Carbapenem-susceptible Isolates Research Articles

Comparative time–kill study of doxycycline, tigecycline, sulbactam, and imipenem against several clones of Acinetobacter baumannii

To assess potential alternative options for the treatment of infections caused by Acinetobacter baumannii, we performed time–kill studies of doxycycline and tigecycline using several isolates recovered from patients residing in 10 different cities in Argentina. Imipenem and sulbactam were also included for comparison purposes. Eleven isolates representing 5 distinctive clones, or isolates with different susceptibility patterns within the same clone, were selected. Tubes containing cation-supplemented Mueller–Hinton broth with and without antibiotics were seeded with a log-phase inoculum of roughly 5 × 10 5 CFU/mL. By using the viable counts determined at 2-, 4-, 6-, 8-, and 24-h intervals after inoculation, a 24-h time–kill curve was constructed for each isolate. No bactericidal activity (defined as a ≥3-log 10 CFU/mL decrease in the viable cell counts with respect to the original inoculum) was observed at any time with sulbactam (4 μg/mL) or tigecycline (1 μg/mL), whereas low bactericidal rate (18% of the isolates) was shown for doxycycline (1 μg/mL) and sulbactam (16 μg/mL) after 24 h of incubation. Doxycycline (4 μg/mL) and tigecycline (8 μg/mL) displayed bactericidal activity at 24 h of incubation against 36% and 54% of the isolates, respectively, including the carbapenem-resistant isolate. Corresponding values for imipenem (1 and 4 μg/mL) against the 10 carbapenem-susceptible isolates were 60% and 90%, respectively. The present study confirms the in vitro efficacy of imipenem against A. baumannii, suggests that doxycycline could be a suitable, cost-effective, alternative option in some instances, and sheds light on the potential role of tigecycline in the treatment of infections with this organism.

A carbapenem-susceptible Pseudomonas aeruginosa strain carrying the blaSPM gene

We studied a carbapenem-susceptible Pseudomonas aeruginosa strain that does not produce carbapenemase but carries the metallo-β-lactamase gene bla SPM identical in sequence to the gene of other fully carbapenem-resistant isolates. Carbapenem-susceptible isolates may be silent reservoirs of the bla SPM gene.

Alterations of Porin, Pumps, and Penicillin-Binding Proteins in Carbapenem Resistant Clinical Isolates of Pseudomonas aeruginosa

Carbapenem-resistant clinical isolates of Pseudomonas aeruginosa from Sweden and Norway (n=27) were characterized regarding transcription of genes encoding OprD, efflux pumps MexAB-OprM and MexCD-OprJ, as well as penicillin-binding proteins (PBP) 2 and 3. Quantification of mRNA was performed with real-time RT-PCR. Levels of mRNA in clinical isolates were compared to ATCC 27853 and average transcription levels of four carbapenem-susceptible clinical isolates of P. aeruginosa. Sequencing of oprD, pbp 2, and pbp 3 was performed in selected isolates. An efflux inhibition assay was performed with Phe-Arg-beta-naphtylamide. Most carbapenem-resistant isolates had decreased levels of oprD mRNA. Among six isolates with normal amount of oprD mRNA, half of them had significant OprD sequence alterations. Increased transcription of mexB was observed in 16 of 23 meropenem-resistant isolates, and one isolate showed mexD hyperproduction. Decreased amount of pbp 2 and pbp 3 was found in two and three isolates, respectively. Sequencing of pbp 2 and 3 revealed no amino acid changes potentially leading to conformational changes. In conclusion, OprD changes were the predominant mechanism of high-level carbapenem resistance, and increased transcription of mexB was often present in meropenem-resistant isolates. Reduced transcription of pbp 2 and 3 may contribute to the carbapenem resistance.

