Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in an Enterobacter sp.

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.

Cefepime, piperacillin-tazobactam, and the inoculum effect in tests with extended-spectrum beta-lactamase-producing Enterobacteriaceae.

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.

Activity of faropenem against resistant isolates of Streptococcus pneumoniae.

An in vitro study of the activity of 9 agents against 181 US pediatric isolates of Streptococcus pneumoniae identified imipenem and faropenem as the most active agents. Overall, faropenem was the most potent oral agent inhibiting 98% of isolates at 1 microg/mL.

Potential impact of the VITEK 2 system and the Advanced Expert System on the clinical laboratory of a university-based hospital.

A study was designed to assess the impact of the VITEK 2 automated system and the Advanced Expert System (AES) on the clinical laboratory of a typical university-based hospital. A total of 259 consecutive, nonduplicate isolates of Enterobacteriaceae members, Pseudomonas aeruginosa, and Staphylococcus aureus were collected and tested by the VITEK 2 system for identification and antimicrobial susceptibility testing, and the results were analyzed by the AES. The results were also analyzed by a human expert and compared to the AES analyses. Among the 259 isolates included in this study, 245 (94.6%) were definitively identified by VITEK 2, requiring little input from laboratory staff. For 194 (74.9%) isolates, no inconsistencies between the identification of the strain and the antimicrobial susceptibility determined by VITEK 2 were detected by the AES. Thus, no input from laboratory staff was required for these strains. The AES suggested one or more corrections to results obtained with 65 strains to remove inconsistencies. The human expert thought that most of these corrections were appropriate and that some resulted from a failure of the VITEK 2 system to detect certain forms of resistance. Antimicrobial phenotypes assigned to the strains by the AES for beta-lactams, aminoglycosides, quinolones, macrolides, tetracyclines, and glycopeptides were similar to those assigned by the human expert for 95.7 to 100% of strains. These results indicate that the VITEK 2 system and AES can provide accurate information in tests for most of the clinical isolates examined and remove the need for human analysis of results for many. Certain problems were identified in the study that should be remediable with further work on the software supporting the AES.

Activity of oral agents against pediatric isolates of Streptococcus pneumoniae.

An in vitro study of the activity of 10 oral agents against 153 pediatric isolates of Streptococcus pneumoniae identified moxifloxacin and levofloxacin as the most active agents regardless of penicillin or macrolide susceptibility. Moxifloxacin inhibited all strains at 0.25 microg/ml and was 8- to 16-fold more potent than levofloxacin.

Controversies about extended-spectrum and AmpC beta-lactamases.

Many clinical laboratories have problems detecting extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. Failure to detect these enzymes has contributed to their uncontrolled spread and sometimes to therapeutic failures. Although National Committee for Clinical Laboratory Standards recommendations exist for detecting ESBL- producing isolates of Escherichia coli and Klebsiella spp., no recommendations exist for detecting ESBLs in other organisms or for detecting plasmid-mediated AmpC beta-lactamases in any organisms. Clinical laboratories need to have adequate funding, equipment, and expertise to provide a rapid and clinically relevant antibiotic testing service in centers where these resistance mechanisms are encountered.

The role of the microbiology laboratory in an era of increasing antibiotic resistance.

In medical institutions where antibiotic resistance is a problem, the situation can be likened to a state of warfare. Antibiotic-resistant bacteria are the enemy, the bodies of infected patients are the battlefield, and the health professionals are the "good guys." In this context, microbiology laboratories can be considered as an intelligence system that has the responsibility to detect the presence of the bacterial enemy, its strengths, its weaknesses (usually by antibiotic susceptibility testing), and any new weapons the enemy may have developed (especially new antibiotic-resistance mechanisms). It is fundamental that if the laboratory cannot provide prompt, accurate, and useful intelligence, it will be much harder to defeat the enemy. That is, without a good laboratory service it will be difficult to choose appropriate therapies for infected patients, and also to prevent the spread of pathogens that are resistant to multiple antibiotics. The laboratory role is critical. Thus, it is worthwhile to review the status of clinical microbiology laboratories for their ability to detect resistance, especially new types of resistance. Laboratories that cannot accurately detect antibiotic-resistant bacteria make it difficult for health professionals to make correct decisions and to respond appropriately.

