone did not owing to a lack of standalone antibacterial activity of the BLI. The elongated cells also had frequent cellular voids thought to be formed by attempted cell divisions and pinching of the cytoplasmic membrane. Additionally, the effect of taniborbactam continued even after its removal from the growth medium. Pretreatment with 4 μg/ml taniborbactam helped to restore the antibacterial action of cefepime by neutralizing the effect of the KPC-3 β-lactamase.IMPORTANCE β-lactam (BL) antibiotics are the most prescribed antimicrobial class. The efficacy of β-lactams is threatened by the production of β-lactamase enzymes, the predominant resistance mechanism impacting these agents in Gram-negative bacterial pathogens. This study visualizes the effects of a combination treatment of taniborbactam, a broad spectrum β-lactamase inhibitor (BLI), and the BL antibiotic cefepime on a carbapenemase-producing E. coli strain. While this treatment has been described in the context of other cephalosporin-resistant bacteria, this is the first description of a microscopic evaluation of a KPC-3-producing strain of E. coli challenged by this BL-BLI combination. Live-cell microscopy analysis of cells treated with taniborbactam and cefepime demonstrated the antimicrobial effects on cellular morphology and highlighted the long-lasting inhibition of β-lactamases by taniborbactam even after it was removed from the medium. This research speaks to the importance of taniborbactam in fighting BL-mediated antibiotic resistance.

Detection of Antibiotic Resistance Genes Among Multiple Drug Resistant Pseudomonas Aeruginosa Isolated from Clinical Sources in Selected Health Institutions in Kwara State.

Pseudomonas aeruginosa (P. aeruginosa) is a frequent nosocomial pathogen that causes severe diseases in many clinical and community settings. Strains of P. aeruginosa are associated with increased morbidity, mortality and healthcare costs. The rapid emergence of antimicrobial resistance among these strains is a public health crisis. Moreover, there is a paucity of data on the characterization of P. aeruginosa isolates from human clinical samples in Kwara State. The objectives of this study are to investigate the occurrence of metallo β-lactamase enzyme, multiple antibiotic resistant P. aeruginosa among clinical samples and detection of antibiotic resistance genes among them. Two hundred and thirty-five samples comprising of 145 males and 90 females human clinical specimens were collected aseptically from five selected health institutions within Kwara State, Nigeria. The samples were cultured immediately using standard microbiological procedures. Multiple drug resistance patterns of the micro-organisms to different antibiotics were determined using the Bauer Kirby disc diffusion technique. Metallo β-lactamase production was determined using E - test strip and the DNA samples of the multiple resistant P. aeruginosa strains were extracted and subjected to Polymerase Chain Reaction (PCR) for resistant genes determination. Data were subjected to descriptive statistics using Statistical Package for Social Sciences (SPSS) software. A total of 145 isolates were identified for P. aeruginosa from the clinical samples. Thirty were positive for metallo β-lactamase production; 11 (8%) males and 19 (13%) females. Absolute resistance to ceftazidime (100%), gentamicin (100%), ceftriaxone (100%) were observed while low resistance to ciprofloxacin (12.4%), piperacillin (6.9%) and imipenem (6.9%). All isolates were sensitive to colistin. The prevalence of various encoding genes blaVIM, , blaCTX-M and blaTEM were 34.4%, 46.7%, 16.7% and 37.7% respectively. This study has shown that there is a high occurrence of metallo β-lactamase enzyme producing and antibiotic-resistant strains of P. aeruginosa in clinical specimens from the studied area. Necessary measures must, therefore be implemented to stop the problems of this antibiotic resistance.

Genomic insights into the diversity, virulence and resistance of Klebsiella pneumoniae extensively drug resistant clinical isolates.

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília’s hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. β-Lactamase production provided the most notable mechanism associated with K. pneumoniae . Each strain presented two or three different β-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D β-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Klebsiella spp. Isolated at the National Reference Laboratory, Kathmandu, Nepal

