Meropenem Minimum Inhibitory Concentration Research Articles

Prevalence of different carbapenemase genes among carbapenem-resistant Acinetobacter baumannii blood isolates in Taiwan

BackgroundAlthough the prevalence of the carbapenem-resistant A. baumannii (CRAB) has increased in Taiwan, few studies have elucidated the prevalence of different carbapenemase genes in Taiwan. The first objective of this study was to identify the types and prevalence of different carbapenemase genes, and the second objective was to determine the carbapenem antimicrobial susceptibility of carbapenemase producing isolates.MethodsIn total, 269 CRAB blood isolates from four medical centres in Taiwan from 1/1/2009 to 31/12/2013 were analysed. Antimicrobial susceptibilities were determined using the Vitek 2 system. Carbapenemase genes were identified by polymerase chain reaction (PCR) and sequencing. Pulsed-field gel electrophoresis (PFGE) was used to identify the different pulsotypes.ResultsAll 269 CRAB isolates had the blaOXA-51-like gene, while 237 (88.1%) had the blaOXA-23-like gene, and 11 (4.09%) had the blaOXA-24-like gene. Twenty-one CRAB isolates (7.81%) contained only the blaOXA-51-like gene. None of the isolates had the blaOXA-58-like gene or the metallo-β-lactamases (MBL)-encoding genes. In 28.69% of isolates with the blaOXA-23-like gene and 90.91% of isolates with the blaOXA-24-like gene, the minimum inhibitory concentrations (MICs) for imipenem were 64 mg/L or more. In 37.55% of isolates with the blaOXA-23-like gene and 100% of isolates with the blaOXA-24-like gene, meropenem MICs were 64 mg/L or more. PFGE analyses indicated that six highly similar genomes which harbored the blaOXA-24-like gene came from three different medical centres.ConclusionOur study determined the prevalence of CRAB, the types and prevalence of carbapenemase genes, carbapenem susceptibility among CRAB isolates, and documented that the blaOXA-24-like gene had greater resistance to carbapenem than the blaOXA-23-like gene. We also demonstrated inter-hospital transmission of the highly resistant blaOXA-24-like gene.

Fluoroquinolone Prophylaxis Selects for Meropenem-nonsusceptible Pseudomonas aeruginosa in Patients With Hematologic Malignancies and Hematopoietic Cell Transplant Recipients.

In Pseudomonas aeruginosa, fluoroquinolone exposure promotes resistance to carbapenems through upregulation of efflux pumps and transcriptional downregulation of the porin OprD. Evidence of this effect among hematologic malignancy (HM) patients or hematopoietic cell transplant (HCT) recipients receiving fluoroquinolone prophylaxis for neutropenia is lacking. We retrospectively evaluated episodes of P. aeruginosa bloodstream infections in HM patients or HCT recipients over a 7-year period at our institution. We determined the association of fluoroquinolone prophylaxis at the time of infection with meropenem susceptibility of P. aeruginosa breakthrough isolates and risk factors for meropenem nonsusceptibility. Whole-genome sequencing (WGS) and phenotypic assessments of meropenem efflux pump activity were performed on select isolates to determine the mechanisms of meropenem resistance. We analyzed 55 episodes of P. aeruginosa bacteremia among 51 patients. Breakthrough bacteremia while on fluoroquinolone prophylaxis was associated with nonsusceptibility to meropenem, but not to antipseudomonal β-lactams or aminoglycosides. The receipt of fluoroquinolone prophylaxis was independently predictive of bacteremia with a meropenem-nonsusceptible isolate. All meropenem-nonsusceptible isolates analyzed by WGS contained oprD inactivating mutations, and all meropenem-nonsusceptible isolates tested demonstrated reductions in the meropenem minimum inhibitory concentration in the presence of an efflux pump inhibitor. A phylogenetic analysis based on WGS revealed several clusters of closely related isolates from different patients. Fluoroquinolone prophylaxis in HM patients and HCT recipients is associated with breakthrough bacteremia with meropenem-nonsusceptible P. aeruginosa strains, likely due to both mutations increasing efflux pump activity and the epidemiology of P. aeruginosa bloodstream infections in our patient population.

