Carbapenem Minimum Inhibitory Concentrations Research Articles

Effect of MIC interpretative breakpoint revision on cephalosporin and carbapenem susceptibility among ESBL-producing Enterobacteriaceae

Background: Minimum inhibitory concentration (MIC) breakpoints of selected cephalosporins and carbapenems against Enterobacteriaceae have been revised by major guidelines including CLSI and EUCAST mainly according to available pharmacokinetic/pharmacodynamic data. A decrease of breakpoint may obviate the need to detect specific resistance mechanisms such as extended-spectrum �-lactamase (ESBL) and carbapenemase, which may be less correlated to treatment outcome than does the actual MIC of each agent. Objective: To analyze cephalosporin and carbapenem MIC distributions among ESBL-producing Enterobacteriaceae at a university hospital against revised interpretative breakpoints. Methods: MIC distributions of selected cephalosporins and carbapenems among 505 isolates of genotypically confirmed ESBL-producing Enterobacteriaceae were determined by E-test TM method and analyzed according to interpretative breakpoints comparing between CLSI and EUCAST guidelines. Results: ESBL-producing Enterobacteriaceae demonstrated a wide range of cephalosporin MIC ( 64). Up to 9.7% of isolates displayed MIC lower than a revised cephalosporin breakpoint. Most isolates remained susceptible to imipenem and meropenem while as high as 24.6% were not susceptible to ertapenem. Lowered breakpoints may result in a change in categorical interpretations. Conclusion: ESBL-producing isolates could be reported as susceptible to a cephalosporin with revised breakpoints although clinical use is uncertain. A higher proportion of isolates would be reported as nonsusceptible to cephalosporins or carbapenems with lowered breakpoints and thus increasing use of broadspectrum antimicrobial agents should be monitored.

Bloodstream infections caused by IMP-8-producing Enterobacteriaceae isolates: the need for clinical laboratory detection of metallo-β-lactamases?

A retrospective study was conducted at a Taiwanese medical center to characterize bloodstream infections caused by IMP-8 metallo-β-lactamase (MBL)-producing Enterobacteriaceae isolates and to assess the need for laboratory detection of IMP producers. We analyzed 37 patients infected with IMP-8 producers (two Escherichia coli, nine Klebsiella pneumoniae, 25 Enterobacter cloacae, and one Citrobacter freundii) and 107 patients infected with non-IMP-8 producers (eight E. coli, 26 K. pneumoniae, 70 E. cloacae, and three C. freundii) that were interpreted as carbapenem-nonsusceptible based on the updated Clinical and Laboratory Standards Institute (CLSI) 2010 guidelines. Only 18 (48.6 %) of the IMP-8 producers were regarded as potential carbapenemase producers based on the CLSI 2012 guidelines. The production of extended-spectrum β-lactamases (ESBLs) was more common in the MBL group (73.0 %) than in the non-MBL group (41.1 %). There were no significant differences in carbapenem susceptibilities, clinical characteristics, carbapenem use for empirical and definitive treatment, and mortality rates between the two groups. Eighteen IMP-8 producers could be deemed as resistant to all carbapenems [minimum inhibitory concentration (MIC) of any carbapenem ≥2 μg/mL]; patients with these isolates had a lower, but non-significant, 28-day mortality rate (27.8 %) than patients infected with non-MBL producers having similar carbapenem MICs (39.0 %) (p = 0.41). A multivariate analysis revealed severity of acute illness as the single independent variable associated with both 7-day and 28-day mortality rates (p < 0.01) for infections caused by Enterobacteriaceae with decreased carbapenem susceptibilities. Our findings suggest that the clinical detection of IMP-producing Enterobacteriaceae is not required even when the "old" CLSI criteria are used.

