Production Of Beta-lactamase Enzymes Research Articles

Detection of Methicillin Resistance and β-Lactamase Production in Staphylococcus aureus Isolates: A Cross Sectional Study from a Tertiary Care Hospital

Nosocomial infections are proving to be a menace for the whole health care system, with methicillin resistant Staphylococcus aureus (MRSA) being a very notorious causative agent. Along with the role of mecA gene producing penicillin-binding protein (PBP2a), production of beta-lactamase enzyme by Staphylococcus aureus makes the organism resistant to all β-lactam agents. This study aims at phenotypic detection of methicillin resistance and β-Lactamase production in all S. aureus isolates by Cefoxitin disk diffusion test and Penicillin zone-edge test, respectively. In this prospective cross-sectional study, samples were obtained from patients admitted to different departments and were processed according to the standard laboratory protocols. As per the CLSI guidelines, phenotypic detection of resistance to methicillin was done by Cefoxitin Disk Diffusion test, whereas production of beta-lactamase enzyme was phenotypically detected by penicillin zone edge test. Among 179 isolates of S. aureus, 116 (64.8%) were MRSA, whereas the remaining 63 (35.2%) isolates were methicillin-sensitive Staphylococcus aureus (MSSA). Staphylococcus aureus infection among ICU and non-ICU patients were found to be 24(13%) and 155(86.6%) respectively. Among 24 ICU patients, 19 had MRSA infection, whereas among 155 non-ICU patients, 97 had MRSA infection. Out of 63 MSSA isolates, only 2 (3.17%) were found to be sensitive to Penicillin by Zone-edge test, 04 isolates showed presence of β-lactamase production, while remaining 57 isolates were resistant to Penicillin. At the same time, several class of antibiotics were found to be ineffective against these MRSA isolates. Cases of methicillin-resistant and b–lactamase producing Staphylococcus aureus infections are on the rise in our hospital settings.

Detection of CTX-M Gene β-Lactamase in Gram Negative Bacteria Isolate from Shendi Health Facilities

Background: The emergence of antibiotic resistance pathogen is an important health risk, usually gram-negative bacteria acquire resistance to antibiotics by the production of beta-lactamase enzyme, CTX-M-type enzymes are a group of class A extended-spectrum B-lactamases (ESBLs) that are rapidly Spreading among gram-negative worldwide. CTX-M-type ESBLs exhibit powerful activity against cefotaxime and ceftriaxone antibiotics but generally not against ceftazidime, in some geographical areas, CTX-M is now the most prevalent ESBLs in Enterobacteriaceae. There are 172 known variants of CTX-M genes identified until August 25, 2016. Objectives: This present study aims to identify the gene, namely: CTX-M, Responsible for extended-spectrum beta-lactamase (ESBL) among Enterobacteriaceae isolated from different samples of Sudanese patients in Shendi city (urine, pus, and wound). Methodology: 200 samples were identified as gram-negative bacteria. Identification of the isolates was done by using conventional biochemical methods, and ESBLs were screened according to (CLSI) guidelines. ESBLs Positive strains were tested for the presence of ESBL encoding CTX-M, Gene by using PCR with specific primers for the detection of CTX-M. Results: ESBL found to be higher in P. aeruginosa n=18(78.3%) Klebsiella spp n=45 (64.2%). Escherichia coli n=51(57.3%) Proteus n=8(53.3%) the total of ESBL from all gram-negative isolate represent n=122 (61%). The presence of the CTX-M, the gene was confirmed in n=57(46.8%) in all isolates. The CTX-M gene were detected in n= 17 (37.8%) Klebsiella spp, n=28(55%) of Escherichia coli, n=3(37.5%) in Proteus and n=9 (50%) in P. aeruginosa isolates. Conclusion: P. aeruginosa isolates represent a high percentage of ESBL producers followed by Klebsiella spp, then Escherichia coli and proteus spp CTX-M gen detected commonly in Escherichia coli followed by P. aeruginosa then Klebsiella spp and Proteus spp. The study concludes that the ESBL production within gram-negative bacteria was high and the CTX-M gene was spread in Shendi city.

