• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.394-401
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• 2016

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/Cas9 technology to kill extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multidrug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among >1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of the CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.

• Journal of Microbiology and Biotechnology
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• v.24 no.3
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• pp.421-426
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• 2014

To characterize the extended-spectrum beta-lactamases (ESBLs) in diarrheagenic Escherichia coli from Korea in 2008-2011, we screened seven enterotoxigenic E. coli (ETEC) and one enteroaggregative E. coli (EAEC) that produce ESBLs from a nationwide survey. All eight isolates produced CTX-M-type ESBLs, including CTX-M-12 (n = 4), CTX-M-14 (n = 2), and CTX-M-15 (n = 2). PCR-based replicon typing indicated that the $bla_{CTX-M-12}$ genes of four ETEC isolates were carried on a conjugative IncF plasmid, whereas the $bla_{CTX-M-14}$ of one EAEC was located on an IncK plasmid. This is the first report of the occurrence of $bla_{CTX-M}$ genes in clinical isolates of EAEC in Korea. The ESBL-producing isolates were shown to be different based on pulsed-field gel electrophoresis and multilocus sequence typing, whereas the four isolates with CTX-M-12 were clonally related. These observations raise an alarm for the spread of plasmid-mediated resistance to ESBL among diarrheagenic E. coli.

• Tuberculosis and Respiratory Diseases
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• v.49 no.1
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• pp.72-81
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• 2000

Backgrounds : Authors evaluated the quantitative culture of bronchoalveolar lavage fluid(BALF) in patients who were being treated with antimicrobial agents and the characteristics of isolated microorganism. Method : A prospective study was done with 25 patients under mechanical ventilation and antimicrobial treatment in ICU and NCU of Yongdong Severance Hospital from Apr. to Sep. 1999. Patients were classified into two groups: control group (n=5) and patients with VAP (n=20). The threshold of quantitative culture of BAL fluid in the diagnosis of VAP was $10^4$ cfu/ml. Results : 1) In gram staining of BALF, one patient in the control group and four in the VAP group showed positive results. Quantitative culture of BALF showed no organisms in the patients in the control group and in 9 VAP patients. Therefore the overall sensitivity was 43.8%. 2) Frequency of isolated organisms cultured above diagnostic threshold was in the following order: E. cloaclae, S. aureus, K. pneumoniae, and A. baumani. S. aureus and Staphylococcus coagulase(-) were a11 resistant to oxacillin. Seven out of 10 isolated G (-) organisms were suspected to be organisms producing extended spectrum $\beta$-lactamase (ESBL). 3) The concurrence between gram staining of sputum aspiration and that of BALF was only in 1 case. And the concurrence of culture results was observed in 3 cases. Conclusion : The sensitivity of gram staining and quantitative culture of BALF from patients under antibiotic therapy and the concordance rate between conventional tracheal aspiration and BAL were low, facts which were important in interpretation the data. Since the frequency of drug resistance organisms was not different from that of foreign data, antibiotics must be prudently selected and used.

• Childhood Kidney Diseases
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• v.19 no.2
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• pp.148-153
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• 2015

Purpose: We investigated trends in antibiotic resistance for gram-negative bacteria in infants with a urinary tract infection (UTI) over 15 years at a single institution. Methods: A retrospective chart review was conducted for children younger than 24 months who visited the emergency room and were diagnosed with a UTI between January 2000 and December 2014. We selected urine culture data that grew Escherichia coli and Klebsiella pneumoniae. Baseline clinical information and results of antimicrobial susceptibility tests were analyzed by dividing the 15-year study time frame into three periods (A: 2000-2004, B: 2005-2009, and C: 2010-2014). Results: During the study period, 478 applicable children were identified (E. coli, 89.7% and K. pneumoniae, 10.3%). Antibiotic resistance to third-generation cephalosporins was increased from period A to period C (A, 2.1%; B, 8.3%; C, 8.8%; P=0.025). Resistance to quinolones also showed a steady pattern during periods A to C, although it was not statistically significant (A, 7.9%; B, 9.7%; C, 12.4%; P=0.221). The incidence of Extended-spectrum ${\beta}$-lactamase (ESBL)-producing gram-negative bacteria increased from period A to period C (A, 1.4%; B, 7.6%; C, 8.2%; P=0.012). Conclusion: This study revealed that the common uropathogens E. coli and K. pneumoniae experienced increasing resistance rates against third-generation cephalosporins and a constant antibiotic resistance to quinolones in children younger than 24 months. We also showed a recent increased incidence of ESBL-producing gram-negative bacteria in patients with community-acquired UTIs. Therefore, it is necessary to actively surveil resistance in order to properly select empirical antibiotics.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1377-1383
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• 2006