OprD mutations and inactivation, expression of efflux pumps and AmpC, and metallo-β-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from South Korea

Among 213 Pseudomonas aeruginosa isolates collected from 10 South Korean hospitals, 57 isolates (26.8%) were carbapenem-resistant. All but three of the isolates had a relevant decrease of oprD expression. However, decreased oprD expression was also detected in five of ten carbapenem-susceptible isolates. Outer membrane protein analysis confirmed porin loss in carbapenem-resistant P. aeruginosa isolates. Based on the mutations of oprD gene sequences, carbapenem-resistant P. aeruginosa isolates could be classified into five oprD mutational groups. However, there was no difference of OprD expression or carbapenem minimum inhibitory concentrations among the five mutational groups. Among the 57 carbapenem-resistant P. aeruginosa isolates, 41 (71.9%) overexpressed efflux systems or ampC. MexAB–OprM and AmpC overexpression (56.1% and 47.4%, respectively) was prevalent and was significantly associated with carbapenem resistance. However, no synergistic effect of efflux systems and AmpC on carbapenem resistance was evident. In conclusion, combination of several mutation-driven mechanisms leading to OprD inactivation and overexpression of efflux systems was the main carbapenem resistance mechanism, but acquisition of a transferable resistance determinant such as metallo-β-lactamase could be problematic in clinical settings in South Korea.

"Silent" Dissemination of Klebsiella pneumoniae Isolates Bearing K. pneumoniae Carbapenemase in a Long-term Care Facility for Children and Young Adults in Northeast Ohio

Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) was identified by PCR amplification and sequencing of the amplicon. In addition, a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla(KPC) carriage. Plasmids from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia coli J-53 recipients. Our findings reveal the "silent" dissemination of bla(KPC-3) as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla(KPC-3) in an LTCF caring for neurologically impaired children and young adults.

Pseudomonas aeruginosa is not just in the intensive care unit any more

To improve empirical therapy for Pseudomonas aeruginosa using susceptibility surveillance by unit type (intensive care unit vs. nonintensive care unit) and to optimize antibacterial dosing using pharmacodynamic profiling. Prospective multicentered surveillance study. Thirteen U.S. hospitals. Seven hundred thirty-six nonduplicate, nonurine P. aeruginosa isolates collected from first quarter, 2009, to second quarter, 2010. None. Isolate minimum inhibitory concentrations (MICs) to ten antimicrobials (three carbapenems-doripenem, imipenem and meropenem-plus three other β-lactams, two fluoroquinolones, and two aminoglycosides) were determined by broth microdilution. Wilcoxon rank sums compared MIC distributions by unit type; chi-square tests compared agents and antibiotic classes. Cumulative fraction of response predicted likelihood of pharmacodynamic target attainment for antimicrobial dosing regimens vs. observed MIC distributions. Nonintensive care units contributed 65% of isolates with identifiable locations (n = 614). Carbapenem class nonsusceptibility nonsusceptible to 1 or more agent) differed by location (35% intensive care unit, 27% nonintensive care unit, p = .03); no other classes differed. Multidrug resistance (nonsusceptible to one or more drug in each of all four classes) was 12% intensive care unit and 5% in nonintensive care units (p < .01). Carbapenem MIC profile in intensive care units was (agent, MIC50, MIC90, percent susceptibility): Doripenem, 1, 8, 69%; imipenem, 2, 16, 67%; and meropenem, 1, 32, 70%; and by nonintensive care units: Doripenem, 0.5%, 8%, 78%; imipenem, 1, 16, 75%; and meropenem, 1, 16, 82%. MIC distributions differed by unit type only for imipenem (p < .01). The remaining nine agents were not different. Standard carbapenem regimens resulted in cumulative fraction of response (regimen, intensive care unit, nonintensive care unit): Doripenem at 500 mg every 8 hrs 1-hr infusion, 73%, 79%; imipenem at 500 mg every 6 hrs 0.5-hr infusion, 62%, 69%; meropenem at 500 mg every 6 hrs 0.5-hr infusion, 67%, 76%. More aggressive doses and prolonged infusion improved cumulative fraction of response: Doripenem at 1000 mg every 8 hrs 4-hr infusion, 92%, 97%; imipenem at 1000 mg every 8 h 3-h infusion, 77%, 83%; meropenem at 2000 mg every 8 hrs 3-hr infusion, 87%, 94%. Although multidrug-resistant and nonsusceptible carbapenem phenotypes were more common in intensive care units, the prevalence of P. aeruginosa among initial cultures of systemic isolates taken elsewhere was high (65%). Unit-specific antibiograms could benefit empirical therapy decisions; consideration of carbapenem, dose, and infusion time may enhance outcomes for P. aeruginosa infection.