Soup with or without meatballs: Impact of nutritional factors on the MIC, kill-rates and growth-rates

BackgroundThe Minimum Inhibitory Concentration (MIC) is a reference value for susceptibility testing of bacteria. However, the MIC is a net result of growth and killing after a certain duration of exposure under standardized favourable in vitro conditions. Killing and growth characteristics of a drug may yield more information on its activity and help to explain discrepancies between efficacy observed in vitro and in vivo. MethodsThe MIC of meropenem was determined for P. aeruginosa ATCC 27853 both by microdilution and the E-test in dilutions of Mueller Hinton (MH) broth from 100% to 1%. Time-kill curves were obtained for twofold dilutions of meropenem. Growth rates and kill rates at each concentration and dilution were obtained by linear regression. The Hill equation was fit to the kill rates vs concentrations. ResultsGrowths rates decreased log linearly from 0.63/h at 100% to 0.29/h at 6% MH. Over the 100–6% MH dilution range, there was a log-linear decrease of the MIC of meropenem of both the E-test and microdilution. The EC50s decreased from 0.29 mg/L to 0.07 mg/L, which is in agreement with the MIC results. There was a log-linear relationship between MIC and EC50 for the various dilutions MH. ConclusionsThe availability of nutritional factors is related to the MIC, and a lower availability is related to both a lower growth rate and higher kill rate. Since nutritional factors are less abundantly available in vivo as compared to in vitro, this should be taken into account when translating in vitro to in vivo pharmacodynamics.

Is meropenem MIC increase against KPC-producing Klebsiella pneumoniae correlated with increased resistance rates against other antimicrobials with Gram-negative activity?

The aim of this study was to assess the minimum inhibitory concentration (MIC) distribution for meropenem and other antimicrobials with Gram-negative activity against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) clinical isolates collected at a tertiary hospital in Italy between 2013-2016. The antimicrobial susceptibility of KPC-Kp strains was tested by the broth microdilution method using customised 96-well plates and the results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Among 169 consecutive KPC-Kp clinical isolates, 45 (26.6%) were susceptible to meropenem (MIC≤2mg/L). Among the 124 meropenem-resistant isolates, 73 (58.9%) had a meropenem MIC between 16-64mg/L. The overall resistance rate for the other antimicrobials tested was very high both for ciprofloxacin and levofloxacin (99.0%), was moderate for amikacin (37.4%) and was low for gentamicin (11.2%), colistin (8.2%) and tigecycline (7.7%). Aminoglycosides had a dichotomous behaviour in relation to meropenem MIC increase. The resistance rate for gentamicin remained <20% across all meropenem MICs; conversely, that for amikacin increased from <20% in the presence of meropenem MIC≤8mg/L up to ca. 80% in the presence of meropenem MIC≥64mg/L. Resistance rates for tigecycline and colistin remained <20% in the presence of meropenem MICs up to 64mg/L. The overall susceptibility rates of antimicrobials with Gram-negative activity may vary greatly among KPC-Kp clinical isolates. A tight relationship between meropenem MIC increase and the resistance rate for amikacin was documented.

