Mutations in gyrA and parC Genes and Plasmid-Mediated Quinolone Resistance in Non-typhoid Salmonella Isolated from Pediatric Patients with Diarrhea in Seoul

A total of 91 non-typhoid Salmonella isolated from pediatric patients with diarrhea in Seoul from 2003 to 2009 was tested for antimicrobial susceptibility of nalidixic acid (NA). Forty strains of NA resistance or intermediate susceptible non-typhoid Salmonella were identified and their minimum inhibitory concentrations (MICs) of NA, ciprofloxacin (CIP), and norfloxacin (NOR) were determined. Of the 40 isolates, 26 were resistant to NA (MIC >256 microg/ml). Only one isolate was high-level resistant to CIP (12 microg/ml) and NOR (48 microg/ml). Mutations in gyrA and parC genes were studied by PCR and sequencing. All NA-resistant isolates carried point mutations in the gyrA quinolone resistance determining regions (QRDR) at codon 83 or 87 (MICs of NA, >256 microg/ml; MICs of CIP, 0.047~0.25 microg/ml; MICs of NOR, 0.38~1.5 microg/ml). A double change in GyrA was found in one Salmonella Enteritidis (MIC of CIP, 12 microg/ml; MIC of NOR, 48 microg/ml). In respect of the ParC protein, a single change at Thr57-->Ser was found in 3 isolates (MICs of NA, >256 microg/ml; MICs of CIP, 0.19~0.25 microg/ml; MICs of NOR, 1 microg/ml). At the same time, these strains changed from Ser83 to Tyr in the gyrA. The result of the investigation for the prevalence of plasmid-mediated quinolone resistance (PMQR) genes, 14 isolates harbored qnr gene among 40 isolates. All of 14 isolates showed decreased susceptibility at NA (MICs 4~16 microg/ml) and except one strain, all of qnr genes were identified as qnrB. Mutations in the gyrA gene and production of PMQR determinants were critical for quinolone resistance and decreased susceptibility to fluoroquinolone in these isolates.

Molecular Epidemiology of Legionella pneumophila Isolated from Bath Facilities of Public Establishments in Seoul

The genus Legionella is common in aquatic environments. Some species of Legionella are recognized as potential opportunistic pathogens for human, notably Legionella pneumophila that causes Legionellosis. During the summer season between June and August in 2010, we isolated 61 L. pneumophila from the bath facilities of public establishments of 25 wards in Seoul. The existence of 16S rRNA and mip gene of L. pneumophila was confirmed in the genome of the isolated strains by PCR. Among the 61 strains of L. pneumophila, thirty three isolates belong to serogroup 1 (54.1%), 13 isolates were serogroup 6 (21.3%), 9 isolates were serogroup 5 (14.8%), 3 isolates were serogroup 3 (4.9%), and 3 isolates were identified in serogroup 2 (4.9%). On pulsed-field gel electrophoresis (PFGE) analysis using SfiI, genetic types of L. pneumophila were classified into 8 (A to H) patterns by the band similarity with excess of 65%. Our results suggest the existence of serological and genetic diversity among the L. pneumophila isolates.

An Outbreak of Astrovirus Infection of Newborns with Hemorrhagic Diarrhea in a Neonatal Unit / 대한임상미생물학회지

BACKGROUND: We investigated the causative agents of hemorrhagic diarrhea which occurred in newborn babies in a hospital nursery in July, 2002. Rotavirus was not confirmed as the cause because only a few patients were positive for rotavirus test while most others with hemorrhagic diarrhea were negative. Therefore, patients with bloody stool were tested for Salmonella, Shigella, rotavirus, adenovirus, enterovirus, astrovirus, and enterohemorrhagic Escherichia coli (EHEC). METHODS: Bloody stools from 12 newborns with hemorrhagic diarrhea were tested. Polymerase chain reaction (PCR) of shiga-toxin gene was performed for EHEC. Rotavirus and adenovirus were tested with latex agglutination kit (Orion Diagnostica). Reverse transcription (RT)-PCR was performed for enterovirus. To detect astrovirus, RNA was extracted with Viral RNA Mini Kit (QIAGEN), reverse-transcribed with random hexamer, and PCR-amplified with specific primers. RESULTS: Of the 12 patients tested, seven (58%) were positive for astrovirus RT-PCR while all were negative for Salmonella, Shigella, EHEC, rotavirus, adenovirus, and enterovirus. CONCLUSIONS: Although diarrhea caused by astrovirus is known to be milder than that caused by rotavirus, our cases showed that astrovirus could cause serious bloody diarrhea in newborn babies.

