Imipenem Minimum Inhibitory Concentrations Research Articles

Molecular epidemiology of Acinetobacter baumannii and Acinetobacter nosocomialis in Germany over a 5-year period (2005–2009)

To investigate the species distribution within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex and the molecular epidemiology of A. baumannii and Acinetobacter nosocomialis, 376 Acinetobacter isolates were collected prospectively from hospitalized patients at 15 medical centres in Germany during three surveillance studies conducted over a 5-year period. Species identification was performed by molecular methods. Imipenem minimum inhibitory concentrations (MIC) were determined by broth microdilution. The prevalence of the most common carbapenemase-encoding genes was investigated by oxacillinase (OXA) -multiplex polymerase chain reaction (PCR). The molecular epidemiology was investigated by repetitive sequence-based PCR (rep-PCR; DiversiLab™). Acinetobacter pittii was the most prevalent Acinetobacter species (n = 193), followed by A. baumannii (n = 140), A. calcoaceticus (n = 10) and A. nosocomialis (n = 8). The majority of A. baumannii was represented by sporadic isolates (n = 70, 50%) that showed unique rep-PCR patterns, 25 isolates (18%) clustered with one or two other isolates, and only 45 isolates (32%) belonged to one of the previously described international clonal lineages. The most prevalent clonal lineage was international clone (IC) 2 (n = 34) and IC 1 (n = 6). According to CLSI, 25 A. baumannii isolates were non-susceptible to imipenem (MIC ≥ 8 mg/L), all of which produced an OXA-58-like or OXA-23-like carbapenemase. The rate of imipenem susceptibility among A. baumannii isolates decreased from 96% in 2005 to 76% in 2009. All other Acinetobacter isolates were susceptible to imipenem. The population structure of carbapenem-susceptible A. baumannii in Germany is highly diverse. Imipenem non-susceptibility was strongly associated with the clonal lineages IC 2 and IC 1. These data underscore the high clonality of carbapenem-resistant A. baumannii isolates.

Synergy of imipenem/colistin methanesulfonate combinations against imipenem-nonsusceptible multidrug-resistant Acinetobacter baumannii

The optimal combination ratio of imipenem to colistin methanesulfonate (CMS) against imipenem-nonsusceptible multidrug-resistant Acinetobacter baumannii (INS-MDRAB) has not been determined in previous studies. To provide an alternative therapeutic option for clinical INS-MDRAB isolates, we investigated whether clinically achievable serum concentrations of CMS in combination with imipenem enhance the in vitro activity of imipenem against the INS-MDRAB isolates. Fifty-nine INS-MDRAB isolates with imipenem minimal inhibitory concentration (MIC) values of ≥8 mg/L were selected randomly from the Clinical Microbiology Laboratory at a university-affiliated medical center between July 1998 and May 2005. The in vitro activity of imipenem among these 59 clinical isolates was explored via serial two-fold dilutions containing a range of imipenem concentration from 0.125 mg/L to 256 mg/L, in combination with two fixed CMS concentrations at 0.5 mg/L and 1 mg/L. Genotype classification was performed using the pulsed-field gel electrophoresis method and infrequent-restriction-site polymerase chain reaction. A significant reversal of imipenem resistance (i.e., MICs ≤ 4 mg/L) was observed in 34 (57.6%) isolates and 44 (74.6%) isolates with the tests of CMS concentrations at 0.5 mg/L and 1 mg/L, respectively (p = 0.041). Genotype 1 was predominant (43 isolates, 72.9%) with imipenem resistance reversal rates of 51.2% and 79.1% (p = 0.004) in the tests of CMS at 0.5 mg/L and 1 mg/L, respectively. The synergy of imipenem/CMS against INS-MDRAB was significantly better for the CMS concentration at 1 mg/L than that at 0.5 mg/L, especially in our predominant clone. Our results provided insightful information for treating INS-MDRAB infections in clinical practice.

Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldtfm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldtfm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldtfm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldtfm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldtfm acylation was followed by rupture of the C5–C6 bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldtfm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test®. These were subjected to Real time PCR for blaIMP-1 and 2, blaVIM-1 and 2, blaOXA23, 24, 51 and 58 using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase blaNDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for blaOXA-51 like gene as well as blaIMP-1 and blaVIM-1 genes. blaOXA 24 and 58 were not detected in any of the isolates. blaIMP-2, blaVIM-2, blaOXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel blaNDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the blaNDM-1 gene. The presence of the novel gene blaNDM-1indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.

Carbapenem susceptibilities and non-susceptibility concordance to different carbapenems amongst clinically important Gram-negative bacteria isolated from intensive care units in Taiwan: Results from the Surveillance of Multicentre Antimicrobial Resistance in Taiwan (SMART) in 2009

To investigate the in vitro susceptibilities to various carbapenems amongst clinical Gram-negative bacteria isolated from patients in intensive care units of ten major teaching hospitals in Taiwan in 2009, a survey was conducted to determine the minimum inhibitory concentrations (MICs) of ertapenem, imipenem, meropenem and doripenem against isolates of Enterobacteriaceae (n=594), Pseudomonas aeruginosa (n=185), Acinetobacter baumannii (n=192) and Burkholderia cepacia (n=23) using the agar dilution method. Susceptibilities were determined according to 2009, 2011 and 2012 MIC breakpoints recommended by the CLSI as well as 2012 MIC breakpoints recommended by EUCAST. Based on CLSI 2012 criteria, the ertapenem susceptible rate was 93%, 81%, 68% and 92% for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens, respectively. All Proteus mirabilis and Morganella morganii isolates were susceptible to ertapenem; however, 64% of P. mirabilis and all M. morganii isolates were non-susceptible to imipenem. Meropenem and doripenem had better activities than imipenem against ertapenem-non-susceptible Enterobacteriaceae isolates. E. coli, K. pneumoniae and E. cloacae with ertapenem MICs≥4mg/L were synchronously not susceptible to imipenem, meropenem and doripenem. Imipenem susceptibility was 65% and 29% for P. aeruginosa and A. baumannii, respectively. Additionally, P. aeruginosa and A. baumannii isolates with imipenem MICs≥8mg/L were also not susceptible to meropenem and doripenem. These data provide a better understanding of choosing appropriate carbapenem agents to treat infections caused by ertapenem-non-susceptible Enterobacteriaceae as well as P. aeruginosa and A. baumannii isolates with imipenem MICs≥4mg/L.

Isolation and molecular characterization of New Delhi metallo-beta-lactamase-1 producing superbug in Bangladesh

New Delhi metallo-beta-lactamse-1 (NDM-1) producing superbugs create a global public health problem because of their resistance to most antibiotics. This study was conducted to determine the presence of MBL producers, including NDM-1 producers, in Bangladesh, along with the antimicrobial resistance patterns of these organisms. Thirty-five isolates resistant to imipenem by disk diffusion technique were investigated for MBL production. Minimum inhibitory concentration (MIC) of imipenem was determined by agar dilution method. MBL producers were phenotypically detected by double disk synergy test and combined disk assay. Gene encoding blaIMP-1, blaIMP-2, blaVIM-1, blaVIM-2, blaNDM-1 and class 1 integron was identified by PCR. Thirty-one (88.57%) MBL producers were detected by PCR, 24 (68.57%) by double disk synergy test, and 30 (85.71%) by combined disk assay. Eight (22.86%) were positive for blaNDM-1, 13 (37.15%) for blaVIM-1, 21 (60.00%) for blaVIM-2, 18 (51.43%) for blaIMP-1, and 9 (25.71%) for blaIMP-2. More than one blaMBL was present in 23 (65.71%) of the isolates. MIC of imipenem of MBL producers ranged from ≥256 µg/ml to ≤8 µg/ml. All the NDM-1 producing isolates carried class 1 integron. NDM-1 producers were 100% resistant to amoxicillin, cephradine, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam, gentamicin and piperacillin, 87.5% to amikacin, 75% to ciprofloxacin, and 62.5% to co-trimoxazole and the combination of tazobactam and piperacillin. All were sensitive to colistin. The results of this study provide insight into the presence of blaMBL, including blaNDM-1, in Bangladesh. Urgent epidemiological monitoring of MBL producers in Bangladesh may combat their rapid dissemination.

