Extended-spectrum Beta-lactam Antibiotics Research Articles

Effectiveness of Double Discs Synergy Test in the Confirmation of Extended Spectrum β-lactamase (ESβL) Production

Extended spectrum beta lactamases (ESβLs) are enzymes which have evolved from bacteria upon exposure to penicillins antibiotics. Such bacteria have developed resistance to Beta lactam antibiotics and their relatives such as cephalosporins (extended spectrum beta lactam antibiotics). The enzymes represent the major source of multidrug resistance in gram negative bacteria. Different methods of confirming ESβL production from bacteria have been described. Most of these methods suggest screening the test isolates in accordance with decrease in susceptibility to extended-spectrum cephalosporins in preliminary susceptibility testing and use any of the available confirmatory tests for ESβL production. However, it is not clear which confirmatory tests are the most sensitive and which extended-spectrum cephalosporins should be tested. In our previous study, disc replacement method was used to detect ESβLs production among Gram negative isolates from Gombe specialist hospital. In this report, the effectiveness of double disc synergy test (DDST) in the confirmation of ESβL- production was studied from the clinical isolates obtained from this hospital for the first time to the best of our knowledge. A total of two hundred and fifty (250) bacteria screened to be suspected ESβL-producers were subjected to confirmatory test using double disc synergy test (DDST). The method effectively confirmed ninety six (96) isolates to be ESβL-producers. Esherichia coli had the highest occurrence (26, 27.08%), followed by Klebsiella pneumonia (20, 20.83%), Providencia spp. (14, 14.58%), Morganella morganii (12, 12.5%), Yersinia enterocolitica (6, 6.25%), Pseudomonas aeruginosa , Shigella spp. and Salmonella paratyphi A each with a total occurrence of 4(4.17%), Citrobacter freundii, Serratia marcescens, and Salmonella typhi each with an occurrence value of 2 (2.08%) while Proteus vulgaris was found to be negative ESβL- producer. In conclusion, the DDST was found to be effective in the confirmation of ESβL-production.

Draft Genome Sequences of Four Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospital Wastewater in Singapore.

Four multidrug-resistant Pseudomonas aeruginosa isolates were cultured from intensive care unit wastewater. All isolates exhibited resistance to carbapenem and extended-spectrum beta-lactam antibiotics. Genome characterization revealed the presence of beta-lactamase resistance genes (bla PAO and bla OXA), and three out of the four isolates carried the bla NDM-1 gene encoding resistance against carbapenems.

The Causes and Treatment Outcomes of 91 Patients with Adult Nosocomial Meningitis

Background/AimsFrequent pathogens of nosocomial meningitis were investigated and the adequacy of empiric antibiotic therapy was assessed. Outcomes of nosocomial meningitis were also evaluated.MethodsNinety-one patients, who were diagnosed and treated for nosocomial meningitis at a single tertiary hospital in Daegu, Korea for 10 years, were included. Medical record and electronic laboratory data on the causative pathogens, antibiotics used, and outcomes were retrospectively investigated.ResultsCoagulase-negative Staphylococcus (40.9%) was the most common pathogen, followed by Acinetobacter (32.5%). Both were cultured as a single organism in cerebrospinal fluid (CSF). Seventy-eight patients (85.7%) had infections related to external ventricular drains (EVD). The most common empirical antibiotics were extended-spectrum beta-lactam antibiotics plus vancomycin (35/91, 38.6%). Of the 27 patients who had cultured Acinetobacter in CSF, 10 (37%) were given the wrong empirical antibiotic treatment. Seven of the 27 patients (26.9%) with cultured Acinetobacter died, and overall mortality of the 91 patients was 16.5%. In the multivariate analysis, the presence of combined septic shock (p < 0.001) and a persistent EVD state (p = 0.021) were associated with a poor prognosis.ConclusionsAcinetobacter is one of the leading pathogens of nosocomial meningitis and may lead to inadequate coverage of empiric antibiotic therapy due to increasing resistance. An EVD should be removed early in cases of suspected nosocomial meningitis, and carbapenem might be required for the poor treatment response.

Aminoglycoside resistance in clinical Escherichia coli and Klebsiella pneumoniae isolates from Western Norway

Resistance to gentamicin in Escherichia coli from blood culture has shown an increase over the past decade in Norway. This study was done to investigate aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae in Western Norway. The material included 49 blood culture isolates which had shown aminoglycoside resistance collected during 2000-2009. To investigate co-resistance to alternative antibiotics and dynamics involved in aminoglycoside resistance 67 isolates (mostly from urine) exhibiting resistance to both aminoglycosides and extended spectrum beta-lactam antibiotics were also included. MIC values were obtained for amikacin, gentamicin, kanamycin, netilmicin, streptomycin and tobramycin and all isolates were screened using PCR for aac(3)-II and aac(6')-Ib, encoding aminoglycoside modifying enzymes. Resistance to ≥3 aminoglycosides was found in 92% of the isolates and 60.3% showed resistance to gentamicin, netilmicin, tobramycin and kanamycin. Amikacin resistance was low. Co-resistance to ciprofloxacin was found in 88% of the isolates with gentamicin resistance. aac(3)-IIa/c was found in 79.3% and aac(6')-Ib in 37.9% of the isolates and 28.4% harboured both genes. aac(6')-Ib-cr, possibly contributing to ciprofloxacin resistance was found mostly in extended spectrum beta-lactamase producers. The aminoglycoside resistance patterns indicate co-existence of multiple resistance mechanisms. The use of ciprofloxacin and third generation cephalosporins is likely to have contributed to the increase in aminoglycoside resistance in Norway.

