ABSTRACT We applied in vitro evolution to an Escherichia coli strain containing blaCTX-M-2 and obtained 10 independent mutant blaCTX-M-2 alleles that confer elevated resistance to ceftazidime (MIC ≥ 32 μg/ml) but lost the ability to confer resistance to cefepime. All alleles had a Pro-to-Ser substitution at position 167.

To determine whether confirmatory tests for extended-spectrum beta-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >/=2 micro g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >/=3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >/=5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one beta-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 micro g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 micro g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC beta-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >/=8 micro g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.

ABSTRACT We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32μ g/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla SHV and bla TEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The bla KPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.

ABSTRACT We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.

TEM-71, a novel extended-spectrum beta-lactamase from a Klebsiella pneumoniae clinical isolate, had an isoelectric point of 6.0 and a substrate profile showing preferential hydrolysis of cefotaxime over ceftazidime. It differed from TEM-1 by two substitutions, Gly238Ser and Glu240Lys, and was under the control of the strong P4 promoter.

Strains of Staphylococcus aureus with reduced susceptibility to glycopeptides have been reported from Japan, the United States, Europe, and the Far East. Although isolates with homogeneous resistance to vancomycin (MICs = 8 microg/mL) continue to be rare, there are increasing reports of strains showing heteroresistance, often with vancomycin MICs in the 1-4 microg/mL range. Most isolates with reduced susceptibility to vancomycin appear to have developed from preexisting methicillin-resistant S. aureus infections. Many of the isolates with reduced susceptibility to glycopeptides have been associated with therapeutic failures with vancomycin. Although nosocomial spread of the vancomycin-intermediate S. aureus (VISA) strains has not been observed in U.S. hospitals, spread of VISA strains has apparently occurred in Japan. Broth microdilution tests held a full 24 hours are optimal for detecting resistance in the laboratory; however, methods for detecting heteroresistant strains are still in flux. Disk-diffusion tests, including the Stokes method, do not detect VISA strains. The Centers for Disease Control and Prevention and other groups have issued recommendations regarding appropriate infection control procedures for patients infected with these strains.

ABSTRACTAKlebsiella pneumoniaeisolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence ofblaSHVandblaTEMgenes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia colishowed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniaecarbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,blaKPC-1, was cloned inE. coliand shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescensS6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parentK. pneumoniaestrain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance inK. pneumoniaestrain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.

ABSTRACTExtended-spectrum β-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs inKlebsiella pneumoniae, Klebsiella oxytoca, andEscherichia coli. To evaluate the confirmation protocol, we tested 139 isolates ofK. pneumoniaethat were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were ≥2 μg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of ≥5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by ≥3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were ≥32 μg/ml, suggesting either the presence of an AmpC-type β-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a β-lactamase with a pI of ≥8.3 suggestive of an AmpC-type β-lactamase; 6 of the 7 isolates were shown by PCR to contain bothampC-type andblaOXAgenes. The IEF profiles of the remaining 16 isolates showed a variety of β-lactamase bands, all of which had pIs of ≤7.5. All 16 isolates were negative by PCR with multiple primer sets forampC-type,blaOXA, andblaCTX-Mgenes. In summary, 83.5% of theK. pneumoniaeisolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained β-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained β-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

ABSTRACT Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-β-lactams. A consistent reduction in the MICs of oxyimino-β-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum β-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single β-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of ablaSHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a blaSHV-specific probe indicated that a blaSHV gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this blaSHV-related gene revealed that it differs from blaSHV-1 at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

ABSTRACT We tested 16 erythromycin-resistant clinical isolates of S. aureus, recovered from patients hospitalized in the United States from 1958 to 1969, for the presence of ermA,ermB, and ermC by using PCR. Fifteen of 16 isolates contained at least one copy of ermA; the remaining isolate, which was also clindamycin resistant, containedermB. Eight of the 15 isolates harboring ermA, all of which were inducible, contained a single copy of the gene in the chromosome, while the remaining seven isolates had two copies of the gene. ermB was plasmid encoded and mediated constitutive resistance to erythromycin.

BACKGROUND:In addition to single-hospital outbreaks, interhospital transmission of extended-spectrum beta-lactam-resistant (ESBLR) Klebsiella pneumoniae has been suspected in some reports. However, these studies lacked sufficient epidemiological information to confirm such an occurrence.METHODS:We reviewed the surveillance data reported to the National Nosocomial Infections Surveillance (NNIS) System during 1986 to 1993 for K pneumoniae isolates and their susceptibility to either ceftazidime, cefotaxime, ceftriaxone, or aztreonam. Pulsed-field gel electrophoresis (PFGE) was used to study available ESBLR K pneumoniae isolates.RESULTS:Among 8,319 K pneumoniae isolates associated with nosocomial infections, 727 (8.7%) were resistant or had intermediate-level resistance to at least one of these antibiotics. One hospital (hospital A) accounted for 321 isolates (44.2%) of ESBLR K pneumoniae. During 1986 to 1993, the percentage of K pneumoniae isolates that were ESBLR increased from 0 to 57.7% in hospital A, from 0 to 35.6% in NNIS hospitals 0 to 20 miles from hospital A (area B), and from 1.6 to 7.3% in NNIS hospitals more than 20 miles from hospital A, including hospitals located throughout the United States. Analysis of PFGE restriction profiles showed a genetic relationship between a cluster of isolates from hospital A and some isolates from one hospital in area B, and consecutive admission in these two hospitals was confirmed for two patients from whom isolates were available.CONCLUSIONS:These data provide evidence of interhospital transmission of ESBLR K pneumoniae in one region of the United States and stress the interrelationship between hospitals when trying to control antimicrobial resistance.

