Survey of Extended-Spectrum β-Lactamases in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae: Prevalence of TEM-52 in Korea

Two hundred ninety isolates of Escherichia coli were investigated for the production of extended-spectrum beta-lactamases (ESBLs). Fourteen (4.8%) of the 290 strains were found to produce ESBLs. Each of the 14 strains produced one or two ESBLs, as follows: 10 strains produced TEM-52, 1 strain produced SHV-2a, 1 strain produced SHV-12, 1 strain produced a CMY-1-like enzyme, and 1 strain expressed SHV-2a and a CMY-1-like enzyme. Another two strains for which the MICs of ceftazidime and cefoxitin were high, were probable AmpC enzyme hyperproducers. Because of the high prevalence of TEM-52 in E. coli isolates, we further investigated the TEM-type ESBLs produced by Klebsiella pneumoniae in order to observe the distribution of TEM-52 enzymes among Enterobacteriaceae in Korea. All TEM enzymes produced by 12 strains of K. pneumoniae were identified as TEM-52. To evaluate the genetic relatedness among the organisms, ribotyping of TEM-52-producing E. coli and K. pneumoniae was performed. The ribotyping profiles of the organisms showed similar but clearly different patterns. In conclusion, TEM-52 is the most prevalent TEM-type ESBL in Korea.     

Identification of Plasmid-Mediated AmpC β-Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results

The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum β-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC β-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC β-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.     

Molecular Epidemiology of Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Isolates in a District Hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum β-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

Nosocomial Outbreak of Klebsiella pneumoniae Producing SHV-5 Extended-Spectrum β-Lactamase, Originating from a Contaminated Ultrasonography Coupling Gel

Klebsiella pneumoniae resistant to ceftazidime was isolated from six adult women and two neonates hospitalized between July and November 1993 in the Department of Obstetrics and Gynecology of Boucicaut Hospital (Paris, France). The epidemiological investigation revealed a notably short delay (less than 48 h) between admission and contamination of the six adults and peripartum transmission to the neonates. The only environmental source of ceftazidime-resistant K. pneumoniae was the ultrasonography coupling gel used in the emergency room. Phenotypic (biotyping and antibiotyping) and genotypic (plasmid profile and pulsed-field gel electrophoresis) analysis of all the clinical isolates indicated the spread of a single strain. It produced SHV-5 and TEM-1 β-lactamases, as demonstrated by isoelectric focusing and gene sequencing. The risk of cross-contamination in ultrasonography procedures is usually low and had not been associated so far with bacteria producing an extended-spectrum β-lactamase (ESBL). Furthermore, this is the first time an epidemic of an SHV-5 ESBL-producing member of the family Enterobacteriaceae has been reported from a French hospital.     

Practical Approach for Detection and Identification of OXA-10-Derived Ceftazidime-Hydrolyzing Extended-Spectrum β-Lactamases

A practical approach to detect and identify ceftazidime-hydrolyzing extended-spectrum mutants of OXA-10 β-lactamase is presented. Large numbers of bacteria were screened by colony hybridization, a 720-bp part of bla_OXA was amplified by PCR from the hybridization-positive isolates, and the products were digested by PvuII and HaeIII.     

βlasEN: Microdilution Panel for Identifying β-Lactamases Present in Isolates of Enterobacteriaceae

A dried investigational use-only microdilution panel named betalasEN (a short named derived from the panel's purpose, to identify beta-lactamases in Enterobacteriaceae) containing 10 beta-lactam drugs with and without beta-lactamase inhibitors was developed to identify beta-lactamases among clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri, Citrobacter freundii group, Enterobacter spp., and Serratia marcescens. The MICs obtained with a collection of 383 organisms containing well-characterized beta-lactamases were used to develop numeric codes and logic pathways for computerized analysis of results. The resultant logic pathways and betalasEN panel were then used to test and identify beta-lactamases among 885 isolates of Enterobacteriaceae recovered in cultures obtained at six different hospital laboratories across the United States. beta-Lactamases present in 801 (90.5%) of the 885 isolates were identified by betalasEN by using the existing logic pathways and codes or after minor modifications were made to the existing codes. The 84 strains that gave codes that betalasEN could not identify were collected, reidentified, and retested by using betalasEN. Three strains had been misidentified, 54 strains gave different codes upon repeat testing that could be identified by betalasEN, and 27 strains repeated new codes. The beta-lactamases in these strains were identified, and the new codes were added to the betalasEN logic pathways. These results indicate that betalasEN can identify clinically important beta-lactamases among most isolates of Enterobacteriaceae. The results also show that good quality control and attention to proper performance of the tests are essential to the correct performance of betalasEN.     

