Isolates Of Klebsiella Spp Research Articles

Prevalence and characterization of extended-spectrum beta-lactamase production in clinical isolates of Klebsiella spp.

Extended-spectrum beta-lactamases (ESBLs) are most prevalent in Klebsiella pneumoniae. This organism is frequently isolated from clinical specimens and can cause septicemia, pneumonia or urinary tract infection. We investigated a spread ofKlebsiella spp. isolates producing ESBL in a university hospital of Sanandaj-Iran.Over one year period, a total of 48 K. pneumoniae isolates, were examined by double disk tests and PCR methods. Ten isolates were defined as ESBLs. The ESBL producer isolates was more resistant to selected antibiotics than ESBL negative isolates. The most frequent ESBL type was CTX-M. This is the first report ofKlebsiella spp. isolates producing ESBL in Sanandaj hospitals. Production of ESBLs by K. pneumoniae is a widespread nosocomial problem. Knowledge about their prevalence is essential to guide towards appropriate infection control and antibiotic management strategies. Key words: Extended-spectrum β-lactamase (ESBL), hospital, Klebsiella spp.

<i>In Vitro</i> Activity of Cephalexin against Community-Acquired Urinary <i>Escherichia coli</i>, <i>Klebsiella</i> and <i>Proteus</i> species Isolates

Problem statement: Resistance among urinary tract pathogens to conventional antibiotics has been considered a health problem worldwide. The aim of this study was to evaluate the in vitro activity of cephalexin on urinary enterobacterial isolates. Approach: Patients with community-acquired urinary tract infections were microbiologically investigated. Urine specimens were collected from each patient. The specimens were cultured on bacteriological media. The isolated urinary pathogens were subjected to antibiotic sensitivity test using modified Kirby-Bauer Disc diffusion method. The method was judged by the National Committee for Clinical Laboratories Standards (NCCLS). Results: A total of 140 enterobacterial urinary isolates were recovered. Of these 93 (66.4%) were identified as E. coli, 20 (14.3%) as Klebsiella spp. and 12 (8.6%) as Proteus spp. The results showed that urinary enterobacterial pathogens had a high rate of resistance to cephalexin amounting to 75 (53.6%), whereas susceptible and intermediate rates were 34 (24.3%) and 31 (22.1%) respectively. Conclusion: It was concluded that E. coli are the predominant organisms isolated from subjects sampled. High resistance to cephalexin activity was noticed with Klebsiella spp isolates.

Antimicrobial Pattern and Clonal Dissemination of Extended-Spectrum Beta-Lactamase Producing <i>Klebsiella Spp</i> Isolates

Problem statement: Gram-negative pathogens harboring ESBLs have caused numerous outbreaks of infections and are becoming an increasing therapeutic problem in many countries. The incidence of ESBL-producing strains among clinical isolates has been steadily increasing over the past years resulting in limitations of therapeutic option. The focus of this study was to examine the molecular epidemiology of ESBL-producing Klebsiella spp, investigate the susceptibility of Klebsiellae spp producing ESBLs towards non-beta-lactam antibiotics in the different seasons, identify the various clonal types of ESBL-producing K.pneumoniae and detect the dominant ESBL clonal types. Approach: Clinical isolates of Klebsiella spp were identified during the period March 2007- April 2008. ESBLs production identified by phenotypic and genotypic methods. MLST was performed for dissemination of ESBLs producing K. pneumoniae. Results: The findings showed that 51.6% of K.pneumoniae were produces ESBLs. 35.8, 21.2 and 38.7% of K. pneumoniae producing ESBLs were resistant to amikacin, ciprofloxacin and cotrimoxazol, respectively. It was found that 40 and 27.3% of K.oxytoca producing ESBLs were resistant to cotrimoxazol and amikacin, respectively. The findings reflected that ESBLs existed in 73% of K. oxytoca. The results showed that the frequency of blaSHV, blaTEM and blaCTX-M due to K.pneumoniae producing ESBLs were 87.5, 12.4 and 24.8%, respectively. Of the eleven K. oxytoca producing ESBLs, 100% blaSHV were obtained. Based on the nucleotide variations of the five genetic loci, twenty-five different STs could be identified among thirty K.pneumoniae producing ESBLs isolates. Among the STs shared by multiple isolates, the most frequently encountered were 14, 16 and ST18. Conclusion: In conclusion, the percentage of K.oxytoca producing ESBLs was higher than K.pneumoniae producing ESBLs. Generally, K.penomoniae produces more ESBLs in winter and fall than in the other seasons.

