Performance evaluation of a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ASTA MicroIDSys system, in bacterial identification against clinical isolates of anaerobic bacteria.

INTRODUCTION: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced for bacterial identification. The ASTA MicroIDSys system (ASTA, Suwon, Korea) is a new MALDI-TOF MS system developed for species identification of microorganisms. We evaluated the performance of MicroIDSys against clinical isolates of anaerobic bacteria. MATERIAL AND METHODS: A total of 370 non-duplicated clinical isolates of anaerobic bacteria were tested in this study. Bacterial identification with MicroIDSys was performed with a direct smear method, and measured spectra were analyzed using respective software. The results of MicroIDSys were compared with the results of Bruker Biotyper and 16S rRNA sequencing. RESULTS: The overall agreement rates for the 370 clinical isolates (34 genera and 99 species) were 95.4% (353/370) at the genus level and 91.6% (n = 340) at the species level. Only 17 isolates were incorrectly identified at the genus level: five misidentifications and 12 unidentifications. The MicroIDSys system exhibited excellent performance in the identification of clinically relevant bacterial species. Most of the Bacteroides isolates (98.0%, 99/101) and all of the Clostridium difficile (100%, n = 11), Clostridium perfringens (100%, n = 10), Finegoldia magna (100%, n = 11), and Parvimonas micra (100%, n = 10) isolates were correctly identified at the species level. CONCLUSION: The MicroIDSys system proved useful in the identification of anaerobic bacteria, especially clinically relevant species. This system could be of use in clinical microbiology laboratories as a primary tool for identifying anaerobic bacteria.

Modification and evaluation of the Triton Hodge test for screening carbapenemase-producing Enterobacteriaceae.

Detection of carbapenemase-producing Enterobacteriaceae (CPE) has become critical for appropriate antimicrobial therapy and for controlling the spread of infection. We evaluated Triton Hodge test (THT) for screening CPE. A spreader can be used to apply more constant volume of Triton on whole surface of Mueller-Hinton agar (MHA), or alternatively, a 10-µL inoculating loop can be used to apply a 20% Triton solution lineally. The THT procedure can be simplified by eliminating the 1/10 dilution step of indicator bacteria from the McFarland 0.5 turbidity suspension. The presence of Triton in the MHA plates significantly increased the enhanced growth size of not only Enterobacteriaceae producing NDM-1-like enzymes but also those producing the most prevalent KPC-2-like enzyme, resulting in 100% sensitivity of the test.

Gene cloning and characterization of MdeA, a novel multidrug efflux pump in Streptococcus mutans.

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

CTX-M-55-type extended-spectrum ß-lactamase-producing Shigella sonnei isolated from a Korean patient who had travelled to China.

We report a case of CTX-M-55-type extended-spectrum ß-lactamase (ESBL)-producing Shigella sonnei infection in a 27-year-old Korean woman who had traveled to China. The patient was admitted to the hospital due to abdominal pain, watery diarrhea, and fever (39.3℃). S. sonnei was isolated from her stool specimens, and the pathogen was found to be resistant to cefotaxime due to CTX-M-55-type ESBL. Insertion sequence (IS)Ecp1 was found upstream of the blaCTX-M-55 gene. The blaCTX-M-55 gene was transferred from the S. sonnei isolate to an Escherichia coli J53 recipient by conjugation. Pulsed-field gel electrophoresis and Southern blotting revealed that the blaCTX-M-55 gene was located on a plasmid of approximately 130 kb.

Clonal change of blaSIM-1-carrying Acinetobacter spp. from 2003 to 2008 in the hospital where it was initially discovered.