Cefotaxime Exposure Selects Mutations within the CA-Domain of envZ Which Promote Antibiotic Resistance but Repress Biofilm Formation in Salmonella.

ABSTRACTCephalosporins are important beta lactam antibiotics, but resistance can be mediated by various mechanisms including production of beta lactamase enzymes, changes in membrane permeability or active efflux. We used an evolution model to study how Salmonella adapts to subinhibitory concentrations of cefotaxime in planktonic and biofilm conditions and characterized the mechanisms underpinning this adaptation. We found that Salmonella rapidly adapts to subinhibitory concentrations of cefotaxime via selection of multiple mutations within the CA-domain region of EnvZ. We showed that changes in this domain affect the ATPase activity of the enzyme and in turn impact OmpC, OmpF porin expression and hence membrane permeability leading to increased tolerance to cefotaxime and low-level resistance to different classes of antibiotics. Adaptation to cefotaxime through EnvZ also resulted in a significant cost to biofilm formation due to downregulation of curli. We assessed the role of the mutations identified on the activity of EnvZ by genetic characterization, biochemistry and in silico analysis and confirmed that they are responsible for the observed phenotypes. We observed that sublethal cefotaxime exposure selected for heterogeneity in populations with only a subpopulation carrying mutations within EnvZ and being resistant to cefotaxime. Population structure and composition dynamically changed depending on the presence of the selection pressure, once selected, resistant subpopulations were maintained even in extended passage without drug.IMPORTANCE Understanding mechanisms of antibiotic resistance is crucial to guide how best to use antibiotics to minimize emergence of resistance. We used a laboratory evolution system to study how Salmonella responds to cefotaxime in both planktonic and biofilm conditions. In both contexts, we observed rapid selection of mutants within a single hot spot within envZ. The mutations selected altered EnvZ which in turn triggers changes in porin production at the outer membrane. Emergence of mutations within this region was repeatedly observed in parallel lineages in different conditions. We used a combination of genetics, biochemistry, phenotyping and structural analysis to understand the mechanisms. This data show that the changes we observe provide resistance to cefotaxime but come at a cost to biofilm formation and the fitness of mutants changes greatly depending on the presence or absence of a selective drug. Studying how resistance emerges can inform selective outcomes in the real world.

Enterobacteria producing beta-lactamase and their distribution in poultry and poultry products

This review article is devoted to the problems of the origin and spreading of antibiotic-resistant enterobacteria producing beta-lactamases in poultry farming.The reason of the spreading of multidrug-resistant bacteria and diseases caused by them among farm animals and poultry is the irrational use of antibacterial agents, determination of enzymatic inactivation of antibiotics by laboratory methods, and the spread of resistance genes.Most gram-negative bacteria such as Klebsiella spp., Escherichia coli, Proteus spp. and others, have tolerance to various antibiotics and disinfectants due to the production of beta-lactamase enzymes. Initially, some strains produced a wide range of beta-lactamases, and these isolates were susceptible to the 1st and 2nd generations of cephalosparins. Over time, the bacteria mutated, producing extended beta-lactamases. Such microorganisms are already resistant to the 3rd generation of cephalosparins. Antimicrobial resistance leads to limited therapeutic options, which increases the difficulty of treating diseased animals.This problem is especially actual all over the world, many scientists conduct research to detect and study this phenomenon. The spread of antimicrobial resistance is one of the most acute problems of our time, carrying biological and economic threats to all countries. Many scientists are conducting research to detect and study the polyresistance of Enterobacteriaceae, such as E. coli and K. pneumoniae.The modern infectious pathology of poultry is characterized by an increase in the frequency of occurrence and spreading of E. coli and K. pneumoniae, producing beta-lactamases, which contributes to ineffective antibiotic therapy.The presence of multidrug-resistant opportunistic microorganisms isolated from poultry food can affect the health of consumers. The protection of the population from antibiotic resistance microorganisms should be based primarily on the protection of people from infection by any multidrug-resistant foodborne pathogens, therefore, it is necessary to improve diagnostic methods, therapeutic and prophylactic measures and the search for new highly effective drugs.