Prevalence and analysis of microbiological factors associated with phenotypic heterogeneous resistance to carbapenems in Acinetobacter baumannii

The objectives of this study were to determine the prevalence of Acinetobacter baumannii with phenotypic heterogeneous resistance (PHR) to carbapenems (colonies inside the halo of inhibition) and to analyse its association with several microbiological variables. Acinetobacter baumannii isolates collected in Spain were used to analyse: (i) minimum inhibitory concentrations (MICs) of carbapenems; (ii) heteroresistance to carbapenems; (iii) genes encoding β-lactamases (bla genes); (iv) insertion sequences; and (v) inactivation of genes encoding porins (CarO, OprD and Omp33–36) and genes associated with the AdeABC efflux system (adeB, adeR and adeS). Polymerase chain reaction (PCR) amplification was used for gene detection. The rate of PHR was 20% to imipenem and 24% to meropenem. Susceptibility to imipenem was observed in 39% of PHR isolates. MICs of carbapenems for colonies were similar (±1log2 dilution) to those of their parental isolates. These colonies growing inside the inhibition halo also reproduced the PHR to carbapenems. Differences observed between PHR isolates and non-PHR isolates were: blaOXA-58-like, 57% vs. 0%; oprD-like, 96% vs. 56%; adeB, 89% vs. 94%; adeR, 82% vs. 94%; adeS, 82% vs. 94%; ISAba2, 61% vs. 31%; and ISAba3, 57% vs. 0%. No interruption of genes encoding porins or the efflux-related genes (adeB, adeR and adeS) was observed. In conclusion, A. baumannii strains with PHR to carbapenems are widespread in Spain. This phenotype is present in carbapenem-susceptible isolates as well as those that are not susceptible to carbapenems. Heteroresistance cannot explain the PHR to carbapenems, which appears to relate more to persistence or tolerance to carbapenems. blaOXA-58-like, blaOXA-51-like, ISAba2 and ISAba3 are associated with PHR to carbapenems. Inactivation of genes encoding porins or genes related to AdeABC is infrequent.

Small Molecule Suppression of Carbapenem Resistance in NDM-1 Producing Klebsiella pneumoniae

The already considerable global public health threat of multi-drug resistant Gram-negative bacteria has become even more of a concern following the emergence of New-Delhi metallo-β-lactamase (NDM-1) producing strains of Klebsiella pneumoniae and other Gram-negative bacteria. As an alternative approach to the traditional development of new bactericidal entities, we have identified a 2-aminoimidazole derived small molecule that acts as an antibiotic adjuvant and is able to suppress resistance of a NDM-1 producing strain of K. pneumoniae to imipenem and meropenem, in addition to suppressing resistance of other β-lactam non-susceptible K. pneumoniae strains. The small molecule is able to lower carbapenem minimum inhibitory concentrations by up to 16-fold while exhibiting little bactericidal activity itself.

Characteristics of carbapenem-resistant Acinetobacter spp. other than Acinetobacter baumannii in South Korea

