Characterization Of Beta-lactamases Research Articles

Characterization of Beta-lactamases among MDR gram negative bacilli from a tertiary care hospital in central Kerala

Infections and mortality associated with multidrug- resistant Gram-negative bacilli (MDR GNB) have dramatically increased in all parts of the world. The present study was conducted to screen the burden of Gram negative multidrug resistance in our tertiary care hospital and to pave a stepping stone for the infection control team to tackle the problem effectively. A total of 300 non-repetitive MDR GNB isolates from the specimens received in our diagnostic Microbiology lab oratory for a period of one year were screened phenotypically for Extended spectrum β -lactamases (ESBL), AmpC betalactamases and Carbapenemases. Colistin and Tigecycline susceptibility E-test were also performed randomly on some carbapenemase producing organisms. Among the 300 MDR GNB isolates, ESBL and AmpC producers were 56% and 24% respectively. E.coli was the most common organism with ESBL (73%) and AmpC (46%) resistance mechanisms. Carbapenemase producers were 14.6% with predominant isolate being Acinetobacter spp (40%). Among the risk factors, 13% patients had immunosuppressive conditions like diabetes, 31% had insitu urinary catheters, 57.7% and 29.4% had prior antibiotic usage and hospitalisations respectively. Twenty five carbapenemase producing MDR GNB tested for Colistin MIC were in the susceptible range. 71% of Acinetobacter spp tested for Tigecycline showed resistant MIC values. Multidrug resistance has a significant adverse impact on clinical outcomes of patients & leads to higher costs due to consumption of health-care resources. Adherence to simple measures like hand hygiene & careful attention to barrier precautions can help in preventing the spread of these infections to a greater extent.

Phenotypic characterization of beta-lactamases producing Gram-negative bacteria in a tertiary hospital, Nepal

Infections caused by beta-lactamases producing Gram-negative bacteria are increasing, thus posing a challenge to the management of such infections. The surveillance data of such bacteria is limited in Nepal so this study aimed to detect the beta-lactamase producing Gram-negative bacteria in a tertiary setting. A total of 604 clinical samples, including urine, blood, sputum and body fluids, were cultured and identified by the routine standard laboratory protocols. Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method following Clinical and Laboratory Standard Institute guidelines (2014). Extended-spectrum beta-lactamases (ESBL) producers were identified by combined disk method and metallo-beta-lactamases (MBL) producers were identified by Imipenem- EDTA combined disk method. Out of 604 samples, 282 (46.7%) samples showed significant growth, of which 229 (81.2%) were Gram-negative bacteria. Of 229 Gram-negative bacteria, 200 (87.3%) were multidrug resistant, 67 (29.3%) were ESBL producers and 16 (7.0%) were MBL producers. Klebsiella pneumoniae were among higher ESBL producers and Pseudomonas aeruginosa were among higher MBL producers. The findings suggest higher antibacterial resistance among Gram-negative bacteria with the added burden of beta-lactamase production. Imipenem was effective against 125 of 229 Gram-negative bacteria tested. Thus, imipenem can be the drug of choice for empirical management. The higher multidrug resistance and higher beta-lactamases production among Gram-negative bacteria warrant the continuous monitoring, surveillance, early detection, and infection control practices of such bacteria

Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms.

The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.

First characterisation of plasmid-mediated quinolone resistance-qnrS1 co-expressed blaCTX-M-15 and blaDHA-1 genes in clinical strain of Morganella morganii recovered from a Tunisian Intensive Care Unit

Purpose: Aim of this study was to show the emergence of the qnr genes among fluoroquinolone-resistant, AMPC and ESBL (extended-spectrum-beta-lactamase) co-producing Morganella morganii isolate. Materials and Methods: A multi resistant Morganella morganii SM12012 isolate was recovered from pus from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. ESBLs were detected using a standard double-disk synergy test. The characterization of beta-lactamases and associated resistance genes were performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Results: The antimicrobial susceptibility testing showed the high resistance to penicillins, cephalosporins (MICs: 64-512 µg/ml) and fluoroquinolones (MICs: 32-512 µg/ml). But M. morganii SM12012 isolate remained susceptible to carbapenems (MICs: 4-<0.25 µg/ml). The double-disk synergy test confirmed the phenotype of extended-spectrum β-lactamases (ESBLs). Three identical β-lactamases with pI values of 6.5, 7.8 and superior to 8.6 were detected after isoelectric focusing analysis. These β-lactamases genes can be successfully transferred by the conjugative plasmid. Molecular analysis demonstrated the co-production of blaDHA-1, blaCTX-M-15 and qnrS1 genes on the same plasmid. The detection of an associated chromosomal quinolone resistance revealed the presence of a parC mutation at codon 80 (Ser80-lle80). Conclusion: This is the first report in Tunisia of nosocomial infection due to the production of CTX-M-15 and DHA-1 β-lactamases in M. morganii isolate with the association of quinolone plasmid resistance. The incidence of these strains invites continuous monitoring of such multidrug-resistant strains and the further study of their epidemiologic evolution.