OXA-181 Beta Lactamase is not a Major Mediator of Carbapenem Resistance in Enterobacteriaceae

Detection of carbapenem hydrolyzing class D beta lactamase OXA-181, (a variant of OXA-48) in Enterobacteriaceae, is important, to institute appropriate therapy and to initiate preventive measures. This study was done to determine the presence of OXA 48 and its derivative OXA-181 in Enterobacteriaceae of pathogenic significance. One hundred and eleven non-repetitive Enterobacteriaceae isolates which were resistant to any of the cephalosporin subclasses III and which exhibited reduced susceptibility to carbapenems were included in the study. Minimum inhibitory concentrations (MICs) to imipenem and meropenem was determined by broth microdilution. Production of carbapenamase was screened by Modified Hodge test (MHT). Polymerase Chain Reaction (PCR) was done to detect the presence of bla OXA-181 and bla OXA-48 .Coexistence of other carbapenemase encoding genes, namely, NDM-1, VIM, IMP and KPC were also looked for, by PCR. Of all the isolates which were tested, only 2 (1.8%) revealed the presence of OXA-181 and OXA-48. These were Klebsiella pneumoniae and Citrobacter freundii. MICs of imipenem and meropenem for Klebsiella pneumoniae were 128mg/l and 64 mg/l and for Citrobacter freundii, they were 32mg/l and 16mg/l respectively. MHT was positive in both isolates. Production of OXA-48 / OXA-181 is not a major mechanism of carbapenem resistance. PCR is the gold standard for its routine identification in clinical microbiology laboratory.

First Survey of Metallo-β–Lactamase Producers in Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province

: Background: Treatment of infectious diseases is becoming more challenging with each passing year. This is especially true for infections caused by Pseudomonas aeruginosa, an opportunistic pathogen with the ability to rapidly develop resistance to multiple classes of antibiotics. Objectives: This study was conducted to determine the prevalence of metallo-β-lactamase (MBL)–producing strains among multidrug-resistant P. aeruginosa strains isolated from burn patients. Materials and Methods: The isolates were identified, tested for susceptibility to various antimicrobial agents, and screened for the presence of MβLs by using the double-disk synergy test. The minimal inhibitory concentration of imipenem was determined by microplate broth dilution method on Mueller-Hinton agar. To detect VIM, SIM, and GIM MBLs, the isolates were subjected to polymerase chain reaction. Results: In this study, we identified 100 P. aeruginosa isolates from 176 clinical specimens obtained from burn patients. The isolates showed maximum resistance to ampicillin (100%), ceftazidime (94%), and ceftriaxone (89%). The CLSI-MBL phenotypic test showed that of the 100 P. aeruginosa isolates, 22 (22%) were positive for MBL production in the double-disk synergy test. Of the 22 MBL-positive P. aeruginosa isolates, 8 were resistant to imipenem. PCR analysis showed that 8 isolates were positive for blaVIM1. The other genes blaSIM1 and blaGIM1 were not detected. Conclusions: The study results demonstrate the serious therapeutic threat of the spread of MBL producers among P. aeruginosa populations. Metallo-β-lactamases were detected in 22% of imipenem-resistant P. aeruginosa isolates. Early detection and infection-control practices are the best antimicrobial strategies for this organism; therefore, systematic surveillance to detect MBL producers is necessary.

Molecular analysis of the carbapenem and metronidazole resistance mechanisms of Bacteroides strains reported in a Europe-wide antibiotic resistance survey

Here we examine the carbapenem and metronidazole resistance mechanisms of 640 Bacteroides strains reported in the 2008–2009 European antibiotic susceptibility survey. Of the 22 strains with elevated imipenem minimum inhibitory concentrations (≥4μg/mL), 10 were cfiA-positive and out of these 5 carried activating insertion sequence (IS) elements in the upstream regions of the cfiA genes. However, resistant strains with cfiA genes but with no activating IS elements were found (n=2) as well as a resistant strain with no cfiA gene. In the former the resistance phenotypes by Etest were heterogeneous, whilst in the latter no carbapenemase production was seen; both mechanisms have been rarely observed, examined and characterised. Interestingly, few (n=3) nim-positive strains were found, including one metronidazole-resistant strain harbouring nimE activated by ISBf6, and two susceptible strains harbouring chromosomally located nim genes.