SIM-1 metallo-ß-lactamase was first discovered from carbapenem-resistant Acinetobacter spp. isolated in a Korean University Hospital in 2003, and was recently reported to have been discovered in A. baylyi isolated in nearby China. The aims of this study were to reveal clonal changes in bla(SIM-1)-harboring Acinetobacter isolates collected from 2003 to 2008 in the same Korean hospital, where they were first discovered to gain further insight into the relation between bla(SIM-1)-harboring plasmids and Acinetobacter spp. Among 1,761 nonduplicated imipenem-resistant Acinetobacter spp. isolates, 29 isolates were identified as bla(SIM-1) carriers. They were categorized into nine types according to pulsed-field gel electrophoresis findings. While most bla(SIM-1)-carrying isolates from 2003 to 2005 belonged to A. pittii, those from 2006 to 2007 were mostly isolates of A. nosocomialis. Most of the bla(SIM-1) genes were carried on ca. 280-kb plasmids and were only discovered in non-baumannii Acinetobacter spp. Integrons carrying the bla(SIM-1) gene were identical in structure in all species. These findings suggest that the plasmids were transferable, but not promiscuous. Further surveillance should be continued to detect and control further spread of the bla(SIM-1) gene, as the appearance of the bla(SIM-1) gene in different Acinetobacter spp. in different countries has already begun.

Molecular mechanisms of carbapenem resistance in Enterobacter cloacae clinical isolates from Korea and clinical outcome.

We investigated the molecular mechanisms of carbapenem resistance in clinical isolates of Enterobacter cloacae and their clinical characteristics. Nonreplicable E. cloacae isolates were recovered from six cancer patients and one patient with liver cirrhosis at a tertiary-care hospital in Korea between 2002 and 2009. Two patients who were considered to have a true infection caused by these microorganisms have died. All isolates produced AmpC ß-lactamases, including ACT-1, ACT-2, MIR-3 and DHA-1, and CTX-M- or SHV-type extended-spectrum ß-lactamase. Two isolates produced plasmid-borne VIM-2 carbapenemase. All probes specific for bla(AmpC) genes hybridized with I-CeuI chromosomal fragments were also recognized by a probe specific for 16S rDNA, suggesting a chromosomal location. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a major outer membrane protein, OmpF, was absent in all isolates. PFGE of XbaI-digested DNA were considered to be unrelated. The results of our study suggest that the chromosomal AmpC ß-lactamase with impermeability in E. cloacae clinical isolates implicated in reduced carbapenem susceptibility. Although carbapenem-resistant E. cloacae isolates were isolated in a few patients in our study, the clinical outcomes were grave. Therefore, the patients colonized or infected by carbapenem-resistant E. cloacae isolates should gain attention of antibiotic therapy.

Comparison of the genetic structures surrounding qnrA1 in Korean Enterobacter cloacae and Chinese Escherichia coli strains isolated in the early 2000s: evidence for qnrA mobilization via Inc HI2 type plasmid.

The flanking genetic structure of qnrA1 in Korean Enterobacter loacae was identical to that of the Chinese Escherichia coli strain, the first qnrA1-carrying strain reported in Asia. Analysis of restriction enzyme sites and Southern blot hybridization results showed that qnrA1 was transferred between E. cloacae and E. coli via Inc HI2 type plasmid.

Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4.

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.

Chromosomal cephalosporinase in Enterobacter hormaechei as an ancestor of ACT-1 plasmid-mediated AmpC ß-lactamase.

In this study of the diversity of AmpC ß-lactamase in clinical isolates of Enterobacter spp., a strain was found carrying the plasmid-mediated AmpC ß-lactamase ACT-1 gene on its chromosome. The strain was identified as Enterobacter hormaechei using phylogenetic analysis of 16S rRNA and hsp60 genes. In addition, the species was confirmed by DNA-DNA hybridization. The genetic environment of the bla(ACT-1) gene was characterized, including the ampR and ampG genes, using a two-step PCR. The amino acid sequences of AmpR at serine 35, arginine 86, glycine 102, aspartic acid 135 and tyrosine 264 were conserved. Measurement of the transcription level of the bla(ACT-1) gene using real-time quantitative PCR showed that it increased 1.98-fold following cefoxitin induction. These results suggest that the plasmid-mediated bla(ACT-1) gene originated from the chromosome of E. hormaechei.

Biochemical characterization of the TEM-107 extended-spectrum ß-lactamase in a Klebsiella pneumoniae isolate from South Korea.