Early emergence of OXA-181-producing Escherichia coli ST410 in China

The aim of this study was to investigate the prevalence and characterisation of OXA-48-like-producing Enterobacteriaceae in Henan Province, China. A total of 339 carbapenem-non-susceptible clinical Enterobacteriaceae isolates [imipenem or meropenem minimum inhibitory concentration (MIC) of >1μg/mL] obtained between January 2013 and December 2016 were screened for the presence of the blaOXA-48-like gene by PCR and sequencing. Antimicrobial susceptibility to various antimicrobials agents was examined by MIC testing. Multilocus sequence typing (MLST) was performed for bacterial genotyping. The plasmid pEC21-OXA-181 was transformed into Escherichia coli DH5α by electroporation and was sequenced using an Illumina MiSeq platform, followed by subsequent annotation and genetic analysis. Among the 339 carbapenem-non-susceptible Enterobacteriaceae isolates, only one (0.3%) E. coli strain EC21, belonging to ST410, was positive for blaOXA-181, a variant of blaOXA-48. This OXA-181-producing E. coli, recovered from a patient without a history of foreign travel, was obtained earlier than the first reported blaOXA-181-positive E. coli (WCHEC14828) in Sichuan Province, China. Plasmid analysis revealed that blaOXA-181 together with the quinolone resistance gene qnrS1 was carried by an IS26-flanked composite transposon on a 51-kb IncX3-type plasmid. These findings indicate the emergence of OXA-181-producing E. coli in China earlier than previously thought. The blaOXA-181 gene is associated with the widely disseminated potentially endemic E. coli ST410 clone and is carried by an IncX3 plasmid, a common vehicle for spreading NDM-type carbapenemases, which might promote the further dissemination of blaOXA-181 among the Enterobacteriaceae in China.

Effect of increasing meropenem MIC on the killing activity of meropenem in combination with amikacin or polymyxin B against MBL- and KPC-producing Enterobacter cloacae

Carbapenem resistant Enterobacteriaceae (CRE) are a growing threat worldwide. Infections caused by these organisms have exhibited high rates of mortality (50%) for which there is no standard of care and a dearth of clinical trials. Most in vitro data on CRE focus on Klebsiella pneumoniae, but it is known that effective therapy may depend on species or even strain. To address this, meropenem, amikacin, and polymyxin B alone and in combination were evaluated by time kill against four carbapenem-producing Enterobacter cloacae clinical isolates representing a range of meropenem nonsusceptibility (2–32 mg/L) and resistance mechanisms (KPC 2 and/or VIM 1). As meropenem minimum inhibitory concentration (MIC) increased, bactericidal activity and synergy were maintained for 48 hours in isolates exposed to meropenem and amikacin, but synergy and bactericidal activity were not maintained in all isolates exposed to meropenem and polymyxin B.

The effect of exposure to sub-inhibitory concentrations of hypochlorite and quaternary ammonium compounds on antimicrobial susceptibility of Pseudomonas aeruginosa

Pseudomonas is a group of medically important species that inhabit a wide range of niches, including hospital environments. Controversies have emerged about the possible link between improper use of disinfectants and the emergence of antibiotic resistance in bacteria. The aim of this study was to assess the effect of exposure of antibiotic-susceptible Pseudomonas isolates to sub-inhibitory concentrations of 2 disinfectants-didecyldimonium chloride and sodium hypochlorite-on their antibiotic susceptibility patterns. This study involved 50 Pseudomonas isolates. The antibiotic susceptibility patterns of the isolates were assessed using broth microdilution method. The minimal inhibitory concentrations (MICs) of each antibiotic were compared before and after exposure to sub-inhibitory concentrations of didecyldimonium chloride and sodium hypochlorite. After overnight incubation with sub-inhibitory concentrations of sodium hypochlorite, a statistically significant increase was observed in the MICs of colistin (P = .012), ceftazidime (P < .001), amikacin (P < .001), meropenem (P < .001), gentamicin (P < .001), piperacillin-tazobactam (P = .003), and ciprofloxacin (P = .004). In contrast, exposure to sub-inhibitory concentrations of didecyldimonium chloride showed a statistically significant increase in the MICs of amikacin (P < .001), gentamicin (P < .001), meropenem (P = .041), and ciprofloxacin (P = .008). The use of suboptimal concentrations of sodium hypochlorite and didecyldimonium chloride can lead to the evolution of antibiotic-resistant Pseudomonas strains.

Molecular characterisation of blaOXA-48 carbapenemase-, extended-spectrum β-lactamase- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India.