Antimicrobial resistance profile of extended-spectrum beta-lactamases, adenosine-monophosphate-cyclic, and carbapenemase-producing Gram-negative bacteria isolated from domestic animals.

Background and Aim:The production of beta-lactamase enzymes, such as extended-spectrum beta-lactamase (ESBL), adenosine-monophosphate-cyclic (AmpC), and Klebsiella pneumoniae carbapenemase (KPC), is one of the most important mechanisms of bacterial resistance to antimicrobials. Gram-negative bacteria show significant resistance due to various intrinsic and acquired factors. These intrinsic factors include low permeability of the outer membrane, various efflux systems, and the production of beta-lactamases, while acquired factors include chromosomal mutation and acquisition of resistance genes by horizontal transfer. Mobile elements such as plasmids, integrative conjugative elements, mobilizable islands, or transposable elements are involved in horizontal transfer. At present, the Gram-negative pathogens of most concern are Acinetobacter baumannii, Pseudomonas aeruginosa, and those belonging to the Enterobacteriaceae family (e.g., Escherichia coli, K. pneumoniae, and Proteus mirabilis). This study aimed to evaluate the profile of antimicrobial resistance and the production of the enzymes ESBL, AmpC, and KPC, in 21 gram-negative bacteria isolated from domestic animals treated at the University Veterinary Hospital (HVU) of the Federal University of Western Bahia (UFOB).Materials and Methods:The biological samples (21) were inoculated to brain heart infusion broth, blood agar, and MacConkey agar and incubated for 24-72 h at 37°C. Gram staining and identification through biochemical tests and matrix-associated laser desorption/ionization time-of-flight mass spectrometry were conducted. To evaluate the antimicrobial resistance profile, the disk diffusion method was used, and 25 antibiotics were employed. For the detection of ESBL, the disk approximation method was applied using chromogenic agar. The presence of KPC was observed using chromogenic agar and the Hodge test. For AmpC evaluation, the disk approximation method was used.Results:The most isolated agent was E. coli (66.66%, 14/21), followed by K. pneumoniae and P. mirabilis (both 14.29%, 3/21), and then Pasteurella spp. (4.76%, 1/21). The bacterial isolates showed high levels of resistance against clindamycin, penicillin, imipenem, polymyxin, cefoxitin, gentamycin, cefotaxime, ceftazidime, cephalothin, ceftriaxone, ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, and tetracycline. The best effectiveness rates were observed for cefepime, streptomycin, amoxicillin-clavulanate, aztreonam, nalidixic acid, tobramycin, levofloxacin, amikacin, and meropenem. All biological isolates showed multiple resistance to at least three of the antibiotics tested (3/25), and some showed resistance to 24 of the antibiotics tested (24/25). Among the 21 pathogens analyzed, 8 were ESBL producers (38.09%); of these, 6 were identified as E. coli (28.57%), and 2 were identified as K. pneumoniae (9.52%). Two strains of K. pneumoniae produced both ESBL and KPC. None of the isolates were producers of AmpC.Conclusion:The results found in the present work raise concern about the level of antimicrobial resistance among pathogens isolated from domestic animals in Brazil. The results highlight the need for the development and implementation of anti-resistance strategies to avoid the dissemination of multiresistant pathogens, including the prudent use of antimicrobials and the implementation of bacterial culture, antimicrobial sensitivity, and phenotypic tests for the detection of beta-lactamase enzymes in bacteria isolated from animals.

