Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Genomic relatedness and dissemination of bla NDM-5 among Acinetobacter baumannii isolated from hospital environments and clinical specimens in Thailand.

Background: Acinetobacter baumannii (A. baumannii) is an important cause of nosocomial infection, especially in intensive care units (ICUs). It has the propensity to tolerate various environments and multiple classes of antibiotics. Our study aimed to characterize the comparative genomes of A. baumannii from hospital environments and clinical isolates. Methods: Clinical and environmental A. baumannii isolates were collected from a university hospital. Antibiotic susceptibility testing was performed, antibiotic resistance genes (ARGs) were characterized, and repetitive element palindromic-PCR (rep-PCR) typing was performed. Eight representative A. baumannii isolated from environmental and clinical samples from the same wards were selected for whole-genome sequencing (WGS) using the Illumina platform. Results: A total of 106 A. baumannii isolates were obtained from 312 hospital environmental samples. A high percentage of samples with A. baumannii colonization were detected from AMBU bags (77.9%), followed by bedrails (66.7%) and suction tubes (66.7%). We found that 93.4% of the environmental isolates were multidrug-resistant A. baumannii (MDRAB), and 44.7% were extremely drug-resistant A. baumannii (XDRAB). bla OXA-23 bla NDM, and bla OXA-58 were present in 80.2%, 78.3%, and 0.9% of all isolates, respectively. Sixty-one A. baumannii isolates were collected from patient specimens in the same ward. Among all A. baumannii clinical isolates, MDRAB and XDRAB accounted for 82% and 55.7%, respectively. The most dominant ARGs identified was bla OXA-23 (80.3%), followed by bla NDM (55.7%). The genetic diversity of all isolates using rep-PCR could be divided into 33 genotypes. The genome size of eight A. baumannii ranged from 3.78-4.01 Mb. We found six of eight strains to be bla NDM-5-harboring A. baumannii. Mobile genetic elements (MGEs), such as integron1 (intl1), located upstream of bla NDM-5 were observed. The phylogenomic relationship of the core and pan genomes as well as the single nucleotide polymorphism (SNP) count matrix revealed the genetic similarity of A. baumannii environmental and clinical strains obtained from the same ward. Conclusion: This study confirmed that A. baumannii colonized in hospital environments were the main reservoir of nosocomial infection and provides critical information to guide the control of A. baumannii infection.

High prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates: A 5-year retrospective study at a Tertiary Hospital in Northern Thailand.

Background: The global emergence and spread of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales, especially Escherichia coli and Klebsiella pneumoniae, have been recognized as a public health concern as severe infections caused by these microorganisms increase morbidity and mortality. This study aimed to assess the prevalence of ESBL-positive E. coli and K. pneumoniae strains isolated from hospitalized patients in Chiangrai Prachanukroh hospital, Chiangrai province, Thailand. Methods: This retrospective analysis was conducted from January 2016 to December 2020. A total of 384,001 clinical specimens were collected aseptically and further cultivated on an appropriate medium. All clinical isolates (one isolate per patient) were identified based on standard laboratory methods. Antibiotic susceptibility testing was performed by the Kirby Bauer disc diffusion technique following CLSI guidelines. ESBL production was screened with ceftazidime and cefotaxime discs based on the CLSI recommendations. Phenotypic confirmation of ESBL production was carried out using a double-disc synergy technique following the CLSI standard. Results: Of a total of 384,001 clinical samples analyzed for bacterial species identification, 11,065 (2.9%) tested positive for E. coli and 5,617 (1.5%) for K. pneumoniae. Approximately 42.5% (4,706/11,065) of E. coli and 30.2% (1,697/5,617) of K. pneumoniae isolates were classified as ESBL producers. A higher proportion of ESBL producers was found in patients older than 60 years and male groups. The highest infection rates of ESBL-positive pathogens were observed among patients in a medical unit. ESBL-producing E. coli and K. pneumoniae isolates were predominantly found in urine and sputum, respectively. ESBL producers exhibited a high resistance rate to ampicillin (99.8-100%), cefazolin (100%), cefotaxime (100%), fluoroquinolones, and trimethoprim/sulfamethoxazole. Conclusions: This study demonstrated the high prevalence and emerging antibiotic resistance of ESBL-positive E. coli and K. pneumoniae isolates from patients admitted to a provincial hospital in northern Thailand. Most ESBL-producing strains were highly resistant to several antimicrobial agents apart from carbapenems and aminoglycosides. These findings indicated that carbapenems and aminoglycosides should be advised as the first-line drugs of choice for serious infections with ESBL-producing Enterobacterales.