Version 2000: the new beta-lactamases of Gram-negative bacteria at the dawn of the new millennium.

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.

Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center.

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.

Clinical efficacy of mesalamine in the treatment of the spondyloarthropathies.

OBJECTIVE: Sulfasalazine (SSZ) has been found to have beneficial effects in the treatment of patients with spondyloarthropathy (SpA) with active disease. The effectiveness of SSZ is limited by both idiosyncratic and dose related side effects when treating SpA. Mesalamine is a drug used to treat inflammatory bowel disease. Case reports have suggested potential efficacy in SpA. We investigated the efficacy and safety of the Pentasa formulation of mesalamine in treating SpA. METHODS: Thirty patients with SpA as defined by the European Spondylarthropathy Study Group were recruited from a rheumatology specialty clinic. All subjects began 16 week open label therapy with mesalamine 1500 mg/day. Dose escalation for lack of efficacy was permitted after 8 weeks of therapy. Clinical, physical, and laboratory data were collected at baseline, at 8 weeks, and at the conclusion of the study at 16 weeks. RESULTS: Twenty-nine of the 30 patients completed the study. There was clinically and statistically significant improvement in all clinical measures (morning stiffness, night awakenings, quality of sleep, severity of stiffness, severity of pain, Dougados functional index, patient and physician global indices). Joint counts and enthesitis counts improved, but measures of axial flexibility did not. Erythrocyte sedimentation rate and C-reactive protein also improved over the duration of the study. CONCLUSION: In this population of patients with SpA, mesalamine was well tolerated in the dose range 1500 to 4000 mg/day. Improvements in clinical, physical, and laboratory measures indicate the efficacy of mesalamine in treating SpA.

Ability of the VITEK 2 advanced expert system To identify beta-lactam phenotypes in isolates of Enterobacteriaceae and Pseudomonas aeruginosa.

The Advanced Expert System (AES) was used in conjunction with the VITEK 2 automated antimicrobial susceptibility test system to ascertain the beta-lactam phenotypes of 196 isolates of the family Enterobacteriaceae and the species Pseudomonas aeruginosa. These isolates represented a panel of strains that had been collected from laboratories worldwide and whose beta-lactam phenotypes had been characterized by biochemical and molecular techniques. The antimicrobial susceptibility of each isolate was determined with the VITEK 2 instrument, and the results were analyzed with the AES to ascertain the beta-lactam phenotype. The results were then compared to the beta-lactam resistance mechanism determined by biochemical and molecular techniques. Overall, the AES was able to ascertain a beta-lactam phenotype for 183 of the 196 (93.4%) isolates tested. For 111 of these 183 (60.7%) isolates, the correct beta-lactam phenotype was identified definitively in a single choice by the AES, while for an additional 46 isolates (25.1%), the AES identified the correct beta-lactam phenotype provisionally within two or more choices. For the remaining 26 isolates (14.2%), the beta-lactam phenotype identified by the AES was incorrect. However, for a number of these isolates, the error was due to remediable problems. These results suggest that the AES is capable of accurate identification of the beta-lactam phenotypes of gram-negative isolates and that certain modifications can improve its performance even further.

Activity of moxifloxacin against pathogens with decreased susceptibility to ciprofloxacin.

A panel of 279 clinical isolates of Gram-positive cocci and Gram-negative bacilli with varying levels of resistance to ciprofloxacin were analysed for susceptibility to moxifloxacin, ciprofloxacin, ofloxacin and nalidixic acid. Moxifloxacin was eight- to 32-fold more potent than ciprofloxacin and ofloxacin against staphylococci and Streptococcus pneumoniae, and equivalent to eight-fold more potent against enterococci. Although ciprofloxacin was intrinsically more potent than the other quinolones against highly susceptible Gram-negative isolates, the percentages of Gram-negative isolates susceptible to 1 mg/L of moxifloxacin or ciprofloxacin, or 2 mg/L of ofloxacin were 78%, 80% and 76%, indicating in-vitro equivalence of the agents against a collection that included isolates with diminished quinolone susceptibility. Staphylococci were analysed according to their ciprofloxacin susceptibility status. As ciprofloxacin resistance increased to high levels, all quinolone MICs increased, but moxifloxacin and ofloxacin MICs increased less than ciprofloxacin MICs. In mutational studies moxifloxacin inhibited more mutants (69%) at a concentration of 1 mg/L than did ciprofloxacin (63%) at 1 mg/L or ofloxacin at 2 mg/L (31%). The study indicated that moxifloxacin is more potent than ciprofloxacin and ofloxacin against Gram-positive pathogens, may be comparable in activity against less quinolone-susceptible Gram-negative isolates (other than Pseudomonas aeruginosa), and is less affected than ciprofloxacin by mechanisms responsible for increasing quinolone resistance in staphylococci.

Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC.

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.

In-vitro activity of levofloxacin against Streptococcus pneumoniae with various levels of penicillin resistance.

This in-vitro study was designed to compare the activity of levofloxacin with that of ciprofloxacin, ofloxacin, erythromycin, penicillin, amoxycillin, loracarbef, cefaclor, cefpodoxime, ceftriaxone, trimethoprim-sulphamethoxazole, clindamycin and vancomycin against a collection of 202 Streptococcus pneumoniae isolates (56% susceptible to penicillin, 34% intermediate, 10% resistant). The isolates (60% nasopharyngeal, 40% middle ear) were obtained from otherwise healthy children at child care centres in urban and rural Nebraska, and at a paediatric clinic in rural Kentucky. MICs were determined by NCCLS agar dilution methodology using an inoculum of 10(4) cfu/spot. Using NCCLS breakpoints, the percentage of penicillin-intermediate and -resistant strains susceptible to the evaluable agents were, respectively, as follows: levofloxacin (99%, 100%), ofloxacin (87%, 100%), erythromycin (52%, 65%), ceftriaxone (93%, 25%), trimethoprim-sulphamethoxazole (7%, 0%), clindamycin (93%, 100%) and vancomycin (100%, 100%). Without NCCLS interpretive criteria, no conclusions could be made concerning the susceptibility of penicillin-intermediate and -resistant strains to the other study drugs. All beta-lactam antibiotics, erythromycin and trimethoprim-sulphamethoxazole were less active against penicillin-resistant strains, indicating that these may be suboptimal agents for empirical therapy for suspected S. pneumoniae infections in these patient populations. However, levofloxacin, ofloxacin, clindamycin and vancomycin were equally active against penicillin-susceptible and -resistant strains. These data suggest that the efficacy of levofloxacin should be examined in both adult and paediatric S. pneumoniae infections involving body sites where levofloxacin concentrations > 2 mg/L can be achieved safely.

Use of microdilution panels with and without beta-lactamase inhibitors as a phenotypic test for beta-lactamase production among Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter freundii, and Serratia marcescens.

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.

The effects of increasing levels of quinolone resistance on in-vitro activity of four quinolones.

A panel of 266 clinically isolated Gram-positive cocci and Gram-negative bacilli with varying levels of resistance to ciprofloxacin were analysed for susceptibility to Du-6859a, ciprofloxacin, ofloxacin, temafloxacin and nalidixic acid. Staphylococci were divided into ciprofloxacin-susceptible, moderately resistant and highly resistant subgroups. Du-6859a was the most potent quinolone against all taxa. As ciprofloxacin resistance increased to high levels, MICs of all quinolones increased but Du-6859a MICs increased least, and ciprofloxacin MICs increased most. Less susceptible single-step mutants were selected from 80% of 15 representative clinical isolates exposed to ciprofloxacin, 71% of isolates exposed to temafloxacin, 67% of isolates exposed to Du-6859a and 53% of isolates exposed to ofloxacin. Du-6859a inhibited more mutants (67%) at a concentration of 1 mg/L than did the other quinolones (26-43%) at their susceptible breakpoints. Du-6859a was the most rapidly bactericidal quinolone in time-kill studies with Enterococcus faecalis and Enterococcus faecium. This study indicated that Du-6859a is more potent than the comparator quinolones, is less affected by the mechanisms responsible for high-level quinolone resistance and may be less likely to select resistant mutants if it has a susceptible breakpoint of 1 mg/L.

Can results obtained with commercially available MicroScan microdilution panels serve as an indicator of beta-lactamase production among escherichia coli and Klebsiella isolates with hidden resistance to expanded-spectrum cephalosporins and aztreonam?

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.