ISAba825 controls the expression of the chromosomal b/aOXA-51-like and the plasmid borne blaOXA-58 gene in clinical isolates of Acinetobacter baumannii isolated from the USA

Four non-repetitive, clonally related (ST114), carbapenem-resistant Acinetobacter baumannii strains isolated in the USA were examined to understand the mechanisms of carbapenem resistance including screening for the presence of an insertion sequence upstream of the b/aOXA-51-like gene, which could be involved in the control and expression of the antibiotic-resistance gene. We observed that the main mechanisms of carbapenem resistance were the result of the over-expression of the b/aOXA-58-like and the b/aOXA-65 gene, both of which had the presence of ISAba825 upstream of the genes. The importance of this element was shown by isolating plasmid-cured isogenic strains that had lost the plasmid with the ISAba825-b/aOXA-58-like genes but during that same process also lost the chromosomal ISAba825 element present upstream of the b/aOXA-65 gene. A 16-fold decrease in minimum inhibitory concentration of imipenem and an eight-fold decrease in the minimum inhibitory concentration of meropenem were seen in the isogenic strains that lost the plasmid. The study presents the first report of ISAba825 simultaneously governing the b/aOXA-65 gene and the b/aOXA-58-like gene expression and also highlights the importance of this element in carbapenem-sensitive isogenic strains, which were once carbapenem resistant.

Evaluation in vitro of the efficacy of colistin methanesulfonate against biofilm-forming multidrug-resistant Pseudomonas aeruginosa (MDRP)

The aim of this study was to evaluate in vitro the efficacy of clinically using colistin methanesulfonate against biofilm-forming multidrug-resistant Pseudomonas aeruginosa (MDRP), with minimum inhibitory concentrations (MICs) of ciprofloxacin, imipenem, and amikacin showing ≥4, 16, and 32μg/ml, respectively, by disk diffusion susceptibility testing (CLSI document M100-S21). The minimum eradication biofilm concentration (MBEC) of colistin methanesulfonate for strain MDRP-YMD isolated from a patient's urine, which formed a biofilm on plastic pegs attached to a microplate lid, was compared with that of P. aeruginosa ATCC27853 for quality control testing with MICs of ciprofloxacin, imipenem, and amikacin showing ≤1, 4, and 16μg/ml, respectively. In an uneven biofilm approximately 10μm thick, as determined with confocal laser scanning microscopy (CLSM), ratios of MBEC to MIC of colistin methanesulfonate against strains MDRP-YMD and ATCC27853 were 10.5 and 8.0, whereas those of minimum bactericidal concentration (MBC) to MIC in planktonic cells were 1.0 and 2.0μg/ml, respectively. Morphological examination using scanning electron microscopy and CLSM verified that embedded cells in biofilm matrices of the two strains were disrupted and died under the MBEC. Therefore, bactericidal effects of colistin methanesulfonate on biofilm-forming cells of strain MDRP-YMD as well as strain ATCC27853 were significantly decreased compared with those on the planktonic cells.

Study of the Correlation of Imipenem Resistance with Efflux Pumps AdeABC, AdeIJK, AdeDE and AbeM in Clinical Isolates of Acinetobacter baumannii

Background: To investigate the role of efflux pumps AdeABC, AdeIJK, AdeDE and AbeM in reduced susceptibility to imipenem in clinical isolates of Acinetobacter baumannii. Methods: Susceptibility testing was conducted by the agar dilution method. The presence and expression of pump genes were analyzed by conventional PCR and real-time RT-PCR, respectively. Results: Most antibiotics, including carbapenem, aminoglycosides, fluoroquinolones and cephalosporins showed high minimum inhibitory concentration (MIC) values in imipenem-resistant A. baumannii (IRAB). After exposure to the efflux pump inhibitor Phe-Arg-β-naphthylamide, a 4- to 32-fold reduction of imipenem MICs was observed in 33 (66%) isolates of IRAB, while no significant decrease occurred in imipenem-susceptible A. baumannii (ISAB). The majority of IRAB isolates (>80%) were found to be positive for adeB, adeR, adeS, adeJ and abeM. Positive adeE was discovered in 6 isolates of IRAB and coexisted with adeB. Approximate 4-, 3- and 11-fold increases in adeB, adeJ and abeM expression were observed, respectively, in IRAB compared to those in ISAB. Conclusions: A variety of efflux pump systems, including AdeABC, AdeIJK and AbeM, seem to play important roles in the resistance to imipenem of A. baumannii. Moreover, this is the first time that adeE has been found to coexist with adeB in a small number of isolates of A. baumannii.