Although many studies have been performed on carbapenem-resistant Acinetobacter baumannii, only a few studies have addressed carbapenem resistance in Acinetobacter spp. other than A. baumannii (non- baumannii Acinetobacter). Amongst 287 Acinetobacter spp. isolates from patients with bacteraemia in a South Korean hospital collected between 2003 and 2010, 160 (55.7%) were non- baumannii Acinetobacter spp. Antimicrobial susceptibility testing was performed and the effect of efflux pump inhibitors was examined. Antimicrobial resistance genes were identified and pulsed-field gel electrophoresis (PFGE) analysis was performed. OprD expression was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), and CarO disruption was investigated by PCR. Seventeen non- baumannii Acinetobacter isolates (10.6%) were resistant to imipenem or meropenem, comprising eight Acinetobacter pittii (or Acinetobacter genomospecies 3), four Acinetobacter nosocomialis (or Acinetobacter genomospecies 13TU), two Acinetobacter genomospecies ‘close to 13TU’, two Acinetobacter bereziniae (or Acinetobacter genomospecies 10) and one Acinetobacter genomospecies 16. bla IMP-1 genes were detected in seven and two carbapenem-resistant A. pittii and A. bereziniae isolates, respectively. PFGE showed that A. pittii isolates carrying bla IMP-1 belonged to the same clone. In addition, bla SIM-1 and bla PER-1 genes were simultaneously identified in two A. nosocomialis isolates. In four isolates (one each of A. pittii, A. nosocomialis, Acinetobacter genomospecies ‘close to 13TU’ and Acinetobacter genomospecies 16), efflux pumps were implicated in the increase in carbapenem minimum inhibitory concentrations. No decreased expression of OprD was identified in any carbapenem-resistant non- baumannii Acinetobacter isolates, and disruption of carO was also not detected. Clonal spread of carbapenem-resistant A. pittii carrying bla IMP-1, which contributes to a high resistance rate in this species, was identified. The bla IMP-1 and bla SIM-1 genes were first identified in A. bereziniae and A. nosocomialis, respectively. Since no carbapenem resistance mechanisms could be identified, further efforts to find the resistance mechanism should be made.

Carbapenem-resistantAcinetobacter baumannii: diversity of resistant mechanisms and risk factors for infection

Carbapenem-resistant Acinetobacter baumannii (CRAB) are an increasing infectious threat in hospitals. We investigated the clinical epidemiology of CRAB infections vs. colonization in patients, and examined the mechanisms of resistance associated with elevated minimum inhibitory concentrations (MICs) for carbapenems. From January to June 2009, 75 CRAB strains were collected. CRAB infection was significantly associated with malignancy and a high APACHE II score. The most dominant resistance mechanism was ISAba1 preceding OXA-51, producing strains with overexpression of efflux pump. Strains carrying blaOXA-23-like enzymes had higher carbapenem MICs than those carrying blaOXA-51-like enzymes; however, the presence of multiple mechanisms did not result in increased resistance to carbapenems. There was no difference in the resistance mechanisms in strains from infected and colonized patients. The majority of strains were genetically diverse by DNA macrorestriction although there was evidence of clonal spread of four clusters of strains in patients.

Evolution of tigecycline resistance inKlebsiella pneumoniaein a single patient

Carbapenemase-producing Klebsiella pneumoniae infections carry serious clinical and infection-control implications. Isolates possessing such hydrolyzing enzymes have been described in the United States and around the world. Besides being resistant to carbapenems, they usually confer resistance to fluoroquinolones, piperacillin-tazobactam, and extended-spectrum cephalosporins. Tigecycline demonstrates in vitro activity against these organisms, but reported resistance raises concern about tigecycline use for these infections. We describe a carbapenemase-producing K pneumoniae evolving resistance to tigecycline in a 75-year-old male after a prolonged stay in a critical care unit.

An Outbreak of Infection due to β‐LactamaseKlebsiella pneumoniaeCarbapenemase 2–ProducingK. pneumoniaein a Greek University Hospital: Molecular Characterization, Epidemiology, and Outcomes

We describe the emergence and spread of Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing K. pneumoniae at a Greek University hospital. Isolates with a carbapenem minimum inhibitory concentration >1 microg/mL and a negative EDTA-imipenem disk synergy test result were submitted to boronic acid disk test and to polymerase chain reaction (PCR) for KPC gene and sequencing. Records from patients who had KPC-2-producing K. pneumoniae isolated were retrospectively reviewed. Clinical isolates were submitted to molecular typing using pulsed-field gel electrophoresis, and the beta-lactamase content was studied using isoelectric focusing and PCR. From January 2007 through December 2008, 50 patients (34 in the intensive care unit [ICU]) were colonized (n = 32) or infected (n = 18) by KPC-2-producing K. pneumoniae. Increasing prevalence of KPC-2-producing K. pneumoniae coincided with decreasing prevalence of metallo-beta lactamase-producing isolates in our ICU. Multidrug resistance characterized the studied isolates, with colistin, gentamicin, and fosfomycin being the most active agents. Besides KPC-2, clinical isolates encoded TEM-1-like, SHV-11, SHV-12, CTX-M-15, and LEN-19 enzymes. Four different clonal types were detected; the predominant one comprised 41 single patient isolates (82%). Sporadic multiclonal cases of KPC-2-producing K. pneumoniae infection were identified from September 2007 through May 2008. The outbreak strain was introduced in February 2008 and disseminated rapidly by cross-transmission; 38 patients (76%) were identified after August 2008. Fourteen cases of bacteremia, 2 surgical site infections, 2 lower respiratory tract infections (1 bacteremic), and 1 urinary tract infection were identified. Most patients received a colistin-containing combination treatment. Crude mortality was 58.8% among ICU patients and 37.5% among non-ICU patients, but attributable mortality was 22.2% and 33.3%, respectively. The emergence of KPC-2-producing K. pneumoniae in Greek hospitals creates an important challenge for clinicians and hospital epidemiologists, because it is added to the already high burden of antimicrobial resistance.