The aim of this study was to characterise carbapenemase-, extended-spectrum β-lactamase (ESBL)- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India. Faecal samples (n=741) from 10 organised pig farms, including non-diarrhoeic (n=546) and diarrhoeic (n=195) piglets, were processed for isolation of carbapenem-resistant and ESBL-producing E. coli. A total of 27 and 243 isolates were phenotypically confirmed as carbapenem-resistant and ESBL-producers, respectively. The meropenem minimum inhibitory concentration (MIC) of carbapenem-resistant isolates ranged from 8-128μg/mL. On genotypic screening of the 27 carbapenem-resistant isolates, 3 isolates were positive for the blaOXA-48 carbapenemase gene; no other carbapenemase genes were detected. The 243 ESBL-producing isolates were positive for blaCTX-M-1 (n=135), qnrA (n=92), qnrB (n=112), qnrS (n=49), tetA (n=42), tetB (n=45) and sul1 (n=43). The Shiga toxin virulence markers stx1 and stx2 were detected in 41 and 38 of the 243 phenotypic ESBL-producing isolates, respectively. Multilocus sequence typing (MLST) of blaOXA-48-positive E. coli isolates showed ST10- and ST5053-like sequence types. This is the first report on the presence of blaOXA-48-carrying E. coli in piglets in India, which pose a potential risk to public health.

Raising the Stakes: Loss of Efflux Pump Regulation Decreases Meropenem Susceptibility in Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance. We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

Risk factors for and role of OprD protein in increasing minimal inhibitory concentrations of carbapenems in clinical isolates of Pseudomonas aeruginosa.

This study examined the risk factors for, and molecular mechanisms underlying, the increase in carbapenem minimum inhibitory concentrations (MICs) in clinical isolates of Pseudomonas aeruginosa. Consecutive clinical isolates of P. aeruginosa were collected. The MicroScan WalkAway system detected more than fourfold increases in the MICs of carbapenems in P. aeruginosa isolates serially recovered from some patients during their clinical course. The clinical risk factors associated with this increase were examined by multiple logistic regression analysis. Western blot analysis and nucleotide sequencing of the oprD gene of 19 clonally related and paired P. aeruginosa isolates from the same patients were undertaken to examine the mechanisms underlying the increase in MICs. The results showed that prior use of carbapenems (OR, 2.799; 95 % CI, 1.088-7.200; P=0.033) and the use of ventilators or tracheostomies (OR, 2.648; 95 % CI, 1.051-6.671; P=0.039) were risk factors for increased carbapenem MICs. Analysis of the underlying mechanisms revealed that loss of functional OprD protein due to mutation of the oprD gene tended to occur in P. aeruginosa isolates with imipenem MICs of more than 8 µg ml-1; a reduction in OprD expression was observed in P. aeruginosa isolates with imipenem MICs of 4 or 8 µg ml-1. This difference in the resistance mechanism was not correlated with the MICs of meropenem. This difference in the resistance mechanism of P. aeruginosa indicates a critical breakpoint at an imipenem MIC of 8 µg ml-1, in accordance with EUCAST criteria. Reducing carbapenem use will prevent P. aeruginosa clinical isolates from developing resistance to carbapenems.

In vitro interaction of ceftazidime–avibactam in combination with different antimicrobials against KPC-producing Klebsiella pneumoniae clinical isolates

ObjectivesCombination therapy has been recommended when using ceftazidime–avibactam (CAZ–AVI) for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp), but the optimal combination is unknown. Six common antimicrobial agents (ertapenem, imipenem, meropenem, gentamicin, tigecycline, and ciprofloxacin) were evaluated for synergy with the recently approved cephalosporin–β-lactamase inhibitor combination CAZ–AVI in this study. MethodsDifferent antimicrobial combinations were tested against 13 KPC-Kp, including CAZ–AVI-susceptible (n=11) and resistant (n=2) clinical isolates. In vitro interactions of CAZ–AVI with different antimicrobials were tested using the gradient synergy test. Changes in the minimum inhibitory concentration (MIC) value were interpreted using the fractional inhibitory concentration (FIC) index and susceptible breakpoint index (SBPI). ResultsThe combination of CAZ–AVI with gentamicin or ciprofloxacin displayed no synergism against any of the KPC-Kp isolates, whereas synergistic activity was observed with imipenem and meropenem against all KPC-Kp isolates. Notably, CAZ–AVI reduced MICs for meropenem and imipenem below the resistance breakpoints against all strains. The SBPI analysis showed that CAZ–AVI in combination with imipenem achieved higher SBPI values than other CAZ–AVI-based combinations. ConclusionsThese data suggest that combinations of CAZ–AVI with imipenem may be considered a useful therapeutic option for the treatment of KPC-Kp infections.