Combination of silver nanoparticles with ineffective antibiotics against extended spectrum beta-lactamases producing isolates at Alexandria Main University Hospital, Egypt

BackgroundThe context and purpose of the study: The spready dissemination of resistance mechanisms among extended spectrum beta-lactamases (ESBL) producing bacterial isolates has increasingly been reported. There is an urgent need to explore the antibacterial property of nontoxic biosafe compounds. This In-vitro experimental study aimed to evaluate the effect of silver nanoparticles (AgNPs) alone and in combination with ineffective antibiotics against ESBL producing isolates.ResultsCiprofloxacin with AgNPs combination had the highest synergistic percentage against 91.43% of ESBL Escherichia coli isolates, and it was additive against 8.57% of them. As regards ESBL K. pneumoniae isolates, AgNPs with cefotaxime were synergistic against 75.00% of them, followed by ceftazidime and ciprofloxacin (62.50%). The least effective combination was ampicillin with AgNPs. The greatest enhancement of activity of the antibiotics was observed at silver minimum inhibitory concentration (MICAg) MICAg/2 and MICAg/4. At lower AgNPs concentrations, enhanced effects were less obvious. AgNPs inhibited the production of beta-lactamase enzymes in 91.43% of E. coli and 75% of Klebsiella pneumoniae isolates.ConclusionAgNPs are a valuable alternative to combat drug resistance, as they had synergistic effects when combined with different ineffective antibiotics against ESBL producing bacteria. AgNPs had lowered MIC values of antibiotics by several folds. Moreover, they inhibited the production of beta-lactamase enzymes.

Prevalensi Gen OXA-23 pada Isolat Klinis Bakter Acinetobacter baumanii di RSUP Dr. M. Djamil Padang

Acinetobacter baumannii is such a big challenge in this modern era because of their multi-drug resistance (MDR) through their resistance mechanism, namely the production of beta lactamase enzymes, one of them is oxacillinases. Several studies have revealed that the OXA-23 gene has an important role in the production of the beta lactamase enzyme so that these bacteria are resistant to antibiotics, especially carbapenem. This study was conducted to determine the description of the resistance of the Acinetobacter baumannii by looking at the prevalence of the OXA-23 gene in clinical isolates of these bacteria at RSUP Dr. M. Djamil Padang.
 This study was a descriptive observational study and used a cross-sectional design using polymerase chain reactions (PCR) and electrophoresis techniques. The samples were clinical isolates of Acinetobacter baumannii bacteria stored in the Microbiology Laboratory of the Faculty of Medicine, Andalas University with inclusion and exclusion criteria.
 Of the 150 clinical isolates of Acinetobacter baumannii studied, most of the bacteria were still sensitive to the antibiotics of Amikacin (84%), Tigecycline (78.76%), and Trimethoprim / Sulfamethoxazole (76.77%). Meanwhile, all of the Acinetobacter baumannii isolates were resistant to cefazolin. In PCR and electrophoresis tests, 50 clinical isolates were positive for the OXA-23 gene. 46 of them were carbapenem resistant, while the other 4 isolates were sensitive to carbapenem. From these results it can be concluded that the prevalence of the OXA-23 gene in Acinetobacter baumannii isolates in RSUP Dr. M. Djamil Padang was 85.18% for carbapenem resistant isolates and 4.30% for carbapenem sensitive isolates.
 Keywords : Acinetobacter baumannii, carbapenem, OXA-23 gene

Prevalence of blaVEB and blaTEM genes, antimicrobial resistance pattern and biofilm formation in clinical isolates of Pseudomonas aeruginosa from burn patients in Isfahan, Iran