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple β-lactamases

Four clinical strains of extended-spectrum β-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C 1 and C 2 were isolated prior to carbapenem therapy, whilst isolates C 3 and C 4 were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). β-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C 1 in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple β-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C 1 and C 2, isolates C 3 and C 4 failed to express OmpE36 owing to insertional inactivation by an IS 903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene ( kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C 3 and C 4 were apparently derived from the previously imipenem-susceptible isolates C 1 and C 2. Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.

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.

Examination of cfiA-mediated carbapenem resistance in Bacteroides fragilis strains from a European antibiotic susceptibility survey

Of 1284 Bacteroides strains collected in Europe in 2000 for antibiotic susceptibility surveillance, 65 isolates displayed imipenem minimum inhibitory concentrations (MICs) ≥1 mg/L and were chosen for a thorough analysis of their resistance mechanism. Twenty-five of the isolates were positive for the cfiA carbapenem resistance gene. The resistance rates were 0.8% and 1.3% for imipenem and meropenem, respectively. In six of the strains, insertion sequence (IS) elements (IS 613, IS 614B, IS 1186 and IS 1187) activated the cfiA gene. However, other strains displayed at least elevated carbapenem MICs or were carbapenem resistant and produced measurable carbapenemase activities but did not harbour IS elements in the region upstream of the cfiA gene. The major determinant of carbapenem resistance in Bacteroides fragilis is production of CfiA metallo-β-lactamase via activation of the cfiA gene by IS elements (higher level resistance) or by activation of its putative own promoter.

Susceptibilities to carbapenems and presence of cphA gene on food-borne Aeromonas

The purpose of this study was to determine the susceptibilities of food-borne Aeromonas to carbapenems, as well as to investigate the presence of a metallo carbapenemase-encoding gene, named cphA. Minimum Inhibitory Concentration (MIC) was determined following NCCLS standards. All the tested microorganisms were susceptible to imipenem, meropenem and biapenem. However, a strong inoculum size effect on carbapenem MICs was observed for most of the strains. Six strains, out of seven, showed the presence of metallo--beta-lactamases but cphA gene was detected in only two strains of A. veronii bv. sobria.

Comparison of activities of β-lactam antibiotics against Streptococcus pneumoniae with recombinant penicillin-binding protein genes from a penicillin-resistant strain

We investigated the antibacterial activities of 19 beta-lactams against three recombinant bacterial strains, in which three penicillin-binding protein genes, pbp2x, pbp1a, and pbp2b, from penicillin-resistant Streptococcus pneumoniae (PRSP), were transformed to a penicillin-susceptible strain. By the acquisition of the pbp2x gene from PRSP, the minimum inhibitory concentrations (MICs) of third-generation cephalosporins were increased more than eight fold. When the strain acquired the PRSP pbp1a gene in addition to pbp2x, the MICs of all tested beta-lactams increased 2- to 16-fold. When the strain acquired the PRSP pbp2b gene in addition to pbp2x and pbp1a, the MICs of penicillins and carbapenems increased 4- to 16-fold. However, two novel carbapenems, ME1036 and L-036, showed excellent antibacterial activities against these recombinant strains, as well as against the parent PRSP.