Protective Roles of γδ T Cells and Interleukin-15 in Escherichia coli Infection in Mice

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.     

Identification of Immunodominant Regions within the C-Terminal Cell Binding Domain of Intimin α and Intimin β from Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) strains are a common cause of infantile diarrhea in developing countries. EPEC strains induce a characteristic attaching and effacing (A/E) lesion on epithelial cells. A/E lesion formation requires intimin, an outer membrane adhesin protein. The cell-binding activity of intimin is localized at the C-terminal 280 amino acids of the polypeptide (Int280). So far, four distinct Int280 types (α, β, γ, and δ) have been identified. The aim of this study was to identify immunodominant regions within the Int280α and Int280β domains. Recombinant DNA was used to construct and express overlapping polypeptides spanning these domains. Rabbit anti-Int280 antisera and human colostral immunoglobulin A were reacted with these polypeptides in Western blots and enzyme-linked immunosorbent assays. The results obtained with the rabbit antisera showed the presence of two separate immunodominant regions which are common to both Int280α and Int280β. The first localized within the N-terminal region of Int280, and the second localized between amino acids 80 and 130. The results with the human colostra revealed one reactivity pattern against the Int280α fragments but two different reactivity patterns against the Int280β domain.     

Characterization of Extended-Spectrum β-Lactamase-Producing Salmonella typhimurium by Phenotypic and Genotypic Typing Methods

During 1994, 10 isolates of extended-spectrum beta-lactamase-producing Salmonella typhimurium were recovered from children transferred to our hospital from two different centers. Two additional isolates were recovered from two nurses from one of these centers. The aim of this study was to determine if there is any relationship between these isolates. The characterization was done by phenotypic and genotypic methods: biotyping, phage typing, antibiotic susceptibility pattern determination, plasmid analysis, ribotyping (by the four endonucleases EcoRI, SmaI, BglII, and PvuII), pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and randomly amplified polymorphic DNA (RAPD) pattern determination (with the three primers 217 d2, B1, and A3). The same biotype, the same serotype, and an identical antibiotype were found. All isolates were resistant to oxyimino-beta-lactams, gentamicin, tobramycin, and sulfamethoxazole-trimethoprim. All isolates showed an indistinguishable pattern by ribotyping and very similar patterns by PFGE and RAPD. The overall results indicated the spread of a closely related strain of S. typhimurium in children and nurses.     

Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat

β-Lactamases continue to be the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum β-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum β-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated β-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these β-lactamases will present a host of therapeutic challenges as we head into the 21st century.     

Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Strains Causing Nosocomial Outbreaks of Infection in the United Kingdom

Representative isolates from 10 distinct extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The β-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 β-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum β-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum β-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.     

Induction of Thymus-Derived γδ T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both γδ and αβ T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of γδ T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing γδ T cells were detected. γδ T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of γδ T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like αβ T cells. γδ T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal γδ and αβ T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. γδ T cells induced by the B subunit are Th2-type cells derived from the thymus. These γδ T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of αβ T cells.     

