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The genetic determinants involved in reduced susceptibility to third-generation cephalosporins and aztreonam were identified in ten canine Enterobacter isolates associated with opportunistic infections in three veterinary hospitals in Brisbane, Australia. All isolates were evaluated by a combination of phenotypic (broth microdilution and disc susceptibility, modified disc diffusion and IEF) and genotypic (PFGE, plasmid analysis, Southern blot hybridization, bacterial conjugation, PCR and sequencing) methods to investigate genetic relatedness and to identify plasmid-mediated resistance genes, in particular beta-lactamase genes responsible for extended-spectrum cephalosporin resistance. The ten canine isolates were genotypically diverse based on PFGE and belonged to either Enterobacter cloacae or Enterobacter hormaechei on the basis of 16S rRNA gene sequence analysis. Plasmid profiles were also diverse. Nine isolates contained a transmissible blaSHV-12-carrying plasmid (approximately 140 kb) that also conferred resistance to chloramphenicol, gentamicin, spectinomycin, tetracycline, trimethoprim and sulfonamides. In all plasmid-mediated extended-spectrum beta-lactamase (ESBL)-producing isolates including transconjugants, blaSHV-12 was shown to reside in a approximately 6.5 kb plasmid fragment. The remaining isolate that was not an ESBL producer possessed an AmpC beta-lactamase gene (blaCMY-2) on a approximately 93 kb transmissible plasmid. This plasmid did not contain any other antimicrobial resistance genes. Additional plasmid-mediated beta-lactamases identified in some isolates included bla(TEM) and blaOXA-10. This is the first report of canine Enterobacter isolates containing transmissible plasmid-mediated blaSHV-12 and blaCMY-2 resistance genes. Therefore, Enterobacter isolated from opportunistic infections in dogs may be an important reservoir of plasmid-mediated resistance genes, which could potentially be spread to other members of the Enterobacteriaceae.

A strain of an Enterobacter sp. with reduced susceptibility to imipenem, which produced a plasmid-mediated class A carbapenem-hydrolyzing enzyme, KPC-2 beta-lactamase, was isolated from a patient with sepsis at a Boston hospital. This is the first report of the production of a plasmid-encoded KPC-2 beta-lactamase by an Enterobacter sp.

Four isolates of Klebsiella pneumoniae obtained from patients at a Maryland medical centre exhibited reduced susceptibility to carbapenems and were found to produce the novel, class A, plasmid-mediated, carbapenem-hydrolysing enzyme, KPC-2. This enzyme has 99% identity with the plasmid-mediated, carbapenem-hydrolysing enzyme KPC-1, reported previously in a North Carolina K. pneumoniae isolate. The KPC-2-producing isolates were either susceptible or intermediate to imipenem and meropenem, unlike the KPC-1-producing isolate, which was resistant to these agents. Detection of KPC-2 may be a problem for clinical laboratories because in this study it was associated with positive extended-spectrum beta-lactamase (ESBL) confirmation tests (clavulanate-potentiated activities of ceftriaxone, ceftazidime, cefepime and aztreonam). Therefore, a failure to recognize the significance of reduced carbapenem susceptibility in the isolates that remained susceptible to imipenem or meropenem could have resulted in the isolates being incorrectly identified as ESBL producers.

There is little information about the clinical effectiveness of cefepime and piperacillin-tazobactam in the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing pathogens. Some inferences have been drawn from laboratory studies, which have usually involved only one or a few strains of ESBL-producing Klebsiella pneumoniae or Escherichia coli that produced only a limited range of ESBLs. Such studies are indirect, sometimes conflicting, indicators of efficacy. To extend previous laboratory findings, a study was designed to investigate organism-drug interactions by determining the in vitro activities of eight parenteral beta-lactam agents against 82 clinical and laboratory strains of Klebsiella, Escherichia, Enterobacter, Citrobacter, Serratia, Morganella, and Proteus species that produced 22 different ESBLs, alone or in combination with other beta-lactamases. Activities were determined in broth microdilution MIC tests using standard and 100-fold-higher inocula. An inoculum effect, defined as an eightfold or greater MIC increase on testing with the higher inoculum, was most consistently detected with cefepime, cefotaxime, and ceftriaxone and least frequently detected with meropenem and cefoteten. Piperacillin-tazobactam was intermediate between these two groups of agents. Although the inoculum effect is an in vitro laboratory phenomenon, if it has any predictive value in identifying increased risk of therapeutic failure in serious infections, these results support suggestions that cefepime may be a less-than-reliable agent for therapy of infections caused by ESBL-producing strains.