The TEM-107 extended-spectrum ß-lactamase detected in a Klebsiella pneumoniae clinical isolate had a Gly238Ser substitution compared to the TEM-43 ß-lactamase. The MIC of ceftazidime was higher (64 µg/ml) than that of cefotaxime (2 µg/ml) for the isolate. Clavulanic acid reduced the MIC of ceftazidime 64-fold.

Comparative in vitro activities of torezolid (DA-7157) against clinical isolates of aerobic and anaerobic bacteria in South Korea.

Resistance of Gram-positive pathogens to first-line antimicrobial agents has been increasing in many parts of the world. We compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic bacteria. Torezolid had an MIC(90) of ≤0.5 µg/ml for the Gram-positive bacterial isolates tested and was more potent than either linezolid or vancomycin.

Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing Gram-negative bacilli.

The detection of carbapenemases in Gram-negative bacilli is important for optimal patient treatment and to control spread of the resistance. The modified Hodge test can detect carbapenemase-producing Gram-negative bacilli. In this study, we compared the performance of MacConkey agar and Mueller-Hinton agar for metallo-ß-lactamase (MBL) and OXA carbapenemase screening. Overall, the performance of Hodge test was better with MacConkey agar due to enhanced release of ß-lactamases from the cells in the presence of bile compounds. Concomitant use of the modified Hodge test could resolve most of the problems with uncertain double-disk synergy tests in MBL detection.

Characteristics of clinical isolates of Acinetobacter genomospecies 10 carrying two different metallo-beta-lactamases.

Acquired metallo-beta-lactamase (MBL) production is an important mechanism of carbapenem resistance. To our knowledge, carriage of two different MBLs has not been described previously in Acinetobacter spp. We present the characteristics of five Acinetobacter isolates carrying two different MBL genes. The species of all five Acinetobacter isolates with two different MBL genes were Acinetobacter genomospecies 10, and bla(IMP-1), bla(VIM-2) and bla(SIM-1) may coexist in this species. Minimum inhibitory concentrations (MICs) of imipenem for all five isolates with two different MBLs were >or=32 microg/mL, whilst those for two segregants that lost both MBLs were 0.5 microg/mL. The presence of MBL gene-carrying integrons with identical structures suggested the easily transferable nature of the elements, whilst instability of the MBL genes indicated potentially erroneous phenotypic and genetic characterisation.

Genetic diversity of chromosomal metallo-beta-lactamase genes in clinical isolates of Elizabethkingia meningoseptica from Korea.

This study was performed to characterize the chromosomal metallo-beta-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5 alpha. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla (GOB) genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla (GOB) gene, including 10 novel variants (bla (GOB-8) to bla (GOB-17)). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient E. coli DH5 alpha. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino acid similarities.

New cfiA variant and novel insertion sequence elements in carbapenem-resistant Bacteroides fragilis isolates from Korea.

Of 276 nonduplicate Bacteroides fragilis clinical isolates recovered from 1997 to 2004, 3 were resistant to carbapenem. cepA and cfiA alleles were detected by polymerase chain reaction in 240 (87.0%) and 11 (4.0%) of the isolates, respectively. Insertion sequence (IS) elements were found only in the 3 carbapenem-resistant B. fragilis isolates, which produced metallo-beta-lactamase at a level detectable by UV spectrophotometry. Sequence analysis showed 1 new cfiA variant, cfiA(11), and 2 novel IS elements. The cfiA(11) gene revealed 5 amino acid substitutions compared to cfiA, with 97.6% amino acid identity. The transposase, terminal inverted repeat sequence, and target site duplication sequence of the 2 novel IS elements were unique. This study reconfirmed the correlation between ISs and carbapenem resistance in B. fragilis.

Role of OXA-23 and AdeABC efflux pump for acquiring carbapenem resistance in an Acinetobacter baumannii strain carrying the blaOXA-66 gene.