Comparison of In Vitro Susceptibility between 7:1 and 4:1 Mixtures of Amoxicillin and Clavulanate against Major Clinically-isolated Bacteria / 대한임상미생물학회지

BACKGROUND: Amoxicillin/clavulanate (A/C) is a combination of a broad spectrum -lactam antibiotic amoxicillin and the potent -lactamase inhibitor clavulanate. A/C 7:1 combination is known to be equal in its clinical efficacy and to have less gastrointestinal adverse effects compared to conventional A/C 4:1 combination. We estimated in vitro antimicrobial activities of the 7:1 combination (AMOCLA Duo) and the conventional 4:1 combination against clinical bacterial isolates known to be the major causes of acute otitis media or sinusitis. METHODS: Total 183 strains isolated from clinical specimens of patients at Seoul National University Hospital were tested for minimal inhibitory concentraion (MIC). Streptococcus pneumoniae and Haemophilus influenzae were tested by microdilution broth method and other bacterial species by agar dilution method according to the recommendations of National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: AMOCLA Duo was compared with the 4:1 combination in respect to MIC50, MIC90 and MIC range. For total 183 strains (30 methicillin-susceptible Staphylococcus aureus, 30 methicillin-sus-ceptible Staphylococcus epidermidis, 25 penicillin-susceptible S. pneumoniae, 42 penicillin-resistant S. pneumoniae, 33 H. influenzae and 23 Moraxella catarrhalis), mean MICs did not show statistically significant difference between the 2 combinations but they did for H. influenzae and M. catarrhalis. CONCLUSIONS: As for the total test strains, in vitro antimicrobial activity of AMOCLA Duo was equal to that of the conventional 4:1 combination. For each species, H. influenzae and M. catarrhalis showed significant difference between mean MICs of the 2 combinations but other species did not. We do not suppose, however, that in case of H. influenzae this difference is of practical and clinical significance according to the NCCLS interpretive criteria for MIC. Although M. catarrhalis showed statistically very significant difference of MICs, this difference can be clinically solved due to the higher dose of amoxicillin in AMOCLA Duo.

A Case of Campylobacter upsaliensis Bacteremia / 대한진단검사의학회지

Campylobacter species are gram-negative, curved or spiral bacteria. Campylobacter upsaliensis is a recently recognized human enteric pathogen associated with enteritis, colitis, bacteremia, and sepsis. This organism is very rarely isolated in clinical laboratories and therefore is little known among clinicians. We isolated C. upsaliensis from a blood culture in a 62-year-old male patient with alcoholic liver cirrhosis and acute pancreatitis, using various phenotypic tests and polymerase chain reaction-restriction fragment-length polymorphism analysis. The patient was treated with broad-spectrum antibiotics and recovered fully. To our knowledge, this is the first case report of C. upsaliensis bacteremia in Korea.

Two cases of Hemolytic Uremic Syndrome Associated with Escherichia coli O114

We report two cases of hemolytic uremic syndrome (HUS) associated with Escherichia coli O114. Two cases were similar and showed the same clinical courses. After prodrome of diarrhea and vomiting lasting 1-2 days, azotemia persisted for about 10 days, and during that period, the patients were on peritoneal dialysis. They recovered without any sequelae after about 15 days. Direct multiplex PCR of stool culture revealed eae and stx2 gene and the result of ELISA done on the colony positive of eae gene confirmed Escherichia coli O114. This is the first report of HUS associated with Escherichia coli O114. We recommend, Shiga toxin producing bacterial infection must be considered and efforts should be made to scrutinize the organism in all diarrhea-prodrome HUS patients.

Detection of Campylobacter jejuni by Multiplex PCR and Patterns of Pulsed-Field Gel Electrophoresis / 대한임상미생물학회지

BACKGROUND: Campylobacter is the most common bacterial cause of food-borne infection in developed countries, and handling or eating of contaminated poultry products was reported as the major cause of human campylobacteriosis in sporadic cases. This study was performed to investigate the prevalence of Campylobacter in patients with diarrhea and raw chickens of grocery, and identify the species by multiplex PCR and determine the genotypes of isolates by SmaI pulsedfield gel electrophoresis(PFGE) profiles. METHODS: Eight hundred and fifty six stool specimens obtained from 773 hospitalized patients with diarrhea and 16 raw chickens purchased from grocery were tested. Karmali's charcoal based selective medium and Campylobacter enrichment broth were used for isolation of Campylobacter from patients and chicken, respectively. And membrane filter method with sheep blood agar was also used in both two cases. Isolates were indentified with PCR, PCR-RFLP, and biochemical test. And genotypes were determined with SmaI PFGE profile analysis. RESULTS: A total of 13 Campylobacter strains(1.7%) were isolated from 856 stool specimens of 773 patients with diarrhea, nine isolates were C. jejuni and four were C. coli. All of 16 raw chickens were contaminated with Campylobacter spp., and both of C. jejuni and C. coli were detected from eight chickens. From the SmaI-digested PFGE profile analysis of nine C. jejuni strains and four C. coli strains isolated from patients, eight types and four types of PFGE profile were obtained, respectively. And 15 types and seven types of PFGE profile were obtained from 23 of C. jejuni and 11 of C. coli which strains were isolated from chicken samples, respectively. The several isolates showing the different PFGE patterns were detected in the same chicken. Three PFGE patterns of C. jejuni isolated from patients were observed in the chickens. One type of C. coli PFGE profiles of the patient's isolates were the same as that of chicken. CONCLUSIONS: The prevalence of Campylobacter infection is not high compared to the other countries, but most of raw chickens are contaminated with Campylobacter spp. Several genotypes of C. jejuni and C. coli are contaminated in the single chicken. The PFGE patterns of some human isolates are the same as those of chicken so that human infection may be originated from the chicken. But the reason of low infection rate in human in spite of the very high contamination rate of chicken should be clarified in the near future