Klebsiella spp. are associated with several nosocomial and opportunistic infections. Increasing antimicrobial resistance of Klebsiella species is aggravated by a number of intrinsic and extrinsic factors. The main aim of this study is to determine antimicrobial resistance due to production of β-lactamase enzymes, extended spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC and Klebsiella pneumoniae carbapenemase (KPC) and biofilm formation in Klebsiella isolates. A total of 2197 non-duplicate specimens of urine, sputum and pus were obtained from the National Public Health Laboratory (NPHL), Kathmandu, Nepal, between February and August 2019. Klebsiella species were isolated, identified and screened for antimicrobial susceptibility testing with the disk diffusion method. Phenotypic detection of ESBL, MBL, KPC and AmpC production was observed and biofilm production was detected by the microtiter plate method. Out of a total of 2197 clinical specimens, bacterial growth was detected in 8% (175/2197) of the specimens. Of the total isolates, 86.3% (151/175) were Gram-negative bacteria and 37.7% (57/151) were Klebsiella spp. Of the total Klebsiella spp., 56% (32/57) were multi drug resistant (MDR), 16% (9/57) were ESBL, 26% (15/57) were MBL, 4% (2/57) were KPC (class A carbapenemase), 16% (9/57) were AmpC producers and 95% (54/57) were biofilm producers. Gentamicin was the most effective antibiotic, followed by cotrimoxazole, as 68% (39/57) and 47% (27/57) of the Klebsiella isolates were susceptible towards these drugs, respectively. The study results show evidence of β-lactamase production, high prevalence of MDR and biofilm producing Klebsiella species. Integrating the test parameters for phenotypic confirmation of ESBL, MBL, AmpC β lactamase and KPC in routine diagnostic procedures can help in the early detection and management of these resistant strains.

Molecular Basis of Bicyclic Boronate β-Lactamase Inhibitors of Ultrabroad Efficacy - Insights From Molecular Dynamics Simulation Studies.

β-Lactam antibiotics represent about 70% of all antibacterial agents in clinical use. They are safe and highly effective drugs that have been used for more than 50 years, and, in general, well tolerated by most patients. However, its usefulness has been dramatically reduced with the spread and dissemination worldwide of multi-drug resistant bacteria. These pathogens elude the therapeutic action of these antibiotics by expressing β-lactamase enzymes that catalyze the hydrolysis of their β-lactam ring to give inactive products, which is one of the most relevant resistance mechanisms in deadly pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. From the drug development point of view, the design of an efficient β-lactamase inhibitor able to block this antibiotic resistance mechanism and restore β-lactam antibiotics efficacy is challenging. This is due to: (1) the huge structural diversity of these enzymes in both the amino acid sequence and architecture of the active site; (2) the distinct hydrolytic capability against different types of substrates; (3) the variety of enzyme mechanisms of action employed, either involving covalent catalyzed processes (serine hydrolases) or non-covalent catalysis (zinc-dependent hydrolases); and (4) the increasing emergence and spread of bacterial pathogens capable of simultaneously producing diverse β-lactamases. Hence, a long-pursued goal has been the development of ultrabroad-spectrum inhibitors able to inhibit both serine- and metallo-β-lactamases. The recent development of taniborbactam (formerly VNRX-5133) and QPX7728, which are bicyclic boronate inhibitors currently under clinical development, represents a huge step forward in this goal. In this article, the molecular basis of the ultrabroad-spectrum of activity of these boron-based inhibitors is analyzed by molecular dynamics simulation studies using the available crystal structures in complex with both inhibitors, or the models constructed from wild-type forms. The efficacy of taniborbactam and QPX7728 is compared with the cyclic boronate inhibitor vaborbactam, which is the first boron-based β-lactamase inhibitor approved by the FDA in combination with meropenem for the treatment of complicated urinary tract infections.

β-Lactams and β-Lactamase Inhibitors: Unlocking their potential to address drug resistance

Antibiotics such as β-lactams are one of the most widely used antibacterial drug classes in the world. The invention of the first β-lactam antibiotic (Penicillin) is regarded as a symbolic landmark in the history of modern chemotherapy. Since that time, several other β-lactam antibiotics have been added to the treatment, revolutionising the treatment of bacterial infections. Antibacterial efficacy of the β-lactams has been kept in check by the emergence of bacterial resistance. One of the most studied and common resistance mechanisms is the expression of β-lactamase enzymes. The invention of β-lactamase inhibitors which restore the efficacy of β-lactam antibiotics, has been a significant advance in the fight against microbial drug resistance. However, many recently identified β-lactamases are not inactivated by the presently available inhibitors. Despite the fact that these inhibitors may not be effective against all β-lactamases, their implementation is still welcome. This review focuses on the development of β-lactam antibiotics and the mechanism of action. It also covers the diversity of β-lactamases with an emphasis on rising bacterial resistance. It provides a summary on β-lactamase inhibitors with a focus on restoring antibiotic efficacy and the various computational approaches used in inhibitor discovery. Finally, we outlined an update on research activities aimed at discovering and developing novel β-lactamase inhibitors.