Resistance of β-lactamase–producing Klebsiella pneumoniae to Imipenem with Ompk36 loss

The present study investigated the mechanisms involved in decreased imipenem susceptibility in Klebsiella pneumoniae. Pulsed field gel electrophoresis was used for homology analysis, and a modified Hodge test was used to detect carbapenemase expression. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for metallo-β-lactamase. An E-test and a conjugation experiment were used to detect drug susceptibility, whereas specific polymerase chain reaction (PCR) amplification and sequencing, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andcarbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibition tests were used to analyze mechanisms underlying the decrease in imipenem susceptibility in K. pneumoniae. Kp1, Kp2, and Kp3 did not belong to the same clone strain and their minimum inhibitory concentrations for imipenem and meropenem were 32, 4, and 24 µg/ml and 4, 8, and 4 µg/ml, respectively. Kp1 and Kp3 were resistant to piperacillin/tazobactam, whereas Kp2 was not. Kp1, Kp2, and Kp3 were highly resistant to cephalosporins and aztreonam. Kp2 and Kp3 were resistant to gentamicin. All produced β-lactamase DHA-1, CTX-M-14, SHV-12, and KPC-2. Kp3 produced CMY-2 and cephalosporinase. Gene sequencing analysis and outer membrane porin SDS-PAGE showed that the Ompk36 gene of Kp1 and Kp3 was inserted by IS5 and IS903, which resulted in the loss of the outer membrane porin Ompk36. CCCP did not increase the sensitivity of Kp1, Kp2, and Kp3 to imipenem. Therefore, the loss of the outer membrane porin Ompk36 in β-lactamase KPC-2, DHA-1, CTX-M-14 and SHV-12 producing K. pneumoniae induces the development of high-level imipenem resistance. Key words: Klebsiella pneumoniae, β-lactamase, imipenem, drug sensitivity and outer membrane porin

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa

Purpose: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. Materials and Methods: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for blaIMP and blaVIM. Results: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of blaVIM. Conclusions: MEM was found to be the best screening and confirmatory agent for MBL detection and blaVIM was found to be the prevalent MBL gene in this part of the country.

First Survey of Metallo-β–Lactamase Producers in Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province

Treatment of infectious diseases is becoming more challenging with each passing year. This is especially true for infections caused by Pseudomonas aeruginosa, an opportunistic pathogen with the ability to rapidly develop resistance to multiple classes of antibiotics. This study was conducted to determine the prevalence of metallo-β-lactamase (MBL)-producing strains among multidrug-resistant P. aeruginosa strains isolated from burn patients. The isolates were identified, tested for susceptibility to various antimicrobial agents, and screened for the presence of MβLs by using the double-disk synergy test. The minimal inhibitory concentration of imipenem was determined by microplate broth dilution method on Mueller-Hinton agar. To detect VIM, SIM, and GIM MBLs, the isolates were subjected to polymerase chain reaction. In this study, we identified 100 P. aeruginosa isolates from 176 clinical specimens obtained from burn patients. The isolates showed maximum resistance to ampicillin (100%), ceftazidime (94%), and ceftriaxone (89%). The CLSI-MBL phenotypic test showed that of the 100 P. aeruginosa isolates, 22 (22%) were positive for MBL production in the double-disk synergy test. Of the 22 MBL-positive P. aeruginosa isolates, 8 were resistant to imipenem. PCR analysis showed that 8 isolates were positive for blaVIM1. The other genes blaSIM1 and blaGIM1 were not detected. The study results demonstrate the serious therapeutic threat of the spread of MBL producers among P. aeruginosa populations. Metallo-β-lactamases were detected in 22% of imipenem-resistant P. aeruginosa isolates. Early detection and infection-control practices are the best antimicrobial strategies for this organism; therefore, systematic surveillance to detect MBL producers is necessary.