The extended-spectrum beta-lactamase, TEM-63, was identified in three separate genera of South African isolates: Proteus mirabilis, Klebsiella pneumoniae, and Escherichia coli. This paper describes identification of the gene in these isolates and compares relative rates of hyrolysis between TEM-63 and other known ceftazidimases.

beta-lactamases of Gram-negative bacteria are evolving dynamically. New developments include the production of enzymes with novel substrate profiles, reduced susceptibility to beta-lactamase inhibitors, and the simultaneous production of multiple types of beta-lactamases. The changes represent evolutionary upgrades which provide modern pathogens with a greater potential to resist beta-lactam antibiotics and cause formidable therapeutic, infection control, and diagnostic challenges. This review is a clinically oriented outline of recent developments in the beta-lactamase production of Gram-negative bacteria.

AmpC beta-lactamases are cephalosporinases that confer resistance to a wide variety of beta-lactam drugs and that may thereby create serious therapeutic problems. Although reported with increasing frequency, the true rate of occurrence of AmpC beta-lactamases in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis remains unknown. We tested a total of 1,286 consecutive, nonrepeat isolates of these three species and found that, overall, 45 (3.5%) yielded a cefoxitin zone diameter less than 18 mm (screen positive) and that 16 (1.2%) demonstrated AmpC bands by isoelectric focusing. Based on the species, of 683 E. coli, 371 K. pneumoniae, and 232 P. mirabilis isolates tested, 13 (1.9%), 28 (7.6%), and 4 (1.7%), respectively, demonstrated decreased zone diameters and 11 (1.6%), 4 (1.1%), and 1 (0.4%), respectively, demonstrated AmpC bands. Cefoxitin resistance was transferred for all but 8 (E. coli) of the 16 AmpC producers. We also describe a three-dimensional extract test, which was used to detect phenotypically isolates that harbor AmpC beta-lactamase. Of the 45 cefoxitin-resistant isolates, the three-dimensional extract test accurately identified all 16 AmpC producers and 28 of 29 (97%) isolates as non-AmpC producers. Interestingly, most (86%) isolates in the latter group were K. pneumoniae isolates. These data confirm that, at our institution, E. coli, K. pneumoniae, and P. mirabilis harbor plasmid-mediated AmpC enzymes.

Organisms encoding multiple antibiotic resistance genes are becoming increasingly prevalent. In this report we describe a multiply resistant Klebsiella pneumoniae which possesses at least five different beta-lactamase genes. Isoelectric focusing, polymerase chain reaction and restriction fragment length polymorphism analysis identified TEM-1, multiple SHVs, OXA-9 and a plasmid-mediated ampC, beta-lactamase. Furthermore, Southern analysis and conjugation experiments established that most of the resistance genes were encoded on one large transferable plasmid. This report demonstrates the complexity of multiply resistant organisms.

