• Journal of Life Science
• /
• v.20 no.8
• /
• pp.1214-1220
• /
• 2010

Imipenem-resistant bacteria were isolated from clinical specimens taken from hospitalized patients in Suncheon, Korea. Fifty-four isolates were phylogenetically analyzed based on 16S rRNA gene and gyrB gene sequence comparisons. Isolates were affiliated with Pseudomonas aeruginosa (30 strains; 55.6%), Acinetobacter baumannii (21; 38.9%), Enterobacter hormaechei (2) and Pseudomonas putida (2). Twenty-two isolates produced metallo-$\beta$-lactamase (MBL); 12 Acinetobacter baumannii strains, 7 Pseudomonas aeruginosa strains, 2 P. putida strains and 1 Enterobacter hormaechei strain. Antibiotic susceptibility of the isolates was determined using the disc diffusion method and Vitek system. Strains producing metallo-$\beta$-lactamase (type IMP & VIM) were more resistant to antibiotics ceftazidime, aztreonam, amikacin and gentamicin than to strains producing OXA and SHV type of $\beta$-lactamase.

• Journal of Life Science
• /
• v.22 no.9
• /
• pp.1268-1276
• /
• 2012

The emergence and dissemination of carbapenem-resistant bacteria have resulted in limitations of antibiotic treatment and potential outbreaks of metallo-${\beta}$-lactamase (MBL) producing Pseudomonas aeruginosa resistant to carbapenems. In this study, we conducted molecular characterization of the MBL genes of the ${\beta}$-lactam drug-resistant P. aeruginosa and prepared basic data for treatment and prevention of proliferation of antimicrobial-resistant bacterial infections. Forty-two P. aeruginosa isolates of 254 were resistant to imipenem or meropenem. Among the 42 isolates, 28 isolates were positive for the Hodge test, and 23 isolates were positive for the EDTA-disk synergy test (EDST). MBLs were detected in 59.5% (25/42) of P. aeruginosa isolates. Eight isolates harbored $bla_{IMP-6}$, whereas 17 isolates harbored $bla_{VIM-2}$. The $bla_{IMP-6}$ gene was in a class 1 integron containing five gene cassettes: $bla_{IMP-6}$, qac, aacA4, $bla_{OXA-1}$, and aadA1. Some strains that produce IMP-6 and VIM-2 showed epidemiological relationships. The $bla_{IMP-6}$ gene in carbapenem-resistant P. aeruginosa showed an identical pattern to a gene cassette that was reported at a hospital in Daegu, Korea. Therefore, MBL-producing P. aeruginosa is already endemic in the community. We are concerned that the existence of carbapenem-resistant bacteria containing the blaMBL gene may increase pressure on antibiotic selection when treating infections. We believe that we should select appropriate antibiotics based on the antibiotic susceptibility test and continue the research to prohibit the emergence and spread of antibiotics resistant bacteria.

• Biomedical Science Letters
• /
• v.25 no.4
• /
• pp.321-330
• /
• 2019

Carbapenem is recently considered as the last resort of the therapeutics for gram negative bacterial infection. Increasing of organisms producing metallo-β-lactamase (MBL), we have difficulty in choosing the antimicrobial agents. Among 345 clinical isolates of Pseudomonas spp., 61 isolates (17.7%) were positive for the modified imipenem or meropenem-Hodge test and 55 isolates (15.9%) were positive for the imipenem-EDTA + SMA double disk synergy test (DDS). PCR and sequencing of bla_VIM-2-allele and bla_IMP-1-allele showed that 17 isolates of Pseudomonas aeruginosa, 9 isolates of Pseudomonas taiwnensis and 2 Pseudomonas plecoglossicida had bla_VIM-2, and 22 isolates of P. aeruginosa and one Pseudomonas otitidis had bla_IMP-6. These MBL genes were all in class 1 integron. The size of class 1 integron with bla_VIM-2 ranged from 3.5 kb to 5.5 kb in clinical isolates of Pseudomonas spp. including P. aeruginosa. bla_VIM-2 was most often located first in the class 1 integron, sometimes in the second or third position, and these integrons often had aacA4 or aadA1. Strict infection control measures are needed to more effectively prevent further spread of these MBL-producing Pseudomonas spp. In addition, MBL-producing Pseudomonas spp. is expected to continue to spread in various countries and regions.