In Vitro Activities of Ertapenem and Imipenem against Clinical Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae Collected in Military Teaching Hospital Mohammed V of Rabat

Objective. To study the sensitivity level of extended spectrum beta-lactamase-producing Enterobacteriaceae to Carbapenems (Imipenem, Ertapenem) marketed in Morocco and discusses the place of Ertapenem in the treatment of extended spectrum-beta-lactamase-producing. Materials and Methods. A retrospective study of 110 extended spectrum beta-lactamase-producing Enterobacteriaceae. Isolates obtained from blood cultures, superficial and deep pus, and catheters were conducted. The minimum inhibitory concentrations of Imipenem and Ertapenem were done by the E-test. The modified Hodge test was conducted for resistant or intermediate strains. Results. 99.1% of isolates were susceptible to Imipenem. For Ertapenem, 4 were resistant and 4 intermediate. The modified Hodge test was positive for all 08 isolates. A minimum inhibitory concentration comparison of K. pneumoniae, E. cloacae, and E. coli for Imipenem has noted a significant difference between E. cloacae on one hand and E. coli, K. pneumoniae on the other hand (P < 0.01). No significant difference was noted for minimum inhibitory concentration of Ertapenem. Conclusion. Our results confirm in vitro effectiveness of Ertapenem against extended spectrum beta-lactamase-producing Enterobacteriaceae as reported elsewhere. However, the emergence of resistance to Carbapenems revealed by production of carbapenemases in this study confirmed a necessary bacteriological documented infection before using Ertapenem.

First Canadian Outbreak of Enterobacteriaceae‐Expressing Klebsiella pneumoniae Carbapenemase Type 3

Organisms expressing Klebsiella pneumoniae carbapenemase (KPC) are found in several regions worldwide but are rarely detected in Canada. The first outbreak of KPC-expressing strains of Enterobacteriaceae clinical isolates in a university-affiliated hospital intensive care unit (ICU) in Canada is described. Enterobacteriaceae isolates that were flagged by the Vitek 2 (bioMérieux, France) system as possible carbapenemase producers were subjected to the modified Hodge test. Modified Hodge test-positive organisms were analyzed by pulsed-field gel electrophoresis, tested for KPC and other beta-lactamase genes by polymerase chain reaction analysis and underwent subsequent nucleic acid sequencing. Antimicrobial susceptibility profiles were determined by Vitek 2 and Etest (bioMérieux, France). A chart review was conducted to establish epidemiological links. During the study period, 10 unique Enterobacteriaceae isolates expressing KPC were detected from nine ICU patients. Five patients had infections (three pneumonias, one surgical site infection, one urinary tract infection). Isolates included Escherichia coli (5), Klebsiella oxytoca (2), Serratia marcescens (2) and Citrobacter freundii (1). Polymerase chain reaction analysis and sequencing confirmed the presence of KPC-3 in all isolates; four also carried TEM, two CTX-M and one CMY-2. The imipenem minimum inhibitory concentrations as determined by Etest ranged from 0.75 μg/mL to ≥32 μg/mL. Pulsed field gel electrophoresis clonal patterns and patient location in the ICU revealed presumptive horizontal transmission events. In the present study, Enterobacteriaceae isolates with KPC are emerging and can result in serious infections. The KPC gene can spread via plasmids to different genera of the Enterobacteriaceae family. The dissemination of KPC in Enterobacteriaceae and the consequences for treatment and infection control measures warrant a high degree of vigilance among clinicians and microbiologists.