Risk factors and outcomes of bloodstream infections with metallo-β-lactamase-producing Acinetobacter

The spread of Gram-negative bacilli with acquired metallo-beta-lactamase (MBL) threatens the successful treatment of major nosocomial infections. The objective of this study was to evaluate the differences in the clinical characteristics of bacteremia caused by MBL-producing Acinetobacter species and MBL non-producing isolates. Two retrospective case-control studies were conducted using data on patients with Acinetobacter bacteremia, who were admitted between January 2001 and December 2005 at a 1500-bed, tertiary-care teaching hospital. Case group 1 (n=27) included patients from whom imipenem-resistant Acinetobacter was isolated in blood culture, and case group 2 (n=7) consisted of those patients from group 1 who yielded MBL-producing isolates. The control group (n=41) included patients from whom carbapenem-susceptible Acinetobacter isolates were isolated in blood culture. Multivariate analysis revealed that the independent risk factors for imipenem-resistant Acinetobacter bacteremia were neutropenia and prolonged use of carbapenem. The independent risk factors for MBL-producing Acinetobacter bacteremia were neutropenia and prolonged use of cephalosporins. The results of this study suggest that a prolonged use of cephalosporins may be associated with MBL-producing Acinetobacter bacteremia.

In vitro activity of tigecycline and comparators against carbapenem-susceptible and resistant Acinetobacter baumannii clinical isolates in Italy.

BackgroundIn a recent multi-centre Italian survey (2003–2004), conducted in 45 laboratories throughout Italy with the aim of monitoring microorganisms responsible for severe infections and their antibiotic resistance, Acinetobacter baumannii was isolated from various wards of 9 hospitals as one of the most frequent pathogens. One hundred and seven clinically significant strains of A. baumannii isolates were included in this study to determine the in vitro activity of tigecycline and comparator agents.MethodsTests for the susceptibility to antibiotics were performed by the broth microdilution method as recommended by CLSI guidelines. The following antibiotics were tested: aztreonam, piperacillin/tazobactam, ampicillin/sulbactam, ceftazidime, cefepime, imipenem, meropenem tetracycline, doxycycline, tigecycline, gentamicin, amikacin, ciprofloxacin, colistin, and trimethoprim/sulphametoxazole. The PCR assay was used to determine the presence of OXA, VIM, or IMP genes in the carbapenem resistant strains.ResultsA. baumannii showed widespread resistance to ceftazidime, ciprofloxacin and aztreonam in more than 90% of the strains; resistance to imipenem and meropenem was 50 and 59% respectively, amikacin and gentamicin were both active against about 30% of the strains and colistin about 99%, with only one strain resistant. By comparison with tetracyclines, tigecycline and doxycycline showed a higher activity. In particular, tigecycline showed a MIC90 value of 2 mg/L and our strains displayed a unimodal distribution of susceptibility being indistinctly active against carbapenem-susceptible and resistant strains, these latter possessed OXA-type variant enzymes.ConclusionIn conclusion, tigecycline had a good activity against the MDR A. baumannii strains while maintaining the same MIC90 of 2 mg/L against the carbapenem-resistant strains.

Molecular Epidemiology and Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa Isolates from Spanish Hospitals

All (236) Pseudomonas aeruginosa isolates resistant to imipenem and/or meropenem collected during a multicenter (127-hospital) study in Spain were analyzed. Carbapenem-resistant isolates were found to be more frequently resistant to all beta-lactams and non-beta-lactam antibiotics than carbapenem-susceptible isolates (P < 0.001), and up to 46% of the carbapenem-resistant isolates met the criteria used to define multidrug resistance (MDR). Pulsed-field gel electrophoresis revealed remarkable clonal diversity (165 different clones were identified), and with few exceptions, the levels of intra- and interhospital dissemination of clones were found to be low. Carbapenem resistance was driven mainly by the mutational inactivation of OprD, accompanied or not by the hyperexpression of AmpC or MexAB-OprM. Class B carbapenemases (metallo-beta-lactamases [MBLs]) were detected in a single isolate, although interestingly, this isolate belonged to one of the few epidemic clones documented. The MBL-encoding gene (bla(VIM-2)), along with the aminoglycoside resistance determinants, was transferred to strain PAO1 by electroporation, demonstrating its plasmid location. The class 1 integron harboring bla(VIM-2) was characterized as well, and two interesting features were revealed: intI1 was found to be disrupted by a 1.1-kb insertion sequence, and a previously undescribed aminoglycoside acetyltransferase-encoding gene [designated aac(6')-32] preceded bla(VIM-2). AAC(6')-32 showed 80% identity to AAC(6')-Ib' and the recently described AAC(6')-31, and when aac(6')-32 was cloned into Escherichia coli, it conferred resistance to tobramycin and reduced susceptibility to gentamicin and amikacin. Despite the currently low prevalence of epidemic clones with MDR, active surveillance is needed to detect and prevent the dissemination of these clones, particularly those producing integron- and plasmid-encoded MBLs, given their additional capacity for the intra- and interspecies spread of MDR.