Molecular characterization of beta-lactamases of Enterobacteriaceae with ertapenem MICS greater than 0.25, and impact of new CLSI carbapenem breakpoints

Molecular characterization of beta-lactamases of Enterobacteriaceae with ertapenem MICS greater than 0.25, and impact of new CLSI carbapenem breakpoints

Detection of Unrelated Escherichia Coli Strains Harboring Genes of CTX-M-15, OXA-1, and AAC(6')-Ib-Cr Enzymes in a Tunisian Hospital and Characterization of Their Integrons and Virulence Factors

Beta-lactamase characterization was carried out in a collection of 18 extended-spectrum beta-lactamase (ESBL)-positive Escherichia Coli isolates from blood (n=8) and urine (n=10) obtained in 2007 in a tunisian Hospital. All isolates were clonally unrelated according to PFGE analysis. Seventeen strains presented the blaCTX-M-15 gene associated with blaOXA-1 and four of these strains with the blatem-1b gene. The remaining ESBL-positive strain contained the blaCTX-m-9 gene associated with the blaOXA-1 and blaTEM-1b genes. The orf477 sequence was identified downstream of the blaCTX-M-15 gene in all 17 blaCTX-M-15-positive strains, and ISEcp1 upstream in 15 of them (in eight cases truncated by IS26). The presence of a class 1 integron was demonstrated in 4 of the 18 ESBL-positive strains (22.2%), with dfrA17 + aadA5 (3 strains) and dfrA12 + orfF + aadA2 (1 strain) being the gene cassettes identified. The variant aac(6')-Ib-cr was found in 15 blaCTX-M-15-containing strains. All 18 ESBL-positive strains were typed as phylogroup b2 and contained at least three of the eight tested virulence genes (fimA, papGIII, hlyA, cnf1, papC, aer, eae and bfp). Six blaCTX-M-15-positive strains were included in the serotype O25b and one of them was typed as ST131. Another blaCTX-M-15-positive strain serotype-O25 was typed as ST638. The blaCTX-M-15, aac(6')-Ib-cr, and aac(3)-II genes were co-transferred by conjugation from 7 donor strains to E. coli CSH26 recipient strain. The blaCTX-M-15 gene is prevalent among ESBL-positive E. coli strains in the studied hospital, that is frequently found together with aac(6')-Ib-cr, and aac(3)-II genes. The detection of the clone O25b-ST131 in a blaCTX-M-15 strain corroborates its worldwide dissemination.

Patterns of Resistance to Beta-Lactams and Characterization of Beta-Lactamases in Escherichia coli Isolates from Children in Tunisia

Seventy-six (75.2%) of 101 Escherichia coli isolates, collected over a 2-month period in 2002 at the Children's Hospital of Tunis (Tunisia), were resistant to at least one beta-lactam. They produced beta-lactamases which were further characterized by spectrophotometry, isoelectric focusing (IEF) and PCR. Seventy-five isolates had a pI 5.4 beta-lactamase and one isolate expressed a pI 5.6 beta-lactamase. These beta-lactamases were active against penicillins and cephalothin. Nineteen of 76 isolates were resistant to ticarcillin-clavulanic acid combination. The blaTEM gene was detected in 71 isolates, all expressed a pI 5.4 beta-lactamase that was assumed to be TEM-1 or inhibitor-resistant TEM (IRT). Thirty-eight of 76 isolates showed one weak band on IEF with pIs ranging from 8.2 to ≥9, suggesting low level expression of the chromosomal AmpC beta-lactamase. Five of 76 isolates produced extended-spectrum beta-lactamases (ESBLs), with a basic pI of 7.9 or 8.7, active on penicillins, extended-spectrum cephalosporins, but not on cefoxitin. blaSHV genes were detected in three isolates producing pI 7.9 ESBLs but not in two isolates expressing pI 8.7 ESBLs. These latter showed strong cefotaxime-hydrolyzing activities. Hence, they might be CTX-M-type ESBLs.

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain.

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.

Carbapenem resistance mechanisms in Pseudomonas aeruginosa clinical isolates.