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of β-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

In Silico Characterization of the Interaction between the PBP2a "Decoy" Protein of Resistant Staphylococcus aureus and the Monomeric Units of Eudragit E-100 and Poly(Maleic Acid-alt-Octadecene) Polymers.

Antimicrobial treatment alternatives for methicillin-resistant Staphylococcus aureus (MRSA) are increasingly limited. MRSA strains are resistant to methicillin due to the formation of β-lactamase enzymes, as well as the acquisition of the mecA gene, which encodes the penicillin-binding protein (PBP2a) that reduces the affinity for β-lactam drugs. Previous studies have shown that the use of ampicillin-loaded nanoparticles can improve antimicrobial activity on resistant S. aureus strains. However, the biological mechanism of this effect has not yet been properly elucidated. Therefore, this short communication focused on characterizing the in silico interactions of the PBP2a membrane receptor protein from S. aureus against the monomeric units of two polymeric materials previously used in the development of different nanoparticles loaded with ampicillin. Such polymers correspond to Eudragit E-100 chloride (EuCl) and the sodium salt of poly(maleic acid-alt-octadecene) (PAM-18Na). For this, molecular coupling studies were carried out in the active site of the PBP2a protein with the monomeric units of both polymers in neutral and ionized form, as well as with ampicillin antibiotic (model β-lactam drug). The results showed that ampicillin, as well as the monomeric units of EuCl and PAM18Na, described a slight binding free energy to the PBPa2 protein. In addition, it was found that the amino acids of the active site of the PBPa2 protein have interactions of different types and intensities, suggesting, in turn, different forms of protein–substrate coupling.

Novel ß-Lactamase Inhibitors: New Weapons in the Arms Race against Antimicrobial Resistance

Antimicrobial resistance is a growing threat in the treatment of infectious diseases. Multidrug-resistant Gram-negative organisms, in particular, have become progressively difficult to manage, due to the increasing diversity and widespread prevalence of β-lactamase enzymes. Novel β-lactamase inhibitors with expanded activity against such enzymes represent a promising strategy to combat Gram-negative resistance. This review discusses the classification of β-lactamases and the history of β-lactamase inhibitor development with a focus on three novel β-lactamase inhibitors—avibactam, relebactam, and vaborbactam—now FDA approved in combination β-lactam/β-lactamase inhibitor products available for clinical use, including their spectrums of inhibition, mechanisms of action, place in therapy, and evidence for use.

Discovery of VNRX-7145 (VNRX-5236 Etzadroxil): An Orally Bioavailable β-Lactamase Inhibitor for Enterobacterales Expressing Ambler Class A, C, and D Enzymes.

A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. The increasing emergence of β-lactamase-producing multi-drug-resistant “superbugs” has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing β-lactamase inhibitor 5 (VNRX-5236). In vitro and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum β-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.

Discovery of Novel Chemical Series of OXA-48 β-Lactamase Inhibitors by High-Throughput Screening.

The major cause of bacterial resistance to β-lactams is the production of hydrolytic β-lactamase enzymes. Nowadays, the combination of β-lactam antibiotics with β-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging β-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC50 = 0.99 μM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies; furthermore, it slightly improved imipenem’s activity in Escherichia coli ATCC BAA-2523 β-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-β-lactam BLIs.

Cefdinir and β-Lactamase Inhibitor Independent Efficacy Against Mycobacterium tuberculosis.

Background: There is renewed interest in repurposing β-lactam antibiotics for treatment of tuberculosis (TB). We investigated efficacy of cefdinir, that withstand the β-lactamase enzyme present in many bacteria, against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (Mtb). Methods: Minimum inhibitory concentration (MIC) experiments were performed with Mtb H37Ra, eight drug-susceptible, and 12 MDR-TB clinical isolates with and without the β-lactamase inhibitor, avibactam at 15 mg/L final concentration. Next, we performed dose-response study with Mtb H37Ra in test-tubes followed by a sterilizing activity study in the pre-clinical hollow fiber model of tuberculosis (HFS-TB) study using an MDR-TB clinical strain. Inhibitory sigmoid Emax model was used to describe the relationship between the drug exposure and bacterial burden. Results: Cefdinir MIC for Mtb H37Ra was 4 and 2 mg/L with or without avibactam, respectively. The MIC of the clinical strains ranged between 0.5 and 16 mg/L. In the test-tube experiments, cefdinir killed 4.93 + 0.07 log10 CFU/ml Mtb H37Ra in 7 days. In the HFS-TB studies, cefdinir showed dose-dependent killing of MDR-TB, without combination of avibactam. The cefdinir PK/PD index linked to the Mtb sterilizing efficacy was identified as the ratio of area under the concentration-time curve to MIC (AUC0–24/MIC) and optimal exposure was calculated as AUC0–24/MIC of 578.86. There was no resistance emergence to cefdinir in the HFS-TB. Conclusion: In the HFS-TB model, cefdinir showed efficacy against both drug susceptible and MDR-TB without combination of β-lactamase inhibitor. However, clinical validation of these findings remains to be determined.

