Sook Shin

Antibacterial Activity and Mechanism of Action of the Silver Ion in Staphylococcus aureus and Escherichia coli

ABSTRACT The antibacterial effect and mechanism of action of a silver ion solution that was electrically generated were investigated for Staphylococcus aureus and Escherichia coli by analyzing the growth, morphology, and ultrastructure of the bacterial cells following treatment with the silver ion solution. Bacteria were exposed to the silver ion solution for various lengths of time, and the antibacterial effect of the solution was tested using the conventional plate count method and flow cytometric (FC) analysis. Reductions of more than 5 log10 CFU/ml of both S. aureus and E. coli bacteria were confirmed after 90 min of treatment with the silver ion solution. Significant reduction of S. aureus and E. coli cells was also observed by FC analysis; however, the reduction rate determined by FC analysis was less than that determined by the conventional plate count method. These differences may be attributed to the presence of bacteria in an active but nonculturable (ABNC) state after treatment with the silver ion solution. Transmission electron microscopy showed considerable changes in the bacterial cell membranes upon silver ion treatment, which might be the cause or consequence of cell death. In conclusion, the results of the present study suggest that silver ions may cause S. aureus and E. coli bacteria to reach an ABNC state and eventually die.

Antifungal activity of the silver ion against contaminated fabric

An anti‐fungal efficacy test of the silver laundry machine, which electrically generates silver ions, was carried out against four fungi –Trichophyton rubrum, Candida albicans, Microsporum canis and Aspergillus flavus– which cause major fungal infection in humans and animals. Compared with the conventional laundry machine, washing with the silver laundry machine regardless of detergent use was effective against most of the fungi with about 4 log10 (CFU ml−1) reduction and eliminated almost all the fungi when using the detergent. Moreover, the cleaning activity of the silver laundry machine with detergent was higher than that of the conventional laundry machine with detergent both after wash and after final spin step against all four examined fungi. The silver laundry machine may be useful in preventing skin irritation caused by fungi‐contaminated fabric in the hospital and in the home.

Occurrence of antimicrobial resistance and virulence genes, and distribution of enterococcal clonal complex 17 from animals and human beings in Korea

Enterococci are major zoonotic bacteria that cause opportunistic infections in human beings and animals. Moreover, pathogenic strains can be disseminated between human beings and animals, particularly companion animals that come into frequent contact with people. Recently, Enterococcus faecium clonal complex 17 (CC17) has emerged as a pandemic clone. Most CC17 strains are ampicillin resistant and possess virulence genes such as esp and hyl. Despite the possible dissemination of CC17 between human beings and animals, prevalence data about CC17 in animals is limited. In the present study, the phenotypes and genotypes of antimicrobial resistance were compared, as well as virulence gene profiles from 184 enterococci strains isolated from chickens, pigs, companion animals, and human patients in Korea. Ampicillin-resistant E. faecium (AREF) strains were selected, and multilocus sequence typing was performed to investigate the dispersion of CC17 among animals and human beings. The companion animal and human isolates showed high resistance rates to ampicillin and ciprofloxacin, whereas food animal isolates showed high tetracycline and erythromycin resistance rates. Ampicillin-resistant E. faecium was only detected in human (21/21 E. faecium, 100%) and companion animal (3/5 E. faecium, 60%) isolates, and all human AREF strains and 1 canine AREF strain were confirmed as CC17. In conclusion, the occurrence of antimicrobial resistance and virulence genes, and the distribution of enterococcal CC17 in companion animal enterococcal strains were similar to those of human strains rather than to those of food animal strains.

Genetic and phenotypic characterization of methicillin-resistant staphylococci isolated from veterinary hospitals in South Korea.