Prevalence of a New Variant OXA-204 and OXA-48 Carbapenemases Plasmids Encoded inKlebsiella pneumoniaeClinical Isolates in Tunisia

Carbapenemase-producing Klebsiella pneumoniae strains have emerged as a major problem for healthcare systems. The aim of this study was to determine the role and diversity of plasmids harboring carbapenemases encoding genes from a collection of K. pneumoniae isolates recovered between July 2011 and January 2012, with decreased susceptibility to carbapenems. Imipenem (IPM), ertapenem (ETP), meropenem (MEM), and doripenem (DOR) minimum inhibitory concentrations (MICs) were determined by E-test. Carbapenemase production was detected with the modified Hodge test. β-Lactamases encoding genes were amplified by PCR and sequenced. Plasmid incompatibility groups harbored by carbapenemases producers were investigated using the PCR-based replicon typing method and the clonal relationship of the isolates was investigated by pulse filed electrophoresis. IMP, ertapenem, meropenem, and doripenem MICs ranged between 0.25 and 16 mg/L. Carbapenemase activity was detected in 14 isolates. Two carbapenemases were identified: OXA-48 in 13 isolates and a new variant OXA-204 in 1 isolate, in combination with extended-spectrum β-lactamases, CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15, and VEB-8. One isolate produced CMY-2. OXA-48 and the new variant OXA-204 were confirmed as transferable plasmid encoded. The carbapenemase-producing K. pneumoniae harbored plasmids of the A/C, LVPK, and L/M replicon types. Thirteen different pulso types were observed. Three pairs of isolates showed a clonal relatedness. This diversity in β-lactamases, in pulso types and in plasmid content, shows the ability of OXA-type carbapenemase to disseminate. This is worrying for the control of the increase in antibiotic resistance frequency and necessitates that continuous investigations in the clinical setting remain a high priority to clarify the contribution of antimicrobial use into multiresistance bacterial dissemination.

Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

AdeABC efflux pump and resistance of Acinetobacter baumannii against carbapenem

To investigate relationship between AdeABC efflux pump and resistance of Acinetobacter baumannii against carbapenem. Methods: Carbapenem-resistant strains were acquired from multistep selection resistance test by meropenem in vitro. The quantitation test for sensitivities of strains before and after induction was determined by the E-test, and carbonylcyanide-m-chlorophenylhydrazone (CCCP) inhibition test was used to screen efflux pump. PCR, sequencing analysis, or real-time PCR was used to analyze the changes of regulatory genes adeR and adeS of the AdeABC efflux pump system, or expressions of adeA, adeB, adeR, and adeS in the strains before and after induction, respectively. Results: The minimal inhibitory concentrations (MICs) of meropenem were at 0.38 μg/mL and 0.25 μg/mL in parental sensitive strain S25595 and S7257, respectively, and the MICs of meropenem for both S25595 and S7257 after induction were more than 32 μg/mL. Compared with parental sensitive strains, the expression level of adeA, adeB, adeR, and adeS mRNA were elevated from 2.45 to 9.44 times, but there were no gene mutations or insertion sequences in the regulatory gene adeS and adeR. Conclusion: High expression of the AdeABC efflux pump system in Acinetobacter baumannii is closely associated with meropenem resistance. The upregulation of adeA and adeB expression is not due to gene mutations in the regulatory gene adeS and adeR and other mechanisms might account for it.