R2314 Disseminated infection with Fusarium solani in an infant with congenital acute lymphoblastic leukaemia

The performance of Oxoid Brilliance™ CRE Agar (BCRE), a new chromogenic medium designed for screening of carbapenem-resistant Enterobacteriaceae, was evaluated on a collection of clinical isolates of enterobacteria (n = 175) and non-fermenters (n = 55) with known β-lactam resistance mechanisms and levels of susceptibility to carbapenems. BCRE supported the growth of 100 of 108 enterobacterial isolates that were non-susceptible to at least one carbapenem, whilst excluding 57 of the 67 carbapenem-susceptible isolates. The eight non-susceptible isolates that did not grow on BCRE were carbapenemase-producers with low carbapenem minimum inhibitory concentrations, mostly exhibiting non-susceptibility only to one carbapenem. In total, of 107 carbapenemase-producing enterobacteria that were included in the study, 16 did not grow, with most of them being either susceptible (n = 8) or intermediate-susceptible (n = 5) to carbapenems. Regarding the 10 carbapenem-susceptible enterobacteria that were not excluded by BCRE, 1 produced a carbapenemase and the rest possessed strong backgrounds of various other β-lactam resistance mechanisms. The medium allowed growth of almost all carbapenem-resistant non-fermenting isolates; nevertheless, non-fermenters were clearly differentiated from Enterobacteriaceae by colony colour and morphology.

In vitro activity of tigecycline against carbapenem-susceptible and -resistant isolates of Klebsiella spp. and Enterobacter spp.