• Biomedical Science Letters
• /
• v.17 no.2
• /
• pp.135-140
• /
• 2011

We compared three EDTA-based phenotypic screening methods for detecting IMP-1 and VIM-2 type metallo-${\beta}$- lactamase (MBL)-producing isolates of Acinetobacter and Pseudomonas spp., EDTA-double disk synergy test (EDTADDST), Etest MBL, and imipenem (IPM)-EDTA disk test. A total of 183 isolates (65 Acinetobacter spp. and 118 Pseudomonas spp. showing IPM resistance), confirmed to MBL genes by PCR, were used. The criteria for MBL production were (i) presence of a synergistic zone between IPM and EDTA disks in EDTA-DDST, (ii) reduction of IPM minimal inhibitory concentration by ${\geq}$ 3 twofold dilutions in the presence of EDTA in the Etest MBL, and (iii) ${\geq}$ 7 mm increase in the inhibition zone around the IPM plus EDTA disks compared with a sole IPM disk in the IPM-EDTA disk test. In this study using 87 MBL-producing and 96 MBL-nonproducing isolates, the sensitivities/specificities of EDTA-DDST, Etest MBL and IPM-EDTA disk tests were 94.3/78.1%, 89.7/91.7%, and 97.7/95.8%, respectively. When the threshold for the increase of the inhibition zone around the IPM plus EDTA disk over a sole IPM disk was altered to ${\geq}$ 5 mm and ${\geq}$ 8 mm for Acinetobacter spp. and Pseudomonas spp., respectively, the sensitivity and specificity of the test were 98.9% and 96.9%, respectively. Of the three EDTA-based phenotypic tests, the IMP-EDTA disk test was superior for detection of MBL-producing isolates.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.7
• /
• pp.1154-1162
• /
• 2015

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-$\beta$-lactamases (MBLs) and AmpC $\beta$lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of $\beta$-lactamases and characterized chromosomal AmpC $\beta$lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various $\beta$-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC₅₀ = $256{\mu}g/ml$) and cefepime (MIC₅₀ = $256{\mu}g/ml$). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC $\beta$-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of $\beta$-lactamases.

• Korean Journal of Microbiology
• /
• v.44 no.4
• /
• pp.339-345
• /
• 2008

This study was to detect MBL (metallo-${\beta}$-lactamase) among glucose non-fermenting Gram-negative bacilli isolated from clinical specimen and to search antimicrobial combination therapy against MBL producing Pseudomonas aeruginosa. Among fifty one isolates of Gram-negative bacilli with reduced imipenem susceptibility ($MIC{\ge}8{\mu}g/ml$), nine isolates have shown positive results in MBL detection test. They were seven Achromobacter xylosoxidans subsp. xylosoxidans and two P. aeruginosa. The results from EDTA-DDST coin-cided with those of PCR and nucleotide sequence analysis which showed the presence of $bla_{VIM-2}$. The combination of aztreonam (AZT) and piperacillin-tazobactarn (TZP) or AZT and amikacin (AN) screened by one disk synergy test showed no synergistic effect. Triple antibiotic combination therapy with AZT, TZP and AN, however, was shown to be effective and the most synergistic after 8 hrs of exposure. This result strongly suggest that the triple combination therapy of AZT, TZP, and AN could be useful for the treatment of infection caused by MBL producing Gram-negative bacilli.

• Biomedical Science Letters
• /
• v.25 no.3
• /
• pp.275-281
• /
• 2019

This study was performed to characterize the chromosomal metallo-${\beta}$-lactamases (MBLs) of Chryseobacterium indologenes isolated from Korea and to propose a clustering method of IND MBLs based on their amino acid similarities. Chromosomal MBL genes were amplified by PCR from 31 clinical isolates of E. indologenes. Nucleotide sequencing was performed by the dideoxy chain termination method using these PCR products. Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. indologenes isolates contained all $bla_{IND}$ genes. DNA sequence analysis revealed that E. indologenes isolates possessed ten types of $bla_{IND}$ gene, including seven novel variants ($bla_{IND-8}$ to $bla_{IND-14}$). The most common combination of MBL was IND-2 (n = 18). Minimum inhibitory concentrations of imipenem and meropenem for the isolates harboring novel IND MBLs were ${\geq}16{\mu}g/mL$. IND MBLs were grouped in three clusters, based on amino acid similarities.