Direct-On-Target Microdroplet Growth Assay for Rapid Detection of Carbapenem Resistance in Pseudomonas aeruginosa using MALDI-TOF Mass Spectrometry

BackgroundVarieties of methods have been suggested for rapid detection of carbapenem resistance in gram-negative rods. However, their clinical application is hampered by either detection of only selected resistance mechanisms or labor-intensive processing. This study investigated a novel easy-to-perform, mechanism-independent method for detection of carbapenem resistance in Pseudomonas aeruginosa.MethodsClinical P. aeruginosa isolates (12 meropenem-susceptible and 12 meropenem-non-susceptible) were included. Meropenem minimum inhibitory concentrations (MICs) were determined by broth microdilution method. For the novel method, microdroplets (2, 4, 6, 8 and 10 µl) were produced by mixing meropenem-containing cation-adjusted Mueller–Hinton broth (CA-MHB) with bacterial suspension directly on a target (MBT Biotargets-96, Bruker) for MALDI-TOF mass spectrometry (MS). The meropenem concentration of 2 µg/mL and the inoculum of 5 × 105 cfu/mL were used. Three targets were inoculated and incubated in a humidity chamber for 4, 5 or 18 hours at 36°C. After incubation and removal of medium, MALDI-TOF MS was performed directly from dried spots. Isolates were interpreted as non-susceptible if MALDI Biotyper (Bruker) provided successful species identification (score ≥1.7) and as susceptible if no identification (score <1.7) was achieved.ResultsMIC50, MIC90and MIC range were 16 µg/mL, 64 µg/mL and 8–64 µg/mL for non-susceptible isolates, and 0.25 µg/mL, 0.5 µg/mL and 0.03–1 µg/mL for susceptible isolates as determined by the reference method. The novel assay demonstrated best performance with 8 µl droplets. Using this volume, 100% agreement with the reference method was achieved after 18 hours. After 4 hours, 62.5% of isolates showed successful detection of growth control without antibiotic; sensitivity and specificity were 83.3% and 100%, respectively. After 5 hours, 91.7% of isolates had successful growth control detection, with 100% sensitivity and 90.9% specificity.ConclusionThis universal and easy-to-perform method showed good performance for determination of carbapenem-non-susceptibility in P. aeruginosa after 5 hours. Longer incubation may be necessary to provide reliable results for isolates with reduced growth rate.Disclosures E. A. Idelevich, Bruker: Inventor of a pending patent which is owned by the University of Muenster and licensed to Bruker, Licensing agreement or royalty; K. Sparbier, Bruker: Employee, Salary; M. Kostrzewa, Bruker: Employee, Salary; K. Becker, Bruker: Inventor of a pending patent which is owned by the University of Muenster and licensed to Bruker, Licensing agreement or royalty

The management of multidrug-resistant Enterobacteriaceae.

Multidrug-resistant (MDR) Enterobacteriaceae are often related to the production of extended-spectrum b-lactamases (ESBLs) and carbapenemase-producing Enterobacteriaceae (CRE), and represent an increasing global threat. Recommendations for the therapeutic management of MDR-related infections, however, are mainly derived from retrospective and nonrandomized prospective studies. The aim of this review is to discuss the challenges in the treatment of patients with infections because of MDR Enterobacteriaceae and provide an expert opinion while awaiting for more definitive data. To avoid the selection of carbapenemase-producing Enterobacteriaceae, carbapenem-sparing strategies should be considered. B-lactams/b-lactamase inhibitors, mainly piperacillin-tazobactam, minimum inhibitory concentration (MIC) 16/4mg/ml or less represents the best alternative to carbapenems for the treatment of ESBL-producing strains. Overall, combination therapy may be preferred over monotherapy for CRE. The combination of a carbapenem-containing regimen with colistin or high-dose tigecycline or aminoglycoside can be administered at high-dose prolonged infusion with therapeutic drug monitoring for the treatment of CRE with MIC for meropenem 8-16 mg/l or less. For MIC higher than 8-16 mg/l, the use of meropenem should be avoided and various combination therapies based on the in-vitro susceptibility of antimicrobials (e.g., colistin, high-dose tigecycline, fosfomycin, and aminoglycosides) should be selected. Carbapenem-sparing strategies should be used, when feasible, for ESBL infections. The majority of available nonrandomized studies highlight that combination for CRE seem to offer some therapeutic advantage over monotherapy. Strict infection control measures toward MDR Gram-negative pathogens remain necessary while awaiting for new treatment options.