Over the past decade, a number of new beta-lactamases have appeared in clinical isolates of Enterobacteriaceae that, unlike their predecessors, do not confer beta-lactam resistance that is readily detected in routine antibiotic susceptibility tests. Because optimal methodologies are needed to detect these important new beta-lactamases, a study was designed to evaluate the ability of a panel of various beta-lactam antibiotics tested alone and in combination with beta-lactamase inhibitors to discriminate between the production of extended-spectrum beta-lactamases, AmpC beta-lactamases, high levels of K1 beta-lactamase, and other beta-lactamases in 141 isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens possessing well-characterized beta-lactamases. The microdilution panels studied contained aztreonam, cefpodoxime, ceftazidime, cefotaxime, and ceftriaxone, with and without 1, 2, and 4 microg of clavulanate per ml or 8 microg of sulbactam per ml and cefoxitin and cefotetan with and without 8 microg of sulbactam per ml. The results indicated that a minimum panel of five tests would provide maximum separation of extended-spectrum beta-lactamase high AmpC, high K1, and other beta-lactamase production in Enterobacteriaceae. These included cefpodoxime, cefpodoxime plus 4 microg of clavulanate per ml, ceftazidime, ceftriaxone, and ceftriaxone plus 8 microg of sulbactam per ml. Ceftriaxone plus 2 microg of clavulanate per ml could be substituted for cefpodoxime plus 4 microg of clavulanate per ml without altering the accuracy of the tests. This study indicated that tests with key beta-lactam drugs, alone and in combination with beta-lactamase inhibitors, could provide a convenient approach to the detection of a variety of beta-lactamases in members of the family Enterobacteriaceae.

A study was designed to determine if an isogenic panel of Escherichia coli strains containing many different beta-lactamases could be used for the preliminary screening of a large number of beta-lactam agents to identify which might be most useful in the development of a definitive test for specific beta-lactamases found among the members of family Enterobacteriaceae. The susceptibilities of 46 strains, comprising the isogenic panel, to expanded-spectrum cephalosporins, cephamycins, and aztreonam were determined in the presence and absence of beta-lactamase inhibitors in broth microdilution tests. The results indicated that strains producing extended-spectrum beta-lactamases (ESBLs) could be distinguished from strains producing other Bush-Jacoby-Medeiros functional group 2 or group 1 beta-lactamases. For strains producing group 1 beta-lactamases, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate did not reduce the MICs more than fourfold. For strains producing group 2 enzymes other than ESBLs, cefpodoxime and ceftazidime MICs were < or = 2 micrograms/ml. With a single exception (ceftazidime for the strain producing SHV-3), among strains producing ESBLs, cefpodoxime and ceftazidime MICs were > or = 4 micrograms/ml and addition of clavulanate reduced the MICs by more than eightfold. Cephamycins could also be used to discriminate between strains producing group 1 beta-lactamases and ESBLs, since only the former required cefotetan concentrations as high as 8 micrograms/ml or cefoxitin concentrations of > 16 micrograms/ml for inhibition. Other cephalosporins provided some discrimination between the various beta-lactamase producers, although they were not as reliable as either cefpodoxime or ceftazidime. These results indicate the utility of an isogenic panel for identification of candidate drugs among many for further testing with clinical isolates of the family Enterobacteriaceae to determine the best agents for detection of specific beta-lactamases in this family.

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of beta-lactamases that may confer resistance to newer beta-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized beta-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of beta-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum beta-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of >/=2 microg/ml were obtained only for isolates producing ESBLs or AmpC beta-lactamases. Cefoxitin MICs of >16 microg/ml were obtained for all strains producing AmpC beta-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of >/=4 microg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of >/= 8 microg/ml were obtained for seven of eight producers of AmpC beta-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain beta-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible beta-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum beta-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum beta-lactamases was evaluated by using the double-disk test, and the beta-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum beta-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum beta-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum beta-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.

A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing. SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL. Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88). In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle. These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.