This study was performed to determine the mechanisms for acquiring carbapenem resistance in six clinical isolates of Acinetobacter baumannii. All isolates showed similar SmaI-macrorestriction patterns with less than 3 band differences by PFGE. The isolates showed a high level resistance (>32 mg/L) to both imipenem and meropenem by Etest. Phe-Arg-beta-naphthylamide lowered the MICs of carbapenems. Real-time PCR experiments showed that expression levels of the adeB gene in the six A. baumannii isolates were 10- to 40-times higher than those of imipenem-susceptible strains. Direct sequencing of PCR products showed that all isolates carried the bla(OXA-23) gene, which was preceded by ISAba1. The bla(OXA-23) probe hybridized with approximately 500-kb I-CeuI chromosomal fragments, but not with a plasmid. These findings suggest that overexpression of the AdeABC efflux pump as well as chromosome-borne OXA-23 may play a role in acquiring carbapenem resistance in our A. baumannii isolates.

Resistance trends of Bacteroides fragilis group over an 8-year period, 1997-2004, in Korea.

BACKGROUND: Bacteroides fragilis group organisms are the most frequently isolated anaerobes in human infections. Increasing resistance to various antimicrobial agents is a significant problem in choosing appropriate antimicrobial agents to treat anaerobic infections. Periodic monitoring of the regional resistance trends of B. fragilis group isolates is needed. METHODS: A total of 466 nonduplicate clinical isolates of B. fragilis group organisms (276 B. fragilis, 106 Bacteroides thetaiotaomicron, and 84 other B. fragilis group organisms) were collected during the 8-yr period from 1997 to 2004 in a Korean university hospital. Minimum inhibitory concentrations to various antimicrobial agents were determined by the CLSI agar dilution method. RESULTS: Eight isolates were resistant to imipenem. Additionally, the resistance rates to cefotetan were decreased in B. thetaiotaomicron, while those for clindamycin were significantly increased compared to the rates found in previous studies. Depending on species, resistance rates were 1-4% for imipenem, 1-6% for piperacillin-tazobactam, 4-11% for cefoxitin, 33-49% for piperacillin, 14-60% for cefotetan, and 51-76% for clindamycin. No isolates were resistant to chloramphenicol or metronidazole. CONCLUSIONS: Piperacillin-tazobactam, cefoxitin, imipenem, chloramphenicol, and metronidazole are still active against B. fragilis group isolates, while clindamycin no longer has a value as an empirical therapeutic agent in Korea. Furthermore, this study identified the first imipenem-resistant B. fragilis group isolates in Korea.

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

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

[In vitro activity of arbekacin against clinical isolates of Staphylococcus species and gram-negative bacilli].

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) and some gram-negative bacilli are very prevalent nosocomial pathogens, commonly causing mixed infections, and are often resistant to multiple drugs. Arbekacin is an aminoglycoside used for the treatment of MRSA infections, but is also active against some gram-negative bacilli. The aim of this study was to determine in vitro activity of arbekacin against recent clinical isolates of staphylococci and gram-negative bacilli. MATERIALS AND METHODS: The strains were isolated from clinical specimens of patients at Severance Hospital in 2003. Antimicrobial susceptibility was tested by the Clinical and Laboratory Standards Institute agar dilution method. The following arbekacin breakpoints were used: susceptible, /=16 microg/mL . RESULTS: All isolates of staphylococci tested were inhibited by 32-fold and >32-128-fold lower than those of amikacin and gentamicin, respectively. The resistance rates of MRSA, methicillin-susceptible S. aureus, methicillin-resistant coagulase-negative staphylococci (CNS) and methicillin-susceptible CNS were 0% to arbekacin, 0-54% to amikacin, and 24-79% to gentamicin. The MIC90s of arbekacin for Escherichia coli and Citrobacter freundii, 1 microg/mL and 16 microg/mL, were 2-4-fold and 8-16-fold lower than those of amikacin and gentamicin, respectively. However, The MIC90s of arbekacin for other species of gram-negative bacilli, 64->128 microg/mL, were similar to those of other aminoglycosides. CONCLUSIONS: Arbekacin may be a useful alternative to glycopeptides for the treatment of monomicrobial methicillin-resistant staphylococcal infections, as well as mixed infections with gram-negative bacilli, as most isolates of E. coli, C. freundii and some other gram-negative bacilli were also susceptible to arbekacin.