Prevalence and Antimicrobial Susceptibility of Enterobacteriaceae Collected from Patients with Wounds at Kenyatta National Hospital, Nairobi, Kenya

Prevalence and sensitivity trends of Enterobacteriaceae isolated from septic wounds were determined through a prospective cross sectional study. One hundred and fifteen specimens isolated from in-patients in the Department of Orthopaedics were studied and antibiotic sensitivity testing performed using the Kirby and Bauer disc diffusion technique. The prevalence of organisms isolated was Proteus spp (33.9%), Eschericia coli (13.2%), Klebsiella spp (7.9%), Alcaligenes (1.7%), Citrobacter freundii (0.9%), Serratia spp (0.9%) and Acinetobacter baumanii (0.9%). The sensitivity rate of ceftriaxone, ceftazidime and ciprofloxacin was above 70% in all cases. Co-amoxiclav, gentamicin, cefuroxime, minocycline and piperacillin showed moderate to high activity. Klebsiella spp isolates portrayed high resistance against several drugs. The sensitivity patterns showed that empirical prescribing should be discouraged since the organisms appear to be developing resistance against commonly used antibiotics.

Detection of Extended-Spectrum β-Lactamases by Using Boronic Acid as an AmpC β-Lactamase Inhibitor in Clinical Isolates of Klebsiella spp. and Escherichia coli

We evaluated highly sensitive methods using boronic acid (BA) to detect extended-spectrum beta-lactamase (ESBL) production. A total of 182 clinical isolates of Klebsiella spp. (n = 118) and Escherichia coli (n = 64) were analyzed: 62 harbored only ESBLs, 80 harbored both ESBLs and plasmid-mediated AmpC beta-lactamases (pAmpCs), and 40 harbored only pAmpCs. The CLSI confirmatory test detected all isolates that produce only ESBLs but detected 85% of isolates that produce both enzymes. When a >/=5-mm increase in the zone diameter of either the cefotaxime (CTX) or the ceftazidime (CAZ) disk in the presence of both clavulanic acid (CA) and BA was considered to be a positive result, the test detected all isolates that harbor ESBLs (+/- pAmpCs) but showed frequent false-positive results (50%) for isolates that produce only pAmpCs. Meanwhile, when a >/=3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs (+/- pAmpCs) and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The latter new interpretive guideline has enhanced detection of ESBLs in clinical isolates of Klebsiella spp. and Escherichia coli and allowed detection of an ESBL even when potentially masked by a pAmpC.

Evaluation of β-Lactamase Inhibitors in Disk Tests for Detection of Plasmid-Mediated AmpC β-Lactamases in Well-Characterized Clinical Strains of Klebsiella spp

The diagnostic utility of the AmpC beta-lactamase inhibitors LN-2-128, 48-1220, and Syn 2190 in combination with cefotetan (CTT) or cefoxitin in a disk test for the detection of clinical isolates of Klebsiella spp. producing plasmid-mediated AmpC beta-lactamases (pAmpCs) was evaluated. The combination of Syn 2190 and CTT had a sensitivity of 91%, a specificity of 100%, and a reproducibility of 100% and showed the best potential of using an inhibitor for detection of Klebsiella spp. producing pAmpCs.

Extended-Spectrum ß-Lactamases in isolates of <i>Klebsiella</i> spp and <i>Escherichia coli</i> from Lagos, Nigeria

Objectives: To investigate the occurrence of Extended-Spectrum Beta-Lactamase (ESBL) enzymes in isolates of Klebsiella spp and E.coli from various health institutions in Lagos. Methods: From December 2000 to October 2001, 356 isolates of Klebsiella spp (200) and Escherichia coli (156) were investigated for ESBL production. These isolates were obtained from the Microbiology laboratories of 7 hospitals in Lagos metropolis. Results: Seventy four (20.8%) were found to be ESBL-producers using the double-disk test (DD test). Amongst the ESBL-producers, Klebsiella pneumoniae (60.8%) was the most represented followed by E.coli (31.1%). All strains positive to DD tests were confirmed for the carriage ESBL genes by polymerase Chain Reaction (PCR amplification). A total of 54 (73%) strains were positive for PCR, of which 12 (22.2%) were of the TEM-type, 26 (48.2%) of SHV-type and 16 (29.6%) of both types. About 27% of the ESBL-producers could not be amplified by PCR using TEM and SHV primers. Isoelectric focusing analysis showed the presence of 2 enzsymes with pI of 7.0 and 8.5 which were non-TEM and non-SHV enzymes in addition to enzymes with pl of 8.2, 7.6 and 5.8 which were of the TEM and SHV-type. A very low number, 10 (13.5%) of the ESBL-producers transferred ESBL resistance by conjugation experiment suggesting that ESBL-resistance in Lagos hospitals, may be generally chromosomal or carried by non-self transferable plasmids. Conclusion: The production of Extended-Spectrum Beta-Lactamase enzymes by clinical isolates compromise the efficacy of beta-lactam antibiotics, particularly the extended-spectrum cephalosporins which are widely used in the treatment of serious gram-negative infections. There are now increasing clinical evidence that shows the importance of detecting strains producing this enzyme in our environment to prevent their spread. Key Words:Extended-Spectrum Beta-Lactamase, Klebsiella spp, E. coli. Nig. J. Health and Biomed. Sciences Vol.2(2) 2003: 53-60

Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States.