Mutations in passive residues modulate 3D-structure of NDM (New Delhi metallo-β-lactamase) protein that endue in drug resistance: a MD simulation approach

The ability of antimicrobial resistance developed by bacteria enhanced the complexity of bacterial treatment leading a serious threat to human health. Production of β-lactamase by bacteria that inactivates β-lactam is a generic cause of resistance. One such β-lactamase enzyme is New Delhi Metallo-β-lactamase (NDM) which is recently reported to have clinically more importance and recognized as an antibiotic resistance marker. Mutations in active and passive residues of NDM protein play a fateful role in the substrate and inhibitor specificity. In this study, in silico point mutations of residues near the active site and flexible regions of protein were investigated. Hybrid modelling and molecular dynamics (MD) simulations were carried to build up the mutant models and monitored structural stability. Molecular docking results articulated that mutant proteins had lesser binding affinities with methicillin, oxacillin and doripenem drugs. Further, to scrutinize the structural alterations and rescore the binding energies per-residue basis, MD simulations of wildtype (WT) and mutant (MT) NDM proteins with methicillin, oxacillin and doripenem were performed. Our results demonstrated that mutations in N193A, S217A, G219A and T262A residues led to protein destabilization and amend their binding affinities with methicillin, oxacillin and doripenem. The present study exploited computational approaches which displayed differential binding of drugs with WT and MT NDM proteins that confer resistance to oxacillin and doripenem. The study features the significance of passive residues, thus provides a clue to accelerate the process of designing an ergastic antibiotic against NDM protein. Communicated by Ramaswamy H. Sarma

Amikacin Combined with Fosfomycin for Treatment of Neonatal Sepsis in the Setting of Highly Prevalent Antimicrobial Resistance.

Antimicrobial resistance (particularly through extended-spectrum β-lactamase and aminoglycoside-modifying enzyme production) in neonatal sepsis is a global problem, particularly in low- and middle-income countries, with significant mortality rates. High rates of resistance are reported for the current WHO-recommended first-line antibiotic regimen for neonatal sepsis, i.e., ampicillin and gentamicin. We assessed the utility of fosfomycin and amikacin as a potential alternative regimen to be used in settings of increasingly prevalent antimicrobial resistance. The combination was studied in a 16-arm dose-ranged hollow-fiber infection model (HFIM) experiment. The combination of amikacin and fosfomycin enhanced bactericidal activity and prevented the emergence of resistance, compared to monotherapy with either antibiotic. Modeling of the experimental quantitative outputs and data from checkerboard assays indicated synergy. We further assessed the combination regimen at clinically relevant doses in the HFIM with nine Enterobacterales strains with high fosfomycin and amikacin MICs and demonstrated successful kill to sterilization for 6/9 strains. From these data, we propose a novel combination breakpoint threshold for microbiological success for this antimicrobial combination against Enterobacterales strains, i.e., MICF × MICA < 256 (where MICF and MICA are the fosfomycin and amikacin MICs, respectively). Monte Carlo simulations predict that a standard fosfomycin-amikacin neonatal regimen would achieve >99% probability of pharmacodynamic success for strains with MICs below this threshold. We conclude that the combination of fosfomycin with amikacin is a viable regimen for the empirical treatment of neonatal sepsis and is suitable for further clinical assessment in a randomized controlled trial.