Enterobacter spp. are becoming increasingly frequent nosocomial pathogens, and beta-lactam-resistant strains are on the increase, especially among isolates recovered from intensive care units. Therefore, a study was designed to characterize the beta-lactamases produced by 80 isolates of E. cloacae, E. aerogenes, E. taylorae, E. gergoviae, E. sakazakii, E. asburiae, and E. agglomerans by induction studies, spectrophotometric hydrolysis assays, and isoelectric focusing. The ability of broth microdilution and disk diffusion susceptibility tests to detect resistance to 16 beta-lactam antibiotics among these species was also assessed. All species except E. agglomerans, E. gergoviae, and some isolates of E. sakazakii were found to produce a Bush group 1 cephalosporinase that was expressed inducibly or constitutively at high levels. In addition, some strains also produced a Bush group 2 beta-lactamase. In comparisons of broth microdilution and disk diffusion tests, disk diffusion tests failed to detect resistance in 1 of 25 isolates resistant to aztreonam and 2 of 30 isolates resistant to ceftazidime. These results indicate that species of Enterobacter can possess a variety of beta-lactamases that are responsible for beta-lactam resistance in this genus and that the disk diffusion test may occasionally miss resistance in some strains.

A three-phase analysis of the Vitek ESBL test and a double-disk (2 disk) test was performed to assess their ability to detect extended-spectrum beta-lactamases (ESBLs) in members of the family Enterobacteriaceae. In the first two phases involving detection of ESBLs in 157 stains processing well-characterized beta-lactamases, sensitivity and specificity were found to be 99.5 and 100%, respectively, for the Vitek ESBl test and 98.1 and 99.4%, respectively, for the 2-disk test. In the third phase, in which the ability of each test to detect ESBLs in 295 clinical isolates was assessed, there was only one false positive (Vitek ESBL test). Across all three phases, the Vitek ESBL test was found to be much easier to perform than the 2-disk test. The latter also involved subjective interpretation of results. There were a total of 176 Escherichia coli and 157 Klebsiella pneumoniae isolates and less than 40 isolates of each of 14 other species evaluated. In a supplemental study of Klebsiella oxytoca, an organism possessing a chromosomal beta-lactamase similar to an ESBL, the Vitek ESBL test was found to be capable of detecting hyperproduction of this enzyme in strains of this species as well. These data indicate that the Vitek ESBL test is reliable for the detection of ESBLs in E. coli and K. pneumoniae, the two species in which ESBLs are most common, and of hyperproduction of the K. oxytoca beta-lactamase, a situation which engenders a level of resistance to this species similar to that seen with ESBLs.

A clinical isolate of Proteus mirabilis was found to produce a new variant of the TEM-10 beta-lactamase gene. This is the first report of TEM-10 production by P. mirabilis and the first report of extended-spectrum beta-lactamase production by an isolate of this species recovered in the United States.

A study was done to determine the genetic locus responsible for altered expression of AmpC beta-lactamase in Enterobacter cloacae 1194E and several mutants derived from E. cloacae 029. These phenotypes were defined by units of enzyme activity found in sonic extracts of cells before and after induction with cefoxitin and included (units uninduced/units induced) the wild-type (7/219), high-level constitutive (10,911/10,862), temperature-sensitive (at 30 degrees C 82/706 and at 42 degrees C 5,031/6,020), and hyperinducible (19/1,688) phenotypes. When the ampD region of each E. cloacae strain was cloned and introduced into an ampD mutant Escherichia coli strain, the altered phenotypes were found to reside within this locus. Furthermore, transformants containing wild-type ampD were poorly inducible at 42 degrees C while those with high-level constitutive or hyperinducible ampD were unaffected by temperature. Since the source of ampD was the only variable in these E. coli transformants, these results suggested that ampD encodes a protein that is involved in sensing the inducer. To test this possibility, the responses to different inducers of E. coli transformants containing various ampD regions were assessed. In the presence of wild-type ampD, transformants responded equally to cefoxitin and cefotetan, regardless of temperature. In the presence of temperature-sensitive ampD, induction by cefotetan was similar to that by cefoxitin at 30 degrees C but greater than that by cefoxitin at 42 degrees C. These results suggest that ampD encodes a protein involved in induction of AmpC beta-lactamase in E. cloacae.

An inhibitor-based characterization system which allowed the identification of beta-lactamases after isoelectric focusing on polyacrylamide gels was developed. This system, using potassium clavulanate and oxacillin, distinguished type I chromosomally mediated enzymes from other beta-lactamases of gram-negative bacteria.