A sample of 752 resistant Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated beta-lactamase involved. Fifty-nine percent of the K. pneumoniae, 24% of the K. oxytoca, and 44% of the E. coli isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type beta-lactamase were found in 8.5% of the K. pneumoniae samples, 6.9% of the K. oxytoca samples, and 4% of the E. coli samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 beta-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 beta-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 beta-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two E. coli strains produced CMY-2, and one K. pneumoniae strain produced DHA-1 beta-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC beta-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum beta-lactamases. A beta-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC beta-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum beta-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of Klebsiella spp. and E. coli, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-beta-lactams.

Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing

His study was performed to compare the methods of detection and to estimate the prevalence of extendedspectrum β-lactamases (ESBL) among Klebsiella spp and E. coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K. pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clinicas de Porto Alegre, using Kirby-Bauer zone diameter (KB), double-disk diffusion (DD), breakpoint for ceftazidime (MIC CAZ), increased zone diameter with clavulanate (CAZ/CAC) and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC). Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E. coli), contrasting with all the other methods (p < 0.05). The confirmatory methods (DD, MIC CAZ, CAZ/CAC and MIC CAZ/CAC) showed a range of ESBL production from 8 to 13% for E. coli and from 33 to 40% for Klebsiella species. Therefore, the KB method was useful only as a screening method as it provided several false positive results. Molecular typing of 17 ESBL K. pneumoniae indicated that the isolates had no clonal relation. We found a good correlation among the confirmatory methods for ESBL detection although the methods which evaluate inhibition of the β-lactamase by clavulanate appeared to be more specific. The high prevalence of ESBL Klebsiella in our hospital is probably due to individual selection of resistant strains rather than the transmission of a common strain.

Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit.

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.

Mechanisms of decreased susceptibility to cefpodoxime in Escherichia coli.

Cefpodoxime is one of five antimicrobial agents recommended by the National Committee for Clinical Laboratory Standards for screening isolates of Klebsiella spp. and Escherichia coli for extended-spectrum beta-lactamase (ESBL) production. In a prior study, we noted that among 131 E. coli isolates for which the MIC of at least one extended-spectrum cephalosporin (ESC) or aztreonam was > or =2 micro g/ml (suggesting the presence of ESBL production), there were 59 isolates (45.0%) for which the MIC of cefpodoxime was 2 to 4 micro g/ml (i.e., a positive ESBL screening test), but the MICs of ceftazidime, cefotaxime, and ceftriaxone were < or =1 micro g/ml (below the ESBL screening breakpoint). Thus, the results appeared to be false-positive ESBL screening tests. These 59 isolates were divided into five phenotypic groups based on the susceptibility patterns of the organisms to a variety of beta-lactam agents and further characterized. The first group (32 isolates) all produced a TEM-1 beta-lactamase, and changes in the major outer membrane proteins were detected in representative strains. The second group (18 isolates) lacked bla(TEM) but showed a number of porin changes; some also showed a modest elevation in production of the AmpC chromosomal beta-lactamase. In the third phenotypic group (seven isolates) all expressed an OXA-30 beta-lactamase. Some also harbored altered porins. The two remaining phenotypes each had a distinct pattern of porin changes with or without beta-lactamase production. These data indicate that several factors are associated with decreased susceptibility to cefpodoxime in E. coli, but none of the mechanisms are related to ESBL production. Current screening methods produced false-positive ESBL results for these isolates. Such isolates should not be classified as containing ESBLs, nor should interpretations of ESCs or aztreonam susceptibility be changed to resistant on test reports for these isolates.

In vitro evaluation of broad-spectrum β-lactams tested in medical centers in Korea: role of fourth-generation cephalosporins

Levels of resistance to the “third-generation” cephalosporins among isolates of clinical bacteria in Korea have been increasing at a rapid rate. This study evaluated the activity of cefepime, a “fourth-generation” cephalosporin, and six other broad-spectrum β-lactam antimicrobials (cefpirome, ceftazidime, ceftriaxone, imipenem, piperacillin/tazobactam [4 μg/mL fixed concentration], oxacillin) against 404 isolates of clinical bacteria from Korea. Susceptibility profiles of each isolate were established using the Etest (AB BIODISK, Solna, Sweden) method of susceptibility testing. Only the carbapenem imipenem was > 90% effective in inhibiting each of the species tested ( Escherichia coli, Klebsiella, spp., Citrobacter spp., Enterobacter spp., indole-positive Proteae, Serratia spp., Acinetobacter spp., Pseudomonas aeruginosa, and oxacillin-susceptible staphylococci). Imipenem was followed by cefepime > cefpirome > piperacillin/tazobactam > ceftazidime > ceftriaxone in overall rank order of usable spectrum against the isolates tested. Extended spectrum β-lactamase producing phenotypes were much more prevalent among the Klebsiella spp. (48.8%) than the E. coli (5.0%) isolates. Cefepime was much more active than cefpirome, 95.1% susceptible as compared with 70.7% susceptible, against the 41 isolates of Klebsiella spp. The results of this study corroborates findings from earlier studies with levels of resistance to the broad-spectrum β-lactams in Korea continuing to rise indicating the need for intervention strategies.