BackgroundPseudomonas aeruginosa is very common in burn wound infections. The biofilm formation and the production of beta-lactamase enzymes have made this bacterium resistant to many antibiotics. This study aimed to evaluate antibiotic resistance, biofilm formation, and the frequency of strains that carry blaTEM and blaVEB. MethodsA cross-sectional study was carried out from March 2017 to March 2018 on 103 clinical isolates of P. aeruginosa, identified from 420 burn wound infection samples by phenotypic tests. Antibiotic resistance and biofilm formation were evaluated by the Kirby-Bauer disk diffusion method and microtiter plate assay, respectively. Double disk synergy test (DDST) was used for phenotypical detection of Extended-spectrum beta-lactamases (ESBL) producing isolates. ESBL genes were detected by polymerase chain reaction (PCR). ResultOf 103 strains isolated from burn wound infection, 91.3% were multiple drug resistance (MDR). The resistance to levofloxacin was the highest (93.2%). The ability of biofilm formation was observed in three groups: 47.6% of isolates had no biofilm formation, 38.8% were weak, and 13.6% showed moderate biofilm formation. The prevalence of DDST-confirmed ESBL was 54 (43.9%). The isolates had 28.15% and 21.35% of blaVEB and blaTEM genes, respectively. ConclusionThis study showed that MDR strains are common in burn infections. Biofilm formation and produce beta-lactamase enzymes also showed a growing trend.

Detection of Extended-Spectrum Beta-Lactamases (ESBLs) and Antibiotic Susceptibility Patterns in Klebsiella pneumoniae in Western, Iran.

Production of Beta-Lactamase enzymes, especially extended- spectrum Beta -Lactamases (ESBL), is a primary mechanism of resistance in these bacteria.The purpose of this study was detection of blaTEM, blaSHV, blaCTXM, blaCTX-M-15, blaPERand blaVEBin K. pneumoniae, isolated from clinical specimens by the PCR method and antibiotic susceptibility patterns in these strains isolated. During a period from October 2015 to July 2016, 52 K. pneumoniae isolates were collected from general hospitals in the city of Sanandaj, Iran. After identifying the strains by biochemical testing, the disc diffusion method was used for determining antimicrobial susceptibility and screening the ESBL-producing isolates. Detection of blaTEM, blaSHV, blaCTX-M, blaCTX-M-15, blaPER and blaVEBESBL-producing K. pneumoniae was carried out by PCR. Out of 52-collected K. pneumoniae, highest and lowest rates of resistance related to co-trimoxazole with 67.3 % and amikacin with 30.7 %. 55.7% identified as MDR and 69.23% were ESBL-producing K. pneumoniae.blaSHV was the most prevalent gene in ESBL-producing K. pneumoniae. blaTEM,blaCTX-M,blaCTX-M-15 producing K. pneumoniae strains showed higher rates of drug resistant compared with negative strains (P < 0.05). Results of this study showed that the prevalence rate of ESBL-producing K. pneumoniae isolates is increasing in MDR strains, which raises concerns regarding the treatment of K. pneumoniae. Therefore, molecular research in the field of antimicrobial resistance of bacteria is essential to prevent the spread of resistant strains.

Emergence of colistin resistance in extended-spectrum beta lactamase producing Enterobacteriaceae isolated from food animals and its public health implication: A review

Antimicrobial resistance as a result of emergence of extended-spectrum beta lactamase producing Enterobacteriaceae is a major health problem of human and animal that requires an intensive global attention. The production of beta lactamase enzymes remains as one of the major factors contributing to the development of resistance to beta lactams. These enzymes hydrolyze the beta lactam ring of the antibiotic and render it ineffective. Extended-spectrum beta lactamase producing bacteria have the ability to develop resistance to a number of antibiotics including the carbapenem and other third generation cephalosporins. In addition, the recent emergence and dissemination of the colistin resistance determinants mcr -1, mcr -2 and mc r-3 poses a serious threat to colistin as a drug of last resort in human medicine. In this review, we utilized words such as “colistin resistance and Escherichia coli ”, “ Klebsiella and colistin resistance”, “colistin resistance and Salmonella ” as well as “detection of mcr -1 genes in Salmonella and E. coli ”. The extended-spectrum beta lactamase producing bacteria under Enterobacteriaceae that are resistant to colistin possess the ability to be transferred resistant determinants to other susceptible cells at a higher frequency. In this paper, the role of manure from food animals and how air travel contributes to the dissemination of mcr- 1 haboring bacteria, resistance determinants and other metabolites that constitute a public health problem was also reviewed. It is concluded that these pathogens have significant consequences to the control of infection and plays key roles in treatment failure with colistin. http://doi.org/10.5455/javar.2018.e246