• Korean Journal of Clinical Laboratory Science
• /
• v.38 no.3
• /
• pp.166-172
• /
• 2006

Metallo-${\beta}$-lactamase (MBL) can hydrolyze all ${\beta}$-lactams except monobactams and frequently coexists with various antibiotic resistance genes such as aminoglycoside resistance, sulfonamide resistance gene, etc. Therefore, the effective antibiotics against infections by these bacteria are markedly limited or can't even be found. We tried to search in-vitro antimicrobial combinations with synergistic effects for a VIM-2 type MBL producing Pseudomonas aeruginosa, isolated from clinical specimen. On the selection of antibiotic combinations with synergistic effects, we performed a one disk synergy test, modified Pestel's method, in agar without aztreonam (AZT). The bacteriostatic synergistic effects of this tests were scored as $S_1$ (by susceptibility pattern in agar without antibiotics), $S_2$ (by the change of susceptibility in agar with or without antibiotics) and $S_3$ ($S_1$ + $S_2$) and was classified into weak (1 point), moderate (2 points) and strong (3 points) by $S_3$ score. Subsequently, we carried out the time-killing curve for the antibiotic combinations with the strong synergistic bacteriostatic effect. One VIM-2 type MBL producing P. aeruginosa confirmed by the PCR showed all resistance against all ${\beta}$-lactams except AZT, aminoglycoside and ciprofloxacin. In the one disk synergy test, this isolate showed a strong bacteriostatic synergistic effect for the antibiotic combination of AZT and piperacillin-tazobactam (PIP-TZP) or AZT and amikacin (AN). On the time-killing curve after six hours of incubation, the colony forming units (CFUs/mL) of this bacteria in the medium broth with both combination antibiotics were decreased to 1/18.7, 1/17.1 of the least CFUs of each single antibiotics. The triple antibiotic combination therapy including AZT, PIP-TZP and AN was shown to be significantly synergistic after 8 hrs of exposure. In a VIM-2 MBL producing P. aeruginosa with susceptibility for AZT, the triple antibiotic combination therapy including AZT, PIP-TZP and AN may be considered as an alternative antibiotics modality against the infection by some MBL type. But the antimicrobial combination therapy for many more MBL producing isolates is essential to know as soon as possible for the selection of effective treatment against the infection by this bacteria.

• Korean Journal of Clinical Laboratory Science
• /
• v.51 no.4
• /
• pp.444-452
• /
• 2019

Carbapenem resistance, mediated by the major acquired metallo-β-lactamase (MBL) genes, has been increasingly reported, particularly for clinical isolates of Acinetobacter spp. Of the 191 nonduplicate clinical isolates of the carbapenem-nonsusceptible Acinetobacter spp. evaluated, 125 isolates (65.4%) were positive for the modified imipenem or meropenem-Hodge test, and 49 isolates (25.7%) were positive for the imipenem-EDTA+SMA double disk synergy test (DDS). PCR and sequencing of the bla_VIM-2-allele and bla_IMP-1-allele showed that 29 A. baumannii isolates and 1 A. calcoaceticus isolate had bla_VIM-2, whereas 16 A. baumannii isolates and 2 A. calcoaceticus isolates had bla_IMP-6; 1 isolate of the A. genomospecies 3 had bla_VIM-2 and bla_AIM-1. All the above MBL genes belong to class 1 integron. The size of class 1 integron encompassing bla_VIM-2 or bla_IMP-6 ranges from 2.8 kb to 3.2 kb in clinical isolates of A. baumannii, and 3.2 kb to 3.5 kb in clinical isolates of A. genomospecies 3. bla_VIM-2 was most often located first or second in the class 1 integron, and these integrons often included aacA4. Due to dispersion of the MBL-producing Acinetobacter spp. as well as integron, which may encompass various resistance genes, there is an expectation for the increase of multidrug resistant Gram-negative bacteria, including resistance of carbapenems such as imipenem or meropenem. Hence, the development of new antimicrobial agents for treating severe Acinetobacter spp. infections is needed.

• Korean Journal of Clinical Laboratory Science
• /
• v.44 no.2
• /
• pp.81-85
• /
• 2012

This study was undertaken to evaluate phenotypic and genotypic methods for detection of Metallo-Beta-Lactamases (MBLs) among nosocomial Pseudomonas aeruginosa. Of the 50 P. aeruginosa isolates from clinical specimens, 20 were evaluated for carbapenem resistance and screened for MBL by double-disk synergy test and combined-disk test. Nineteen strains (95%) were found to be MBL producers among the 20 P. aeruginosa. MBL positives were further confirmed by Polymerase Chain Reaction (PCR). For the IMP and VIM types of MBLs, PCR analysis was performed on 19 of the 20, and 10 were positive for VIM MBL type. This study reports the validation of a simple and accurate MBL detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of MBL-carrying organisms, including those with susceptibility to carbapenems, is of paramount clinical importance, as it allows rapid initiation of strict infection control practices as well as therapeutic guidance for confirmed infection.Key Words : Hepatitis A virus (HAV), Anti-HAV, Hospital workers, Prevalence, Vaccination