Survey of antimicrobial activity of four commonly used third generation cephalosporins tested against recent bacterial isolates from ten American medical centers, and assessment of disk diffusion test performance

Over 2,000 clinical isolates from ten American medical centers were tested for susceptibility to cefotaxine, ceftriaxone, ceftizoxime, and ceftazidime by both broth microdilution and disk diffusion methods. Typically resistant (e.g. enterococci) and highly susceptible (e.g. streptococci) isolates whowed no change in susceptibility patterns compared to previous surveys. Pseudomonas aeruginosa and Stenotrophomonas maltophilia exhibited significant decreases in susceptibility to these cephalosporins. Among Escherichia coli and Klebsiella spp isolates, two to three percent were resistant to one of the four drugs whereas they were very susceptible to the other three. Of the four antibiotics, the cefotaxime disk diffusion test results correlated best with the microdilution results. With the other three drugs the disk diffusion test yielded 1 to 9% more susceptible test results and 1 to 20% fewer resistant test results than broth microdilution when testing gram negative bacilli. The clinical significance of such discrepancies is not known, but the impact on antibiotic susceptibility surveys and antibiogram comparisons could be significant.

Klebsiella species: antimicrobial susceptibilities, bactericidal kinetics, and in vitro inactivation of beta-lactam agents.

In vitro properties of 19 antimicrobial agents were tested with 56 isolates of Klebsiella spp. The aminoglycosides and the new beta-lactam compounds cefotaxime and moxalactam were the most inhibitory drugs tested. Chloramphenicol, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole were moderately active, whereas piperacillin, mezlocillin, and furazlocillin were ineffective against 25% of the isolates. Gentamicin was the only agent tested that was uniformly bactericidal in time-kill experiments with drug concentrations of four times the minimal inhibitory concentration. In combination studies with gentamicin, moxalactam and furazlocillin each increased the rate of bacterial killing for three of five isolates as compared with gentamicin alone, whereas chloramphenicol significantly retarded the rate of bacterial killing for the same number of strains. Furazlocillin was completely inactivated after 24 h of incubation with each of five selected strains. The inactivation of moxalactam, cefoxitin, and cephalothin was 36, 56, and 72%, respectively. In all instances in which these four agents were inactivated to levels below the minimal bactericidal concentration, there was accelerated growth after initial inhibition. However, regrowth also occurred in three instances in which drug levels were higher than the minimal bactericidal concentration. Retesting after drug exposure revealed a 4- to 32-fold rise in the minimal inhibitory concentration and minimal bactericidal concentration in two of these isolates.

In vitro studies on netilmicin, a new aminoglycoside antibiotic.

Netilmicin, a semisynthetic derivative of sisomicin, was tested in vitro against 600 clinical bacterial isolates. At a concentration of 1.56 mug/ml, over 90% of gram-negative bacilli were inhibited. Netilmicin was substantially more active against isolates of Serratia marcescens and Enterobacter spp. than gentamicin, sisomicin, tobramycin, or amikacin. Isolates of Staphylococcus aureus (both penicillin G susceptible and resistant) were quite susceptible to netilmicin. Most isolates of Klebsiella spp. and Serratia spp. and some of the isolates of Pseudomonas aeruginosa that were resistant to gentamicin proved to be susceptible to netilmicin.

Pirbenicillin, a new semisynthetic penicillin with broad-spectrum activity.

Pirbenicillin is a new semisynthetic penicillin which inhibited 67% of isolates of Proteus aeruginosa tested in our laboratory, 93% of P. mirabilis, 31% of Enterobacter spp., 41% of Serratia spp., and 58% of Escherichia coli at a concentration of 6.25 mug/ml. Its activity appeared to be inoculum dependent and it was virtually inactive against 10(7) inocula of P. aeruginosa. It was more active than carbenicillin or ticarcillin, but less active than BL-P1654 against P. aeruginosa. Carbenicillin and ticarcillin appeared to be more active than pirbenicillin against Proteus spp., but pirbenicillin was active against some isolates of Klebsiella spp.