Comparative genome analysis of Escherichia coli bacteriophages isolated from sewage and chicken meat.

Escherichia coli, a bacterium that causes severe foodborne diseases, is transmitted to humans primarily through the consumption of contaminated foods. These foodborne pathogens are causing a public health problem that requires alternative control approaches, such as bacteriophage (phage) biocontrol. In this study, we characterized vB_EcoM_Tw01 (vTw01) isolated from sewage and vB_EcoM_Tcm05 (vTcm05) isolated from chicken meat. Both vTw01 and vTcm05 were assigned to the family Myoviridae based on their morphology, with the former exhibiting a narrow host range with low minimum inhibitory multiplicity of infection (miMOI), and the latter exhibiting a broad host range with high miMOI. The latent periods of these phages were 20 and 30 min for vTw01 and vTcm05, while the burst sizes were ∼140 and ∼300 PFU/cell, respectively. They were relatively stable over a wide range of pH values and temperatures. The bioinformatics analysis of the genomic sequence suggests that vTw01 and vTcm05 have double-stranded DNA with genome sizes of 170,107 bp and 149,059 bp, respectively. Bacteriophage encoded enzymes, such as tail-lysozyme, spanin Rz, holin, cell wall hydrolase (CWH), and endolysin, were identified in the genome of both phages. In conclusion, this study investigated the morphological, physiological, and genomic features of two E. coli phages isolated from different sources. It was confirmed that these phages and their enzymes can serve as potential candidates for phage biocontrol.

Insight into Molecular Epidemiology, Antimicrobial Resistance, and Virulence Genes of Extensively Drug-Resistant Acinetobacter baumannii in Thailand.

Extensively drug-resistant Acinetobacter baumannii (XDR-AB) is a major threat to public health worldwide. A retrospective study for 27 XDR-AB isolates from four tertiary hospitals in Thailand was conducted. Beta-lactamase and virulence genes were characterized by PCR. The blaADC, blaOXA-51, and blaOXA-23 were detected in all isolates, whereas blaPER-1 and blaNDM-1 genes were present in 7.4% and 3.7% of isolates. All isolates had virulence genes, including genes in iron acquisition system, biofilm formation and secretion systems. The plasmids in XDR-AB belonged to GR2 (100%), GR6 (40.7%), and GR1 (7.4%). Multilocus sequence typing sequence types (STs) were further investigated. The data demonstrated that XDR-AB isolates had nine STs: ST195 (n = 4), ST208 (n = 4), ST368 (n = 1), ST451 (n = 5), ST457 (n = 2), ST1947 (n = 1), ST1166 (n = 7), including two novel STs namely ST1682 (n = 2) and ST1684 (n = 1). We observed that the majority ST1166 (25.9%) was associated with the prevalence of GR2 and GR6 plasmids and traU virulence gene. Genome-based single nucleotide polymorphism phylogenetic analysis of the isolates with two novel ST types indicated that the two isolates belonged to the international clone II (IC2) within the same cluster. In conclusion, our data showed the dissemination of XDR-AB isolates harbored virulence genes and antibiotic resistance genes among four hospitals in Thailand. The results highlighted the difficulty posed for the empirical treatment of the patients with the A. baumannii infection.

Genomic analysis reveals high virulence and antibiotic resistance amongst phage susceptible Acinetobacter baumannii.

In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value < 0.05). We also found association between biofilm formation and the presence of ompA gene among phage susceptible A. baumannii strains (p-value < 0.05). A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.

Biofilm Formation of Methicillin-resistant Coagulase-Negative Staphylococci Isolated from Clinical Samples in Northern Thailand.