Among 46 Acinetobacter baumannii isolates collected in 2004, two imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Republic of Korea. Two carbapenemase-producing isolates were further investigated to determine the mechanism of resistance. These isolates were analyzed by antibiotic susceptibility testing, microbiological tests of carbapenemase activity, determination of pI, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Two cases of infection by A. baumannii producing the IMP-1 ${\beta}$-lactamase were detected. The isolates were characterized by a modified cloverleaf synergy test and EDTA-disk synergy test. Isoelectric focusing of crude bacterial extracts revealed nitrocefin-positive bands with a pI value of 9.0. PCR amplification and characterization of the amplicons by direct sequencing indicated that the isolates carried a $bla_{IMP-l}$ determinant. The isolates were characterized by a multidrug resistance phenotype, including penicillins, extended-spectrum cephalosporins, carbapenems, and aminoglycosides. These results indicate that the observed imipenem resistance of two Korean A. baumannii isolates was due to the spread of an IMP-1-producing clone. Our microbiological test of carbapenemase activity is simple to screen class B metallo-${\beta}$-lactamase-producing clinical isolates to determine their clinical impact and to prevent further spread. This study shows that the $bla_{IMP-l}$ resistance determinant, which is emerging in Korea, may become an emerging therapeutic problem, since clinicians are advised not to use extended-spectrum cephalosporins, imipenem, and aminoglycosides. This observation emphasizes the importance of having effective control measures in Asian hospitals, such as early detection of colonized patients, isolation procedures, and a judicious use of antibiotics.

• Journal of Microbiology
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• v.44 no.4
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• pp.423-431
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• 2006

Among 53 Acinetobacter baumannii isolates collected in 2004, nine imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Korea. Nine carbapenemase-producing isolates were further investigated in order to determine the mechanisms underlying resistance. These isolates were then analyzed via antibiotic susceptibility testing, microbiological tests of carbapenemase activity, pI determination, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. One outbreak involved seven cases of infection by A. baumannii producing OXA-23 ${\beta}-lactamase$, and was found to have been caused by a single ERIC-PCR clone. During the study period, the other outbreak involved two cases of infection by A. baumannii producing IMP-1 ${\beta}-lactamase$. The two clones, one from each of the outbreaks, were characterized via a modified cloverleaf synergy test and an EDTA-disk synergy test. The isoelectric focusing of the crude bacterial extracts detected nitrocefin-positive bands with pI values of 6.65 (OXA-23) and 9.0 (IMP-1). The PCR amplification and characterization of the amplicons via direct sequencing showed that the clonal isolates harbored $bla_{IMP-1}$ or $bla_{oxA-23}$ determinants. The two clones were characterized by a multidrug resistance phenotype that remained unaltered throughout the outbreak. This resistance encompassed penicillins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. These results appear to show that the imipenem resistance observed among nine Korean A. baumannii isolates could be attributed to the spread of an IMP-lor OXA-23-producing clone. Our microbiological test of carbapenemase activity is a simple method for the screening of clinical isolates producing class D carbapenemase and/or class B $metallo-{\beta}-lactamase$, in order both to determine their clinical impact and to prevent further spread.

• Childhood Kidney Diseases
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• v.20 no.2
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• pp.37-44
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• 2016