We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin-resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same.

WHITTINGTON, W. L. BA; RICE, R. J. MD; BIDDLE, J. W. MS; KNAPP, J. S. PHD Author Information

Neisseria gonorrhoeae isolates were studied to determine their patterns of antimicrobial susceptibility and possible chemotherapeutic implications. Of 370 consecutive isolates, 32 (8.7%) were penicillinase-producing N. gonorrhoeae (PPNG). The remaining 338 were subjected to disk-diffusion tests, and those apparently resistant to penicillin, tetracycline, or spectinomycin were tested by an agar-dilution method. The dilution test showed that 5.4% (20/370) were penicillin-resistant, non-PPNG strains, of which 100%, 90%, and 45% were also resistant to tetracycline, cefoxitin, and erythromycin, respectively. No resistance to spectinomycin or ceftriaxone was demonstrated, although there was an association between minimum inhibitor concentrations (MICs) of penicillin of greater than or equal to 1.0 microgram/ml and increased MICs of ceftriaxone. The overall incidence of penicillin resistant isolates, including PPNG, was 14.1% (52/370). Of the 20 penicillin-resistant, non-PPNG strains, all were also resistant to tetracycline, and another 21 exhibited tetracycline resistance but were sensitive to penicillin. The in-vitro data suggested that: (1) neither penicillin, tetracycline, nor cefoxitin were acceptable drugs for routine treatment of gonorrhea in our population during the study period; (2) spectinomycin and ceftriaxone continue to demonstrate adequate in-vitro activity against N. gonorrhoeae despite increasing in-vitro resistance to penicillin; and (3) non-plasmid-mediated resistance to penicillin may predict future resistance to ceftriaxone.

Journal Article Frequency and Distribution in the United States of Strains of Neisseria gonorrhoeae with Plasmid-Mediated, High-Level Resistance to Tetracycline Get access Joan S. Knapp, Joan S. Knapp Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Please address requests for reprints to Dr. Joan S. Knapp, Center for Infectious Diseases, Neisseria Research Laboratory, Sexually Transmitted Diseases Laboratory Program, Building 1, Room 3254, Centers for Disease Control, Atlanta, Georgia 30333. Search for other works by this author on: Oxford Academic PubMed Google Scholar Jonathan M. Zenilman, Jonathan M. Zenilman Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar James W. Biddle, James W. Biddle Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar Goldie H. Perkins, Goldie H. Perkins Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar Wallis E. DeWitt, Wallis E. DeWitt Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar Myrtle L. Thomas, Myrtle L. Thomas Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar Steve R. Johnson, Steve R. Johnson Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar Stephen A. Morse Stephen A. Morse Sexually Transmitted Diseases Laboratory Program, Center for Infectious Diseases, and the Epidemiology Research Branch, Center for Prevention Services, Centers for Disease Control, Atlanta, Georgia Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 155, Issue 4, April 1987, Pages 819–822, https://doi.org/10.1093/infdis/155.4.819 Published: 01 April 1987 Article history Received: 09 June 1986 Revision received: 05 September 1986 Published: 01 April 1987

Recently, strains of Neisseria gonorrhoeae have been isolated which are highly resistant to tetracycline (MICs of 16 to 64 micrograms/ml). This resistance was due to the acquisition of the resistance determinant tetM, a transposon-borne determinant initially found in the genus Streptococcus and more recently in Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella vaginalis. In N. gonorrhoeae, the tetM determinant was located on a 25.2-megadalton plasmid. This plasmid arose from the insertion of tetM into the 24.5-megadalton gonococcal conjugative plasmid. The tetM determinant could be transferred to suitable recipient strains of N. gonorrhoeae by both genetic transformation and conjugation.

Between January 1983 and October 1984, 446 cases of infection due to chromosomally mediated resistance in Neisseria gonorrhoeae (CMRNG) were reported in 23 states. Eighty percent were detected as primary penicillin or ampicillin treatment failures. Gonococcal isolates were submitted from 175 (40%) for confirmation of resistance, susceptibility testing, gonococcal strain typing using monoclonal antibodies specific for outer membrane Protein I, and auxotyping. All were typed as Protein I serogroup IB (WII/WIII), and the majority were proline or prototrophic auxotypes. All were resistant in vitro to less than 1 microgram/ml of either penicillin or tetracycline. Comparing CMRNG with penicillinase-producing Neisseria gonorrhoeae (PPNG), we found that CMRNG were significantly more resistant to tetracycline and erythromycin, but PPNG were more resistant to penicillin (P less than .01). Because of increasing reports of gonococcal resistance in the United States, improved surveillance of clinical and laboratory resistance is needed in support of control and treatment recommendations for gonorrhea.