Detection of SHV-Type Extended-Spectrum Beta-Lactamase in Enterobacter Isolates

One hundred four Enterobacter isolates were tested by standard CLSI disk diffusion methods for detecting extended-spectrum beta-lactamases (ESBLs) and with cefepime-clavulanate disk combinations. SHV-12 was produced by 8.7% of isolates. The cefepime-clavulanate combination provided 88% sensitivity and 91% specificity for the detection of SHV-12 ESBL.Detection of extended-spectrum beta-lactamase (ESBL) production in Enterobacter spp. has not been undertaken by most clinical laboratories due to a lack of recommendations from the Clinical and Laboratory Standards Institute (CLSI) and potential interference in test interpretation from high-level production of an AmpC beta-lactamase (5). This inability to readily identify ESBLs in pathogens that potentially coproduce AmpC beta-lactamase and an ESBL presents a concern for providers considering cefepime therapy for these patients, since there is an inoculum effect with ESBLs that affects all cephalosporins (2, 6, 7, 13, 16). Detection of an ESBL could lead to selection of a carbapenem for therapy rather than a cephalosporin like cefepime due to a potentially poorer clinical response (4, 11, 14, 17), whereas Enterobacter isolates that produce only AmpC have been treated reliably with cefepime (12).Enterobacter isolates recovered from blood cultures at the University Health System in San Antonio, TX, between 1 October 2004 and 31 December 2007 were examined in this study. These represented the first isolates from each patient and were nonrepetitive, with the exception of one patient, from whom two different Enterobacter spp. were identified. Initial identification and susceptibility testing were performed using a Vitek 2 instrument (GN13 cards; bioMerieux, Durham, NC) and/or standard CLSI disk diffusion methodologies (3). All isolates underwent ESBL phenotypic confirmatory disk testing utilizing the cefotaxime- and ceftazidime-clavulanate combinations as described by CLSI for Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis (3). In addition, cefepime disks (30 μg) were tested and zone diameters were recorded in a comparison with cefepime disks to which 10 μg of clavulanate was added in an attempt to improve the ability to detect ESBL production in organisms producing native AmpC beta-lactamase. E. coli ATCC 25922, E. coli ATCC 35218, and K. pneumoniae ATCC 700603 were included with each day''s tests for quality control purposes. Zone diameters were recorded for the antimicrobial agent combinations with each isolate. Any isolate with a zone diameter that increased by at least 3 mm (the minimum zone difference that we felt could be reliably measured) with the addition of clavulanate for any of the three cephalosporins or for which no zone of inhibition was seen around any of the three cephalosporin disks was considered a possible ESBL producer and was subjected to further examination.Using previously described methods, those isolates meeting the screening criteria and a random sampling of 15 isolates not meeting those criteria (control group) were further examined by PCR and gene sequencing to detect possible ESBLs of the three main families, i.e., CTX-M, SHV, and TEM (10). Isoelectric focusing was performed on selected isolates to confirm that all enzymes were detected.One hundred four Enterobacter isolates were examined. Enterobacter cloacae represented 81/104 (77.9%) of those tested, with the remaining 23 (22.1%) being Enterobacter aerogenes. Sixteen percent (n = 17) of isolates were identified as either having a zone diameter change of ≥3 mm or greater or no zone of inhibition around any of the three cephalosporin disks (study group). Ten isolates in the study group had a zone diameter change of ≥5 mm for one or more of the three cephalosporins. Based upon PCR and sequencing, nine (8.7%) of the isolates harbored an ESBL; all harbored a single enzyme, SHV-12. Eight of the nine (89%) SHV-12-producing strains were E. cloacae. No CTX-M or TEM ESBLs were identified in the study group. In addition, no isolates in the control group were found to have an ESBL following molecular characterization.Sensitivity and specificity were evaluated for each of the disk diffusion tests at each of the zone diameter changes (Table ​(Table1).1). Sensitivity analyses revealed that both ceftazidime and cefepime exhibited excellent sensitivity for detection of SHV-12 if a zone diameter change of >3 mm was employed as opposed to the usual ≥5-mm change advocated by the CLSI for other organisms. Specificity was highest overall in the cefotaxime arm (100%), but poor sensitivity (66%) limits the applicability of this substrate for the detection of ESBL in Enterobacter spp. The cefepime-clavulanate combination at a zone diameter change of ≥3 mm exhibited the highest combination of sensitivity (88%) and specificity (91%) of all combinations. Unexpectedly, tests utilizing ceftazidime and ceftazidime-clavulanate also provided very good results, especially if a zone diameter change of ≥3 mm was used (sensitivity of 88%; specificity of 82%).

TABLE 1.

Sensitivity and specificity of ESBL detection based upon disk diffusion zone diameter changes with addition of clavulanate^a