Although asymptomatic bacteriuria, cystitis, and acute pyelonephritis (APN) have been categorized as urinary tract infections (UTIs), the immunopathogenesis of each disease is different. APN shows an age predilection; the majority of children (over 70-80%) with APN are under 1-2 years of age, with a male predominance. After 1-2 years of age, female predominance has been reported. This finding suggests that the immature immune state of infancy may be associated with the pathogenesis of APN. Escherichia coli is the most common etiologic agent; other uropathogens associated with UTIs originate from the host and comprise normal flora that are continuously altered by environmental factors. Therefore, uropathogens may have characteristics different from those of extraneous bacterial pathogens. Although antibiotic-resistant uropathogens, including extended-spectrum beta-lactamase-producing strains, are increasing in Korea and worldwide, treatment failure is rare in immune-competent children. The immunopathogenesis of APN remains unknown. Intact bacteria may not be the causative substances in renal cell injury; rather, smaller substances produced during bacterial replication may be responsible for renal cell injury and scarring. Moreover, substances from host cells such as proinflammatory cytokines may be involved in renal cell injury. A dimercaptosuccinic acid scan is used to detect the site of bacterial replication in the renal parenchyma, and may be influenced by the size of the focus and the stage of APN. Traditional aggressive studies used to identify vesicoureteral reflux after the first episode of APN have been modified because of rare cases of chronic kidney disease in patients with recurrent UTI.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.765-770
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• 2014

This study aimed to characterize CTX-M producers of urinary E. coli and K. pneumoniae isolates and to determine the prevalence of plasmid-mediated antimicrobial resistance genes among them. Minimum inhibitory concentrations (MICs) were determined, and PCR and sequencing were performed. Among the 42 (82.3%) E. coli and 24 (77.4%) K. pneumoniae isolates containing $bla_{CTX-M}$, $bla_{CTX-M-14}$ and $bla_{CTX-M-15}$ were detected in 23 and 19 E. coli isolates, respectively, and in 7 and 17 K. pneumoniae isolates, respectively. CTX-M producers of urinary E. coli and K. pneumoniae were resistant to multiple antibiotics and contained other antimicrobial resistance genes. CTX-M-15 producers contained more antimicrobial resistance genes than did CTX-M-14 producers.

• Korean Journal of Clinical Pharmacy
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• v.23 no.1
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• pp.26-32
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• 2013

Objective: This study was to define the clinical effect on the clinical decision support system (CDSS) for prescribing antibiotics integrated with the order communication system in a National Hospital. Method: We extracted data collected before integrating the CDSS of 4,406 adult patients in 2007 and data collected after integrating the CDSS of 4,278 adult patients in 2009. These patients were 50.4% and 45.2% of all patients admitted in 2007 and 2009, respectively. The clinical effect was defined as the proportion of prescribed antibiotics, the length of antibiotics use, and the DDDs (defined daily doses) of antibiotics per 1,000 patient-days using these retrospective data. Results: There were a significant change in the proportion of patient prescribed penicillins with extended spectrum (OR=0.55, p=001), penicillins included beta-lactamase inhibitors (OR=0.75, p<.001), 3rd cephalosporin (OR=1.47, p<.001). The mean of the length of antibiotics use was decreased statistically from $6.09{\pm}5.48$ to $5.85{\pm}5.51$ days (p=.003). The DDD of glycopeptides was decreased from 24.43 DDD to 19.55 DDD per 1000 patient-days. The DDD of 3rd cephalosporins was also decreased from 15.88 to 11.65. Conclusion: Therefore, the clinical decision support system for prescribing antibiotics was effective for the clinical outcomes.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.465-472
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• 2019

Several reports describe antimicrobial-resistance transfer among children and the community in outbreak situations, but transfer between a child and a caregiver has not been examined in child care facilities under normal circumstances. We investigated the transfer of antimicrobial-resistance genes, resistant bacteria, or both among healthy children and teachers. From 2007 to 2009, 104 Escherichia coli isolates were obtained from four teachers and 38 children in a child care center. Twenty-six cephem-resistant isolates were obtained from children in 2007 and 2008. In 2009, cephem-resistant isolates were detected in children as well as a teacher. Nalidixic acid-resistant isolates from the same teacher for 3 years showed low similarity (<50%) to each other. However, an isolate from a teacher in 2007 and another from a child in 2008 showed high similarity (87%). Pulsed-field gel electrophoresis revealed 100% similarity for four isolates in 2007 and one isolate in 2008, and also similarity among seven isolates carrying the virulence gene (CNF1). This study yielded the following findings: (1) a gene for extended-spectrum ${\beta}$-lactamase was transferred from a child to other children and a teacher; (2) a nalidixic acid-resistant isolate was transferred from a teacher to a child; and (3) a virulent bacterium was transferred between children.