Bulgecin A as a β-lactam enhancer for carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii clinical isolates containing various resistance mechanisms.

Genetic screening of Pseudomonas aeruginosa (PSDA) and Acinetobacter baumannii (ACB) reveals genes that confer increased susceptibility to β-lactams when disrupted, suggesting novel drug targets. One such target is lytic transglycosylase. Bulgecin A (BlgA) is a natural product of Pseudomonas mesoacidophila and a lytic transglycosolase inhibitor that works synergistically with β-lactams targeting PBP3 for Enterobacteriaceae. BlgA also weakly inhibits di-Zn2+ metallo-β-lactamases like L1 of Stenotrophomonas maltophilia. We hypothesized that because of its unique mechanism of action, BlgA could restore susceptibility to carbapenems in carbapenem-resistant PSDA (CR-PSDA) and carbapenem-resistant ACB, as well as ACB resistant to sulbactam. A BlgA-containing extract was prepared using a previously published protocol. CR-PSDA clinical isolates demonstrating a variety of carbapenem resistance mechanisms (VIM-2 carbapenemases, efflux mechanisms, and AmpC producer expression) were characterized with agar dilution minimum inhibitory concentration (MIC) testing and polymerase chain reaction. Growth curves using these strains were prepared using meropenem, BlgA extract, and meropenem plus BlgA extract. A concentrated Blg A extract combined with low concentrations of meropenem, was able to inhibit the growth of clinical strains of CR-PSDA for strains that had meropenem MICs ≥8 mg/L by agar dilution, and a clinical strain of an OXA-24 producing ACB that had a meropenem MIC >32 mg/L and intermediate ampicillin/sulbactam susceptibility. Similar experiments were conducted on a TEM-1 producing ACB strain resistant to sulbactam. BlgA with ampicillin/sulbactam inhibited the growth of this organism. As in Enterobacteriaceae, BlgA appears to restore the efficacy of meropenem in suppressing the growth of CR-PSDA and carbapenem-resistant ACB strains with a variety of common carbapenem resistance mechanisms. BlgA extract also inhibits VIM-2 β-lactamase in vitro. BlgA may prove to be an exciting adjunctive compound to extend the life of carbapenems against these vexing pathogens.

Characterization of carbapenem-resistant Gram-negative bacteria from Tamil Nadu

Carbapenem resistance is disseminating worldwide among Gram-negative bacteria. The aim of this study was to identify carbapenem-resistance level and to determine the mechanism of carbapenem resistance among clinical isolates from two centres in Tamil Nadu. In the present study, a total of 93 Gram-negative isolates, which is found to be resistant to carbapenem by disk diffusion test in two centres, were included. All isolates are identified at species level by 16S rRNA sequencing. Minimal inhibitory concentrations (MICs) of isolates for Meropenem were tested by agar dilution method. Presence of blaOXA, blaNDM, blaVIM, blaIMP and blaKPC genes was tested by PCR in all isolates. Amplicons were sequenced for confirmation of the genes. Among 93 isolates, 48 (%52) were Escherichia coli, 10 (%11) Klebsiella pneumoniae, nine (%10) Pseudomonas aeruginosa. Minimal inhibitory concentration results showed that of 93 suspected carbapenem-resistant isolates, 27 had meropenem MICs ≥ 2 μg/ml. The MIC range, MIC50 and MIC90 were < 0.06 to >128 μg/ml, 0.12 and 16 μg/ml, respectively. Fig. 1. Among meropenem-resistant isolates, E. coli were the most common (9/48, 22%), followed by K. pneumoniae (7/9, 77%), P. aeruginosa (6/10, 60%), Acinetobacter baumannii (2/2, 100%), Enterobacter hormaechei (2/3, 67%) and one Providencia rettgeri (1/1, 100%). PCR results showed that 16 of 93 carried blaNDM, three oxa181, and one imp4. Among blaNDM carriers, nine were E. coli, four Klebsiella pneumoniae, two E. hormaechei and one P. rettgeri. Three K. pneumoniae were OXA-181 carriers. The only imp4 carrier was P. aeruginosa. A total of seven carbapenem-resistant isolates were negatives by PCR for the genes studied. All carbapenem-resistance gene-positive isolates had meropenem MICs >2 μg/ml. Our results confirm the dissemination of NDM and emergence of OXA-181 beta-lactamase among Gram-negative bacteria in South India. This study showed the emergence of NDM producer in clinical isolates of E. hormaechei and P. rettgeri in India.