Staphylococci were isolated from veterinary staff, hospitalized animals, and medical equipment from 2 major tertiary veterinary hospitals in South Korea to investigate antimicrobial resistance and genetic relatedness. The detection rate for staphylococci was 55.2% (111/201 samples), and 11 species were identified among the collected staphylococcal strains. The most prevalent species were Staphylococcus pseudintermedius (52/111, 46.8%), Staphylococcus epidermidis (21/111, 18.9%), and Staphylococcus aureus (19/111, 17.1%). The methicillin-resistance rates of staphylococci isolated from veterinary staff and medical equipment were higher than those from hospitalized animals. The genotype of methicillin-resistant S. aureus (MRSA) strains in the current study was sequence type (ST)72-SCCmec IVc-t324, which is similar to the genotype of prevalent MRSA strains in human beings and food animals in South Korea. Among the mecA-positive S. pseudintermedius isolates, SCCmec V was most prevalent in strains originating from both veterinary staff and hospitalized animals. SCCmec IVa was detected in methicillin-resistant S. epidermidis, whereas SCCmec IVc was found in other methicillin-resistant, coagulase-negative staphylococci. The SCCmec typing, antimicrobial susceptibility tests, and pulsed field gel electrophoresis results showed that methicillin-resistant staphylococci dissemination between hospitalized animals and veterinary staff is possible in South Korean veterinary hospitals.

Prevalence and Mechanism of Fluoroquinolone Resistance in Escherichia coli Isolated from Swine Feces in Korea

In this study, we investigated the prevalence and fluoroquinolone (FQ) resistance mechanisms in Escherichia coli isolated from swine fecal samples. E. coli isolates were collected from 171 (72.2%) of 237 swine fecal samples. Of these, 59 isolates (34.5%) were confirmed as FQ-resistant E. coli by the disk diffusion method. Of the FQ-resistant isolates, three major FQ resistance mechanisms were investigated. Of the 59 isolates, plasmid-mediated quinolone resistance genes were detected in 9 isolates (15.3%). Efflux pump activity was found in 56 isolates (94.9%); however, this was not correlated with the increased FQ resistance measured by determining the MIC. Point mutations in quinolone resistance-determining regions were the main cause of FQ resistance. All 59 ciprofloxacin-resistant isolates had mutations in quinolone resistance-determining regions; of these 59 isolates, all (100%) had mutations in gyrA, 58 (98.3%) had mutations in parC, 22 (37.3%) had mutations in parE, and none had mutations in gyrB. The predominant mutation type was double mutation in gyrA (Ser83Leu plus mutation in aspartic acid 87), and all FQ-resistant isolates (except one) that had mutations in parC or parE also had double mutations in gyrA. Importantly, the frequencies of multidrug-resistant and extended-spectrum β-lactamase-producing E. coli were significantly higher in the high ciprofloxacin MIC group in this study. Compared with previous studies in Korea, the prevalence of FQ resistance and plasmid-mediated quinolone resistance genes had increased considerably in swine. Although the use of FQ as a feed additive is prohibited in Korea, use for self-treatment and therapeutic purposes has been increasing, which may be responsible for the higher FQ resistance rate observed in this study. Therefore, prudent use of FQ on animal farms is warranted to reduce the evolution of FQ-resistant bacteria in the animal industry.

Characterisation of Pseudomonas aeruginosa related to bovine mastitis.

Pseudomonas aeruginosa is one of the causative pathogens of bovine mastitis. Most P. aeruginosa strains possess the type III secretion system (TTSS), which may increase somatic cell counts (SCCs) in milk from mastitis-affected cows. Moreover, most of P. aeruginosa cells can form biofilms, thereby reducing antibiotic efficacy. In this study, the presence and effect of TTSS-related genotypes on increase of SCCs among 122 P. aeruginosa isolates obtained from raw milk samples from mastitis-affected cows and their antibiotic susceptibility at planktonic and biofilm status were investigated. Based on the presence of TTSS-related genes a total of 82.7% of the isolates were found to harbour exoU and/or exoS genes, including the invasive (exoU-/exoS+, 69.4%), cytotoxic (exoU+/exoS-, 8.3%) and cytotoxic/invasive strains (exoU+/ exoS+, 5.0%). Milk containing exoS-positive isolates had higher SCCs than those containing exoS-negative isolates. The majority of isolates showed gentamicin, amikacin, meropenem and ciprofloxacin susceptibility at planktonic status. However, the susceptibility was decreased at the biofilm status. Based on minimum biofilm eradication concentration (MBEC)/minimum inhibitory concentration (MIC) ratios, the range of change in antibiotic susceptibility varied widely depending on the antibiotics (from ≥ 3.1-fold to ≥ 475.0-fold). In conclusion, most P. aeruginosa isolates studied here had a genotype related to increase in SCCs. The efficiency of antibiotic therapy against P. aeruginosa-related bovine mastitis could be improved by analysing both the MBEC and the MIC of isolates.