Bacteriological profile and antibiotic susceptibility patterns of wound infections in a tertiary care hospital in South India

Infections caused due to injuries are due to microbial proliferation at the wound site following skin damage. Initial testing of micro-organisms in terms of culturing and sensitivity leads to appropriate antibiotic selection and prevents escalation of antimicrobial resistance.To evaluate the bacteriological profile and antibiotic susceptibility patterns of wound infections in this single-centered study.The study included 160 patients suspected to have wound infection. Pus or tissue samples collected from patients were subjected to microbiological processing including Gram staining, culture and antibiotic susceptibility testing. Their demographic data and wound related factors (duration, nature, type) were recorded. The isolated organisms were evaluated for β-lactamase production using Extended spectrum β-lactamase (ESBL) test, Modified Hodge test for Carbapenemase and AmpC β-lactamase enzyme detection tests. Most participants were 41-60 years old (45.63%). The majority had surgical site infections (SSI, 91.25%), early infected (91.1%) and clean (67.12%) wounds. The bacterial isolation rate was 80% and 45.27% (n=67) were Gram positive isolates, out of which 47 (70.14%) were multidrug resistant. was identified as the predominant organism (n=40), where 33 among 40 were methicillin sensitive, followed by (n=30, 23.43%). Amongst the Gram negative isolates (n=81, 54.73%), 60 (74.07%) were multidrug resistant with majority being susceptible to imipenem, meropenem and amikacin. The most common pathogen associated with wound infection was Methicillin sensitive with SSI being the most common type of wound infection.

Phenotypic Identification and Genotypic Characterization of Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates in Iran.

One of the mechanisms of Klebsiella pneumoniae and Escherichia coli resistance to β-lactam antibiotics is the production of β-lactamase enzymes. Among these are the AmpC β-lactamases, which confer resistance to a class of antibiotics. However, little is known about the AmpC β-lactamases of K. pneumoniae and E. coli clinical isolates in Qazvin, Iran. This study was designed to assess the AmpC β‑lactamases-producing strains and also identify the prevalence of AmpC β‑lactamases genes. Antimicrobial susceptibility tests were performed on 435 K. pneumoniae and E. coli isolates using disk diffusion technique. Plasmid-mediated AmpC genes were studied using a multiplex PCR assay. The AmpC β-lactamase-producer isolates were studied by employing cefoxitin disk diffusion test, AmpC induction test, AmpC cefoxitin-EDTA test, and boronic acid disk test. Our results showed that of 46 (18.4%) cefoxitin-insensitive E. coli isolates, 10 (21.7%) were positive for AmpC β-lactamase genes, among them 4 (8.69%) isolates were positive for blaDHA genes and 6 (13%) for blaCIT genes. Of 57 (30.4%) cefoxitin-insensitive K. pneumoniae isolates, 10 (17.5%) were positive for AmpC gene with 4 (6.34%) and 6 (9.5%) isolates positive for blaDHA and blaCIT genes, respectively. However, no MOX, ACC, FOX, or EBC genes were detected in the isolates. Considering the results of different confirmatory phenotypic tests, the AmpC cefoxitin-EDTA test showed a higher discriminatory power for detecting AmpC β-lactamase-producing strains. The specificity and sensitivity of AmpC cefoxitin-EDTA were 77%, 100% for K. pneumonia and 70%, 90% for E. coli higher than the other two tests, respectively. Also, the authors demonstrated high prevalence rate for resistance to certain antibiotics, such as cefuroxime, trimethoprim-sulfamethoxazole, ampicillin, and cefotaxime. In conclusion, our study provided valuable information regarding the plasmid-mediated AmpC β-lactamase gene content, antibiotic resistance, and confirmatory phenotypic tests for AmpC β-lactamases in E. coli and K. pneumoniae isolates from clinical sources.

Detection Of β-lactamases Enzymes Responsible For β-lactam Resistance In Some Gram Negative Bacteria By PCR