Sir, Carbapenems are often the last active antibiotics for serious infections caused by Gram-negative opportunist pathogens. Resistance is still extremely rare among Enterobacteriaceae, but is increasingly detected in isolates of Klebsiella and Enterobacter in the UK. In a few cases it is mediated by class A or B carbapenemases, but more often results from combinations of a b-lactamase [often a CTX-M extended-spectrum b-lactamase (ESBL) in Klebsiella or an AmpC enzyme in Enterobacter] acting together with impermeability and/or increased efflux. Among the three available carbapenems, ertapenem is most affected and, hence, is also the best indicator. The public health importance of this resistance type, which prompted the UK’s first National Resistance Alert in December 2005 (http://www.hpa.org.uk/infections/ topics_az/antimicrobial_resistance/alert.htm), reflects the limited options for treating serious infections caused by such multiresistant isolates: many of which are susceptible only to polymyxins among established antibiotics. Tigecycline is a new glycylcycline with broad-spectrum activity, including against most Enterobacteriaceae except Proteeae. Previously, we noted a trend toward decreased susceptibility to tigecycline among ESBL-producing Klebsiella spp. and AmpC-producing Enterobacter spp. collected in a survey of cephalosporin-resistant isolates from south-east England, with a raised modal MIC for the Klebsiella spp. and with increased ‘trails’ of resistant isolates among both genera. Here we investigated whether this pattern was general for ESBL and AmpCproducing isolates of Klebsiella and Enterobacter submitted from 6. Bradford PA, Yang Y, Sahm D et al. CTX-M-5, a novel cefotaxime-hydrolyzing b-lactamase from an outbreak of Salmonella typhimurium in Latvia. Antimicrob Agents Chemother 1998; 42: 1980–4. 7. Edelstein M, Pimkin M, Dmitrachenko T et al. Multiple outbreaks of nosocomial salmonellosis in Russia and Belarus caused by a single clone of Salmonella enterica serovar Typhimurium producing an extended-spectrum b-lactamase. Antimicrob Agents Chemother 2004; 48: 2808–15. across the UK; we also assessed whether the in vitro activity of tigecycline was further reduced against those isolates with the permeability and efflux changes that, acting in concert with an ESBL or AmpC enzyme, can confer carbapenem resistance. Eight hundred and sixty-nine clinical isolates of Klebsiella spp. (n 1⁄4 540) and Enterobacter spp. (n 1⁄4 329) were included. These had been referred to the Antibiotic Resistance Monitoring and Reference Laboratory from UK clinical laboratories between June 2004 and July 2006 on the basis of resistance, mainly to cephalosporins. The isolates included 89 Klebsiella spp. and 65 Enterobacter spp. that were resistant to ertapenem (MIC .2 mg/L). Among ertapenem-resistant isolates, 7 Klebsiella spp. and 21 Enterobacter spp. were also resistant to imipenem; 19 Klebsiella spp. and 15 Enterobacter spp. also to meropenem. Even when not considered resistant, MICs of the latter carbapenems for these ertapenem-resistant isolates were above the modal values for the genera. Among referred carbapenemsusceptible isolates, 240 (91%) Enterobacter spp. and 380 (84%) Klebsiella spp. were resistant to cephalosporins, defined here as cefotaxime MIC .1 mg/L. Tigecycline was supplied as powder of known potency by the manufacturer (Wyeth Pharmaceuticals, Taplow, UK). MICs were determined and interpreted in accordance with BSAC and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; values 1 mg/L indicate susceptibility for Enterobacteriaceae and those .2 mg/L indicate resistance (http://www.bsac.org.uk). Statistical comparisons were performed using Fisher’s exact test (http://www.graphpad.com/ quickcalcs/contingency2.cfm). MIC distributions of tigecycline for referred carbapenemsusceptible and -resistant isolates are shown in Table 1, together with EUCAST data for susceptible wild-type populations (http:// www.srga.org/eucastwt/WT_EUCAST.htm) and data from the BSAC Bacteraemia Surveillance for 2002–2004 (http://www. bsacsurv.org.uk). Among Klebsiella spp., the modal MIC was 0.5 mg/L for referred carbapenem-resistant isolates and for the BSAC and EUCAST datasets, but was raised to 1 mg/L for referred carbapenem-susceptible isolates; the reason for this requires further investigation. The modal MIC for all groups of Enterobacter spp. was 0.5 mg/L (Table 1). Among referred carbapenem-susceptible Enterobacter spp. isolates, 205 (78%) were susceptible to tigecycline, 35 (13%) were fully resistant, and 24 (9%) had intermediate susceptibility. No differential was seen between the tigecycline susceptibility of these carbapenem-susceptible isolates and isolates in the BSAC dataset (P 1⁄4 0.37). However, referred carbapenemresistant Enterobacter spp. isolates were less often susceptible to tigecycline (32/65, 49%; P , 0.0001); with 20 (31%) fully resistant, and 13 (20%) intermediate. In contrast, there was no significant difference in the proportions of tigecycline-susceptible Klebsiella spp. isolates among referred carbapenem-susceptible (303/451; 67%) and carbapenem-resistant isolates (54/89; 60%) (P 1⁄4 0.27). However, tigecycline non-susceptibility (MICs 2 mg/L) was more frequent in both groups when compared with BSAC bacteraemia isolates (P , 0.0001). Among new agents, tigecycline is unique in having good activity against Gram-negative bacteria in general. However, these data support the observation that many cephalosporinresistant (i.e. mostly ESBL-producing) Klebsiella spp. isolates require slightly raised tigecycline MICs, but refute the hypothesis that further rises occur in this genus contingent on the Correspondence

Phenotypic Detection of Carbapenem-Susceptible Metallo-β-Lactamase-Producing Gram-Negative Bacilli in the Clinical Laboratory