In order to define the contributions of the mechanisms for carbapenem resistance in clinical strains of Pseudomonas aeruginosa, we investigated the presence of OprD, the expressions of the MexAB-OprM and MexEF-OprN systems, and the production of the beta-lactamases for 44 clinical strains. All of the carbapenem-resistant isolates showed the loss of or decreased levels of OprD. Three strains overexpressed the MexAB-OprM efflux system by carrying mutations in mexR. These three strains had the amino acid substitution in MexR protein, Arg (CGG) --> Gln (CAG), at the position of amino acid 70. None of the isolates, however, expressed the MexEF-OprN efflux system. For the characterization of beta-lactamases, at least 13 isolates were the depressed mutants, and 12 strains produced secondary beta-lactamases. Based on the above resistance mechanisms, the MICs of carbapenem for the isolates were analyzed. The MICs of carbapenem were mostly determined by the expression of OprD. The MICs of meropenem were two- to four-fold increased for the isolates which overexpressed MexAB-OprM in the background of OprD loss. However, the elevated MICs of meropenem for some individual isolates could not be explained. These findings suggested that other resistance mechanisms would play a role in meropenem resistance in clinical isolates of P. aeruginosa.

Susceptibility patterns and characterization of beta-lactamases in clinical isolates ofBacteroids fragilis

Strains of Bacteroides fragilis isolated from clinical specimens are known to be frequently resistant to penicillins or cephalosporins. The production of beta-lactamases is the major mechanism in this resistance. Up to now at least five types of beta-lactamase enzymes have been described in the Bacteroides fragilis group. The purpose of this study was to evaluate the susceptibility to different antibiotics and the prevalence of the beta-lactamase types among 100 Bacteroides fragilis strains isolated from clinical specimens in Italy. MICs of 11 antibiotics were determined by the reference agar dilution method and by the new E test (AB Biodisk, Sweden). Results showed an acceptable correlation between the MICs determined for most strains, although those obtained by the E test tended to be lower. In the beta-lactam resistant strains the enzyme activity and isoelectric points were determined on crude enzyme extract.

Antimicrobial susceptibilities and beta-lactamase characterization of Capnocytophaga species.

Capnocytophaga species have been associated with a wide variety of infections in both immunocompetent and immunocompromised patients. On the basis of data from antimicrobial susceptibility studies, beta-lactam antibiotics have been considered efficacious therapy. Six of 19 isolates from primarily clinical sources across Canada demonstrated beta-lactamase production, and agar dilution susceptibility testing showed broad resistance to beta-lactam antibiotics. For the beta-lactamase producing isolates, clavulanate reduced the MIC of amoxicillin for 90% of the strains tested by 64-fold. Isolates were highly susceptible to clindamycin, imipenem, and ciprofloxacin. Characterization of the beta-lactamases produced by two of these isolates (Van1 and Van2) was performed. Isoelectric focusing revealed an identical isoelectric point of 5.6 for both enzymes, but they had markedly different relative hydrolysis efficiencies, and different conditions were required to extract the enzymes. This study demonstrates the production of different types of beta-lactamases by Capnocytophaga spp. and suggests the need to screen all clinical isolates of Capnocytophaga spp. for the presence of beta-lactamases.

Characterization of beta-lactamases from non-Bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in beta-lactam resistance

Twelve beta-lactamase-positive non-Bacteroides fragilis group Bacteroides spp. belonging to seven different species were examined by MIC determination and enzyme characterization. MICs of most beta-lactams except cefoxitin, cefotetan, imipenem, and meropenem were relatively high or very high. All enzymes hydrolyzed cephaloridine (Vmax, 100%; Km, 12 to 70 microM), cephalothin (Vmax, 25 to 826%; Km, 8 to 143 microM), cefamandole (Vmax, 13 to 158%; Km, 17 to 170 microM), and cefuroxime (hydrolysis rate, 19 to 98%), and 11 of 12 hydrolyzed cefotaxime (Vmax, 26 to 145%; Km, 13 to 127 microM); no hydrolysis of cefoxitin or moxalactam was observed. Penicillins were hydrolyzed at lower rates, with Vmax values less than or equal to 20% of that obtained with cephaloridine. Addition of clavulanate, sulbactam, or tazobactam led to a 4- to 2,048-fold lowering of MICs of penicillins as well as cephalosporins. All enzymes were inhibited by clavulanate (50% inhibitory concentration [IC50], 0.01 to 1.8 microM), sulbactam (IC50, 0.02 to 1.9 microM), tazobactam (IC50, 0.001 to 0.9 microM), cefoxitin (IC50, 0.002 to 0.35 microM), and moxalactam (IC50, 0.03 to 6.6 microM). No enzymes were inhibited by 100 microM EDTA or p-chloromercuribenzoic acid; an enzyme of one strain of B. loescheii was inhibited by 100 microM cloxacillin (IC50, 2.35 microM). Ten enzymes had optimal activity at pH 5.0 to 6.0, and two had optimal activity at pH 8.0. Isoelectric focusing revealed pIs between 4.2 and 5.6. These enzymes seem to belong to a previously unclassified group of beta-lactamases, related (but not identical) to beta-lactamases of the B. fragilis group.