First Survey of Metallo-β–Lactamase Producers in Clinical Isolates of Pseudomonas aeruginosa From a Referral Burn Center in Kurdistan Province

: Background: Treatment of infectious diseases is becoming more challenging with each passing year. This is especially true for infections caused by Pseudomonas aeruginosa, an opportunistic pathogen with the ability to rapidly develop resistance to multiple classes of antibiotics. Objectives: This study was conducted to determine the prevalence of metallo-β-lactamase (MBL)–producing strains among multidrug-resistant P. aeruginosa strains isolated from burn patients. Materials and Methods: The isolates were identified, tested for susceptibility to various antimicrobial agents, and screened for the presence of MβLs by using the double-disk synergy test. The minimal inhibitory concentration of imipenem was determined by microplate broth dilution method on Mueller-Hinton agar. To detect VIM, SIM, and GIM MBLs, the isolates were subjected to polymerase chain reaction. Results: In this study, we identified 100 P. aeruginosa isolates from 176 clinical specimens obtained from burn patients. The isolates showed maximum resistance to ampicillin (100%), ceftazidime (94%), and ceftriaxone (89%). The CLSI-MBL phenotypic test showed that of the 100 P. aeruginosa isolates, 22 (22%) were positive for MBL production in the double-disk synergy test. Of the 22 MBL-positive P. aeruginosa isolates, 8 were resistant to imipenem. PCR analysis showed that 8 isolates were positive for blaVIM1. The other genes blaSIM1 and blaGIM1 were not detected. Conclusions: The study results demonstrate the serious therapeutic threat of the spread of MBL producers among P. aeruginosa populations. Metallo-β-lactamases were detected in 22% of imipenem-resistant P. aeruginosa isolates. Early detection and infection-control practices are the best antimicrobial strategies for this organism; therefore, systematic surveillance to detect MBL producers is necessary.

Synergistic anti-bacterial and proteomic effects of epigallocatechin gallate on clinical isolates of imipenem-resistant Klebsiella pneumoniae

Imipenem-resistant Klebsiella pneumoniae (IRKP) were used to explore the synergistic anti-bacterial and proteomic effects of imipenem alone or in combination with epigallocatechin gallate (EGCg). The minimal inhibitory concentrations (MICs) of EGCG for 12 clinically isolated IRKP strains ranged from 300 to 650 μg/ml. Each of the 12 IRKP strains experienced a 4- to 64-fold reduction in the MIC of imipenem upon co-incubation with 0.25 × MIC level of EGCg. The time-kill method was used on the 12 IRKP clinical isolates to evaluate the bactericidal activities of imipenem alone or with EGCg. Compared to imipenem alone, EGCg with imipenem demonstrated enhanced bactericidal activity. Two-dimensional polyacrylamide gel electrophoresis identified eight down-regulated and four up-regulated proteins in the IRKP strain upon exposure to 1 × MIC of EGCg. Analysis of the outer membrane protein profiles of IRKP cultures treated with EGCg revealed unique changes in outer membrane proteins. In addition, scanning electron microscopic analysis demonstrated the presence of cells with wrinkled surfaces containing perforations and irregular rod-shaped forms after treatment with EGCg or imipenem. These studies demonstrate that EGCg can synergize the bacterial activity of imipenem and differentially stimulate the expression of various proteins in IRKP.

Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility

Purpose: The present study was carried out to characterize the ESBL types and evaluated their in vitro activity against a collection of Gram negative bacteria (GNB) from a multicentric Indian surveillance study. Material and Methods: During January 2005 to June 2006, six tertiary care centres in India forwarded 778 non-duplicate GNB to our reference laboratory. Three hundred GNB from this collection were selected based on clinical significance and were used in the present study. Tested isolates included Escherichia coli (167), Klebsiella spp. (122) and Enterobacter spp. (11). ESBL screening and confirmation was performed for all the isolates. Minimum inhibitory concentration of imipenem, meropenem, ertapenem, levofloxacin, amikacin, piperacillin/tazobactam and ceftriaxone was determined by the E-test method. Molecular typing of the ESBLs was performed by polymerase chain reaction among the 121 selected isolates. Results: The study showed excellent susceptibility among the strains to imipenem (100%), meropenem (100%) and ertapenem (98.7%); good susceptibility to amikacin (89.7%) and piperacillin/tazobactam (85.3%) was observed. TEM and CTX-M were predominantly found in E. coli (39.2%) while, among the Klebsiella spp., TEM, SHV and CTX-M occurred together in 42.6% of the isolates. Conclusion: More than one ESBL was produced by many strains, and this was correlated with increased resistance levels. Carbapenems continue to show good in vitro activity and ertapenem is a potential alternative to imipenem and meropenem. Continued antimicrobial resistance surveillance is warranted in light of these findings.