Meropenem for treating KPC-producing Klebsiella pneumoniae bloodstream infections: Should we get to the PK/PD root of the paradox?

ABSTRACTThe objective of this study was to assess the achievement of pharmacokinetic/pharmacodynamic (PK/PD) targets of meropenem (MEM) in critically-ill patients with bloodstream infections (BSI) due to Klebsiella pneumoniae-carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) with MEM minimum inhibitory concentrations (MICs) ≥16 mg/L. Nineteen critically-ill patients with KPC-Kp BSI were given combination therapy including MEM, tigecycline, plus colistin or gentamicin (according to susceptibility testing). MEM was administered as an extended 3-hour infusion of 2 g every 8 hours, or adjusted according to renal function. MEM plasma concentrations were determined by high-performance liquid chromatography. PK/PD targets for MEM were defined as T > 40% 1×MIC and T > 40% 4×MIC. Possible synergisms between MEM and coadministered agents were assessed by time-kill assays based on plasma levels for MEM and on fixed plasma concentrations for the other agents. In none of 19 patients MEM reached any PK/PD target. The actual MEM MICs were 256, 512, and 1024 mg/L in 1, 3, and 15 isolates, respectively. However, theoretically, the PK/PD target of T > 40% 1×MIC could have been achieved in 95%, 68%, 32% and 0% of the isolates for MIC equal to 8, 16, 32, and 64 mg/L, respectively. No synergisms were observed between MEM and coadministered agents. In conclusion, high-dose MEM failed to reach PK/PD targets in 19 patients with BSI due to KPC-Kp with very high MEM MICs. On a theoretical basis, our results suggest a possible usefulness of MEM against resistant blood isolates with MICs up to 32 mg/L.

Activity of Colistin in Combination with Meropenem, Tigecycline, Fosfomycin, Fusidic Acid, Rifampin or Sulbactam against Extensively Drug-Resistant Acinetobacter baumannii in a Murine Thigh-Infection Model.

Few effective therapeutic options are available for treating severe infections caused by extensively drug-resistant Acinetobacter baumannii (XDR-AB). Using a murine thigh-infection model, we examined the in vivo efficacy of colistin in combination with meropenem, tigecycline, fosfomycin, fusidic acid, rifampin, or sulbactam against 12 XDR-AB strains. Colistin, tigecycline, rifampin, and sulbactam monotherapy significantly decreased bacterial counts in murine thigh infections compared with those observed in control mice receiving no treatment. Colistin was the most effective agent tested, displaying bactericidal activity against 91.7% of strains at 48 h post-treatment. With strains showing a relatively low minimum inhibitory concentration (MIC) for meropenem (MIC ≤ 32 mg/L), combination therapy with colistin plus meropenem caused synergistic inhibition at both 24 h and 48 h post-treatment. However, when the meropenem MIC was ≥64 mg/L, meropenem did not significantly alter the efficacy of colistin. The addition of rifampin and fusidic acid significantly improved the efficacy of colistin, showing a synergistic effect in 100% and 58.3% of strains after 24 h of treatment, respectively, while the addition of tigecycline, fosfomycin, or sulbactam did not show obvious synergistic activity. No clear differences in activities were observed between colistin-rifampin and colistin-fusidic acid combination therapy with most strains. Overall, our in vivo study showed that administering colistin in combination with rifampin or fusidic acid is more efficacious in treating XDR-AB infections than other combinations. The colistin-meropenem combination may be another appropriate option if the MIC is ≤32 mg/L. Further clinical studies are urgently needed to confirm the relevance of these findings.