Rapid detection of metallo-beta-lactamase (MBL)-producing gram-negative pathogens is critical to prevent their widespread dissemination. Thus far, no standardized phenotypic method is available, and previously reported techniques have poor sensitivity for detecting carbapenem-susceptible MBL-carrying isolates, an increasingly described phenomenon. We developed a phenotypic detection method using both a double-disk synergy test and a combined-disk test with imipenem and 292 microg EDTA on one agar plate. Genotypic confirmation was used for validation. Of the 134 clinical isolates, 84 were confirmed to carry an MBL. Of these, 51 (61%) were susceptible to at least one carbapenem, and 22 (26%) were isolated from blood. The phenotypic method correctly differentiated all MBL-producing isolates (sensitivity, 100%). Fifty-one of the 52 MBL-negative isolates were correctly differentiated (specificity, 98%). This study reports the validation of a simple and accurate MBL detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of MBL-carrying organisms, including those with susceptibility to carbapenems, is of paramount clinical importance, as it allows rapid initiation of strict infection control practices as well as therapeutic guidance for confirmed infection.

Molecular epidemiology and mechanisms of carbapenem resistance in Acinetobacter baumannii endemic in New York City.

Multidrug-resistant Acinetobacter baumannii has emerged as a serious nosocomial pathogen in certain areas. In Brooklyn, New York, citywide surveillance revealed that approximately 2 of every 3 isolates were resistant to carbapenem antibiotics. Genetic fingerprinting revealed that 2 strains accounted for 82% of these resistant isolates. Compared with carbapenem-susceptible isolates, carbapenem-resistant isolates had reduced expression of 47-, 44-, and 37-kDa outer-membrane proteins. No specific carbapenemase was found; however, carbapenem-resistant isolates expressed greater levels of a class C cephalosporinase. Although expression of penicillin-binding proteins varied among strains, no consistent pattern appeared to account for carbapenem resistance. An efflux pump, present in several strains, did not appear to contribute to carbapenem resistance. Clonal spread of carbapenem-resistant A. baumannii has occurred in hospitals in Brooklyn. The preliminary findings for a small number of strains suggest that diminished production of outer-membrane porins, together with increased expression of a class C cephalosporinase, appear to be important factors leading to carbapenem resistance in this region.

A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation

High-level carbapenem-resistant (CpmR) mutants, with MICs for imipenem and carbapenem of > 128 µg/ml, were selected in vitro from four carbapenem-susceptible (CpmS) clinical isolates of Bacteroides fragilis. The CpmS strains produced very low levels of β-lactamase activity, which was increased approx. 50- to 100-fold in the CpmR mutants. Isoelectric focussing and enzyme kinetic analysis (Km and Vrel) of the ‘carbapenemases’ from the CpmR mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584–2593) hybridized with DNA from the CpmR mutants, their CpmS parental strains as well as clinical CpmR isolates, but not from randomly chosen carbapenem-susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.

A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation

High-level carbapenem-resistant (Cpm R) mutants, with MICs for imipenem and carbapenem of > 128 μ g/ml , were selected in vitro from four carbapenem-susceptible (Cpm S) clinical isolates of Bacteroides fragilis. The Cpm S strains produced very low levels of β-lactamase activity, which was increased approx. 50- to 100-fold in the Cpm R mutants. Isoelectric focussing and enzyme kinetic analysis ( K m and V rel ) of the ‘carbapenemases’ from the Cpm R mutants and similarly resistant clinical isolates suggested a close relatedness of the enzymes. A probe covering most of the cfiA gene encoding such an enzyme (Thompson, J.S. and Malamy, M.H. (1990) J. Bacteriol. 172, 2584–2593) hybridized with DNA from the Cpm R mutants, their Cpm S parental strains as well as clinical Cpm R isolates, but not from randomly chosen carbapenem-susceptible strains. The possibility is considered that mutations leading to expression of the silent carbapenemase gene, and thereby to clinically relevant carbapenem resistance, may also occur in the clinical setting.