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.

Mechanisms of quinolone resistance in Escherichia coli isolated from companion animals, pet-owners, and non-pet-owners

The present study investigated the prevalence and mechanisms of fluoroquinolone (FQ)/quinolone (Q) resistance in Escherichia (E.) coli isolates from companion animals, pet-owners, and non-pet-owners. A total of 63 E. coli isolates were collected from 104 anal swab samples, and 27 nalidixic acid (NA)-resistant isolates were identified. Of those, 10 showed ciprofloxacin (CIP) resistance. A plasmid-mediated Q resistance gene was detected in one isolate. Increased efflux pump activity, as measured by organic solvent tolerance assay, was detected in 18 NA-resistant isolates (66.7%), but was not correlated with an increase in minimum inhibitory concentration (MIC). Target gene mutations in Q resistance-determining regions (QRDRs) were the main cause of (FQ)Q resistance in E. coli. Point mutations in QRDRs were detected in all NA-resistant isolates, and the number of mutations was strongly correlated with increased MIC (R = 0.878 for NA and 0.954 for CIP). All CIP-resistant isolates (n = 10) had double mutations in the gyrA gene, with additional mutations in parC and parE. Interestingly, (FQ)Q resistance mechanisms in isolates from companion animals were the same as those in humans. Therefore, prudent use of (FQ)Q in veterinary medicine is warranted to prevent the dissemination of (FQ)Q-resistant bacteria from animals to humans.

Genotypic and Phenotypic Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Bovine Mastitic Milk in Korea

Staphylococcus aureus is a major etiological pathogen for bovine mastitis, foodborne illness, and various clinical infections. Methicillin-resistant S. aureus (MRSA) has been isolated from bovine mastitic milk, and the presence of MRSA in milk is a major public health concern. We investigated the frequency of MRSA isolation from mastitic raw milk in Korea and characterized the patterns of antimicrobial resistance, virulence, and genotypes of the MRSA isolates. A total of 1,222 raw milk samples were collected from 47 dairy farms in Gyeonggi province from 2011 to 2012. Of these samples, 649 were considered mastitic milk based on somatic cell counts of more than 200,000 cells per ml, and 165 S. aureus isolates (from 25.4% of samples) were obtained from these samples. Of these isolates, 23 (13.9%) collected from five farms were confirmed as MRSA by detection of the mecA gene. Disk diffusion and MIC tests for antibiotic resistance revealed that all MRSA isolates were resistant to four or more antimicrobial agents. All MRSA isolates had staphylococcal enterotoxin genes, and two clusters of these genes were identified: seg-sei-sek-sem-sen-seo (20 isolates, 87%) and sed-seg-sei-sej-sem-sen-seo (3 isolates, 13%). Each MRSA-positive farm had only one spa-SCCmec type. Nine MRSA isolates (39.1%) with the t324-IVa genotype, which is related to community-acquired MRSA infection, were isolated from three dairy farms. Additional genotypes of t148-IVa and t002-II were detected and related to human MRSA strains. Most MRSA isolates had distinct pulsed-field gel electrophoresis subtypes, indicating they were not the same clones. Only two isolates collected from the same farm during different years had an identical electrophoresis type, indicating persistence of the clone at this farm. Taken together, these findings may indicate an increased virulence and risk of MRSA strains on dairy farms. Therefore, an efficient surveillance and control program is needed to prevent the transmission of MRSA from animals to humans.