Phenotypic detection of beta-lactamases production in enterobacteriaceae

Beta-lactam antibiotics are the most commonly used antibacterial drugs. However, many bacteria have developed resistance to these antibiotics, and the most common form of resistance is the production of beta-lactamase enzymes. Many members of the Enterobacteriaceae family produce different types of these enzymes. The aim of this study was to perform phenotypic detection of production and identification of beta-lactamase type in Enterobacteriaceae isolated from different clinical specimens from patients hospitalized in the Clinical Center of Serbia. The strains of Enterobacteriaceae were collected between November 2011 and January 2012 in the laboratory of the Clinical Center of Serbia.The isolates were identified according to the standard microbiology procedures and confirmed by the Vitek2 automated system. Antimicrobial susceptibility testing was performed by the disk diffusion method, and the phenotypic detection of production and identification of beta-lactamases was performed according to previously described methodologies. In this study, a total of 172 Enterobacteriaceae strains were isolated. Further testing was performed on 54/145 (37.2%) strains showing decreased susceptibility to beta-lactam antibiotics: 13/85 (15.3%) Escherichia coli, 31/46 (67.4%) Klebsiella pneumoniae and 10/14 (71.4%) Proteus mirabilis. Among them, 40/145 (27.6%) strains produced extended spectrum beta-lactamases (ESBLs), 9/145 (6.2%)--AmpC, 1/145 (0.7%)--K1 beta-lactamase and 4/145 (2.8%)--carbapenemases. Carbapenemases were predominantly detected in K. pneumoniae (75%). Enterobacteriaceae produce different types of beta-lactamases, and the most common type in our study was ESBLs. Production of carbapenemases detected in Enterobacteriaceae is also an associated problem.

Beta-lactamases: a survey of protein diversity.

Bacterial resistance to beta-lactam antibiotics and beta-lactamase inhibitors is an ever increasing problem that threatens the clinical utility of drugs that form the cornerstone of the antibiotic armamentarium. Especially among Gram-negative pathogens, elaboration of structurally and mechanistically novel beta-lactamase enzymes is the most important means by which resistance occurs. An appreciation of the tremendous diversity of these drug-modifying enzymes will assist in understanding why so few generally effective inhibitory agents exist for these unique drug targets. This review will give a general background on the reaction mechanisms and classification schemes of the more than 340 beta-lactamase enzymes described to date. A discussion will follow highlighting the emerging Class A SHV and TEM-derived extended-spectrum (ESBLs), and inhibitor-resistant enzymes, non-TEM, non-SHV Class A ESBLs, and carbapenemases, Class B metallo-beta-lactamases and some of their novel inhibitors, plasmid and chromosomally encoded Class C enzymes, and finally, the OXA-type oxacillinases, ESBLs, and carbapenemases of Class D. The clinical importance of multiple resistance mechanisms in conjunction with the production of beta-lactamase enzymes is emphasized.

Molecular analysis of beta-lactamase structure and function

AbstractThe extensive and sometimes irresponsible use of beta-lactam antibiotics in clinical and agricultural settings has contributed to the emergence and widespread dissemination of antibiotic-resistant bacteria. Bacteria have evolved three strategies to escape the activity of beta-lactam antibiotics: 1) alteration of the target site (e.g. penicillin-binding protein (PBPs), 2) reduction of drug permeation across the bacterial membrane (e.g. efflux pumps) and 3) production of beta-lactamase enzymes. The beta-lactamase enzymes inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring rendering the antibiotic ineffective. The inactivation of the antibiotic provides resistance to the bacterium. Currently, there are over 300 beta-lactamase enzymes described for which numerous kinetic, structural, computational and mutagenesis studies have been performed. In this review, we discuss the recent work performed on the four different classes (A, B, C, and D) of beta-lactamases. These investigative advances further expand our knowledge about these complex enzymes, and hopefully, will provide us with additional tools to develop new inhibitors and antibiotics based on structural and rational designs.