Bacterial Load of Fresh Vegetables and Their Resistance to the Currently Used Antibiotics in Saudi Arabia

This study was carried out to describe the bacterial load and the occurrence of some disease-causing enteric bacteria on raw vegetables sold in Saudi markets. The study further aimed to analyze antibiotic resistance rates, production of extended-spectrum beta lactamase, and plasmid carriage among bacterial population of raw vegetables. Results revealed that none of them contained Bacillus cereus, Salmonella, and Escherichia coli O157:H7. However, Staphylococcus aureus and Shigella were detected in 11.8% and 4.4% of the samples, respectively. The bacterial loads ranged from 3 to 8 log(10) CFUg(-1) for aerobic bacteria and 1 to 4 log(10) CFUg(-1) for coliforms as well as Enterobacteriaceae. The isolates exhibited resistance in decreasing order for ampicillin (76.5%), cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline (17.2%), fluoroquinolones (17.2%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Maximum resistance to extended-spectrum beta-lactam antibiotics occurred in 14.8% of isolates and the production of extended-spectrum beta-lactamase was achieved by 2.3% of isolates. Multiple resistances to four or more antimicrobial agents along with plasmid with varied sizes were documented. These investigations indicate the occurrence of antibiotic resistance and plasmid carriage among bacterial isolates populating raw vegetables.

Prevalence of extended spectrum beta lactamase and AmpC beta lactamase producers among Escherichia coli isolates in a tertiary care hospital in Jaipur

Resistance to broad spectrum beta lactams, mediated by extended spectrum beta lactamase (ESbetaL) and AmpC betaL enzymes is an increasing problem worldwide. Presence of these in clinical infections can result in treatment failure if one of the second or third generation cephalosporins is used. Therefore, it is recommended that any ESbetaL-producing organism according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria can be reported as resistant to all extended spectrum beta lactam antibiotics regardless of the susceptibility test results. In this study, a total of 250 Escherichia coli (E. coli) isolates were subjected to Double disc test and AmpC disc test for the detection of ESbetaL- and AmpC betaL-producing strains, respectively. Prevalence of ESbetaL- and AmpC betaL-producing strains among E. coli isolates, over a 3-month-period in the hospital-based population of Jaipur, was 64.80% (162/250). AmpC betaL producers were 24.00% (60/250) and co-existence of ESbetaL and AmpC betaL was detected in 8.00% (20/250) of the isolates.

A secondary drug resistance mutation of TEM-1 beta -lactamase that suppresses misfolding and aggregation

In Gram-negative bacteria, TEM-1 beta-lactamase provides the major mechanism of plasmid-mediated beta-lactam resistance. Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum beta-lactam antibiotics (e.g., ceftazidime) and beta-lactamase inhibitors (e.g., clavulanic acid). Some of the variant enzymes are more efficient at catalyzing beta-lactam hydrolysis, whereas others are more resistant to inhibitors. M182T is a substitution observed in both types of variant TEM-1 beta-lactamases. This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity. An engineered core mutation, L76N, which diminishes the periplasmic beta-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation. Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta-lactamase. Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance.

Analysis of a carbapenem-hydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein.

Carbapenems such as imipenem are extended-spectrum beta-lactam antibiotics, which are not hydrolyzed by the beta-lactamases commonly found in Enterobacteriaceae. Here we report a gene encoding a carbapenemase, which was cloned from the chromosome of a clinical isolate of Enterobacter cloacae, strain NOR-1, into pACYC184 plasmid in Escherichia coli. Unlike all the sequenced carbapenemases, which are class B metallo-beta-lactamases, the mature protein (NmcA) is a class A serine beta-lactamase. NmcA shares the highest amino acid identity (50%) with the extended-spectrum class A beta-lactamase MEN-1 from E. coli. In the opposite orientation from the nmcA promoter, an overlapping and divergent promoter was detected, along with an open reading frame, which encoded a 33.5-kDa protein (NmcR). The NmcR amino acid sequence displays homology with LysR-type transcriptional regulatory proteins, including the conserved residues near its N terminus within a helix-turn-helix motif. Deletion of nmcR resulted in decreased carbapenem resistance and a loss of beta-lactamase inducibility, demonstrating a positive role of NmcR in NmcA expression.

Cloning and expression in Enterobacteriaceae of the extended-spectrum beta-lactamase gene from a Pseudomonas aeruginosa plasmid.

An extended-spectrum beta-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum beta-lactam antibiotics. We cloned the pMS350 beta-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum beta-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the beta-lactamase and MICs of various beta-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other beta-lactam antibiotics, with the exception of aztreonam.

Effect of clavulanic acid on activity of beta-lactam antibiotics in Serratia marcescens isolates producing both a TEM beta-lactamase and a chromosomal cephalosporinase

An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 beta-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum beta-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-1-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal beta-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these beta-lactam antibiotics, at least modest increases in chromosomal beta-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal beta-lactamase, much higher levels of chromosomal beta-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate.

Mechanisms of resistance to extended-spectrum cephalosporins.

The extended-spectrum beta-lactam antibiotics represent a major advance in antimicrobial therapy, but a newly recognized resistance mechanism may rapidly negate their usefulness. Judicious clinical use will prolong their efficacy.