Abstract: &#x0D; Objective(s): β- lactamases enzymes are well studied in literature since they lead to β-lactam resistance among many pathogens, hence global concern of human and animal health issues is rising. The aim of study is to detect these kinds of enzymes among some gram negative bacteria isolated locally.&#x0D; Material and methods: 30 isolate of each gram negatives Citrobacter spp., Serratia spp., and Enterobacter spp. were obtained from different clinical samples in Baghdad and Al-Najaf cities through March 2020 until January 2021. Isolates were identified biochemically and by Vitek2 system. β- Lactamases encoding genes like (blaSHV (231bp), blaIMP-1 (500), blaVIM (382), blaAmp (1150), blaCMY (1014), bla oxa23 (501), and bla oxa51 (353bp) were detected by PCR.&#x0D; Results: It was found that (80%, 75%, 30%, 50%, 38%, 55%, and 69%) of Citrobacter spp isolates had β- Lactamases encoding genes (blaSHV, blaIMP-1, blaVIM, blaAmp, blaCMY, bla oxa23, and bla oxa51), respectively. Serratia spp. showed having (blaSHV, blaIMP-1, blaVIM, blaAmp, blaCMY, bla oxa23, and bla oxa51) among (20%, 85%, 60%, 28%, 40%, 67%, and 58%), respectively. There were also (60%, 50%, 25%, 33%, 60%, 81%, and 73%) of Enterobacter spp. isolates harbored (blaSHV, blaIMP-1, blaVIM, blaAmp, blaCMY, bla oxa23, and bla oxa51), respectively.&#x0D; Conclusion: Our findings revealed that most of gram negative bacteria isolated locally had different β- Lactamases encoding genes, leading to increase health problems among patients infected with those pathogens.

The Effect of Some Factors on Virulence of E.coli Bacteria Isolated from UTI Infection. (Review study)

The study aims to demonstrate the effect of some traits on the virulence of E.coli bacteria, the important of which are the number and type of bacterial isolates, their sensitivity to some antibiotics and the production of the β-lactamase enzyme. We adopted in this research a multi-studies relative to some countries and different governorates inside Iraq to show the prevalence range of this gender and its great impact on urinary tract infection. It was found through various previous studies that E.coli is present in a large extent in the urinary tract compared to other isolates, as it is a suitable environment for causing infection after seizing the opportunity and reaching to the urinary tract after ascending from the digestive system, which are endemic organisms in which the normal flora. And it is found more often in women than in men for physiological reasons.It also contains multiple resistance to MDR antibiotics. This resistance may be depend on the presence of the plasmid that carries the genetic information to produce the enzyme β-lactamase, which works to break the β-lactamase cycle and create the bio resistance to the antibiotic. This resistance varies from gender to another and from one environment to another. Various studies around the world, especially penicillin’s, the difference in the degree of sensitivity to this antibiotic or the degree of resistance according to the place in which the patient is located and according to the manufacture of the antibiotic and its chemical composition. This type of infection depends on its rapid identification and taking the appropriate antibiotic as soon as possible and for the required time, lest it lead to other complications, the most important of which is the kidney failure caused by this genus, and it is preferable to make a sensitivity test for antibiotics in order to be able to determine the appropriate antibiotic.

Designing Inhibitors of β-Lactamase Enzymes to Overcome Carbapenem Resistance in Gram-Negative Bacteria.

Ever since the first β-lactam antibiotic, penicillin, was introduced into the clinic over 70 years ago, resistance has been observed because of the presence of β-lactamase enzymes, which hydrolyze the β-lactam ring of β-lactam antibiotics. Early β-lactamase enzymes were all of the serine β-lactamase (SBL) type, but more recently, highly resistant Gram-negative strains have emerged in which metallo-β-lactamase (MBL) enzymes are responsible for resistance. The two types of β-lactamase enzymes are structurally and mechanistically different but serve the same purpose in bacteria. The SBLs use an active serine group as a nucleophile to attack the β-lactamase ring, forming a covalent intermediate that is subsequently hydrolyzed. In contrast, the MBLs use a zinc ion to activate the β-lactam toward nucleophilic attack by a hydroxide anion held between two zinc ions. In this Account, we review our recent contribution to the field of β-lactamase inhibitor design in terms of both SBL and MBL inhibitors. We describe how we have approached these challenges from the particular perspective of a small biotechnology company, identifying new inhibitors when faced with either a paucity of starting points for medicinal chemistry (MBL inhibitors) or else an abundance of prior research necessitating a search for novelty, improvement, and differentiation (SBL inhibitors). During the journey from the beginning of lead optimization to successful identification of a preclinical candidate for development, we encountered and solved a range of issues. For example, in the MBL inhibitor series we were able to prevent metabolic cleavage of a glycinamide moiety by circulating amidases while still retaining the activity by converting the amino group into a guanidine. In the SBL inhibitor series, the structure-activity relationship led us to consider introducing a fluorine substituent adjacent to a urea functionality. At first sight this grouping would appear to be chemically unstable. However, deeper theoretical considerations suggested that this would not be the case, and in practice the compound is remarkably stable. Both examples serve to illustrate the importance of scientific insight and the necessity to explore speculative hypotheses as part of the creative medicinal chemistry process.