BACKGROUND: Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are multidrug-resistant bacteria that are difficult to treat because of their ability to form biofilms. OBJECTIVES: In the present study, we evaluated the antibiotic-resistant phenotypes, biofilm-forming ability, and biofilm associated genes of 55 clinical MR-CoNS isolates obtained from two hospitals in Thailand. MATERIALS AND METHODS: MALDI-TOF-MS and tuf gene sequencing were performed to determine the species of all isolates. Biofilm production was determined using Congo red agar (CRA) and the microtiter plate (MTP) assay. Biofilm-associated genes were characterized using polymerase chain reaction (PCR). RESULTS: Among the 55 MR-CoNS isolates, five species were identified as Staphylococcus haemolyticus (34.5%), Staphylococcus epidermidis (32.7%), Staphylococcus capitis (18.2%), Staphylococcus cohnii (9.1%), and Staphylococcus hominis (5.5%). The antimicrobial susceptibility pattern of MR-CoNS isolates indicated high resistance to cefoxitin (100%), penicillin (98.2%), erythromycin (96.4%), ciprofloxacin (67.3%), sulfamethoxazole/trimethoprim (67.3%), gentamicin (67.3%), and clindamycin (63.6%). All the isolates were susceptible to vancomycin and linezolid. The biofilm production was detected in 87.3% isolates through the CRA method and in 38.1% isolates through the MTP assay. The prevalence rates of icaAD, bap, fnbA, and cna were 18.2%, 12.7%, 47.3%, and 27.3%, respectively. There were significant differences in the presence of these biofilm-associated genes among the MR-CoNS isolates. Moreover, quantitative biofilm formation was significantly different among MR-CoNS species. CONCLUSION: The present study revealed that biofilm-associated genes are important for biofilm biomass in MR-CoNS isolates, and the findings of this study are essential for finding new strategies to control biofilm formation and prevent the spread of MR-CoNS infectious diseases.

Molecular Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary Hospital in Northern Thailand.

Methicillin-resistant staphylococci are now recognized as a major cause of infectious diseases, particularly in hospitals. Molecular epidemiology is important for prevention and control of infection, but little information is available regarding staphylococcal infections in Northern Thailand. In the present study, we examined antimicrobial susceptibility patterns, detection of antimicrobial resistance genes, and SCCmec types of methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from patients in a hospital in Northern Thailand. The species of MRSA and MR-CoNS were identified using combination methods, including PCR, MALDI-TOF-MS, and tuf gene sequencing. The susceptibility pattern of all isolates was determined by the disk diffusion method. Antimicrobial resistance genes, SCCmec types, and ST239 were characterized using single and multiplex PCR. ST239 was predominant in MRSA isolates (10/23). All MR-CoNS (N=31) were identified as S. haemolyticus (N=18), S. epidermidis (N=3), S. cohnii (N=3), S. capitis (N=6), and S. hominis (N=1). More than 70% of MRSA and MR-CoNS were resistant to cefoxitin, penicillin, oxacillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin. In MRSA isolates, the prevalence of ermA (78.3%) and ermB (73.9%) genes was high compared to that of the ermC gene (4.3%). In contrast, ermC (87.1%) and qacA/B genes (70.9%) were predominant in MR-CoNS isolates. SCCmec type III was the dominant type of MRSA (13/23), whereas SCCmec type II was more present in S. haemolyticus (10/18). Ten MRSA isolates with SCCmec type III were ST239, which is the common type of MRSA in Asia. This finding provides useful information for a preventive health strategy directed against methicillin-resistant staphylococcal infections.

Dissemination of blaOXA-23, blaOXA-24, blaOXA-58, and blaNDM-1 Genes of Acinetobacter baumannii Isolates from Four Tertiary Hospitals in Thailand.

Acinetobacter baumannii is a major threat to public health due to the emergence and dissemination of antibiotic-resistant strains. The purpose of this study was to determine the molecular epidemiology of antibiotic-resistant A. baumannii isolates collected from four tertiary hospitals in Thailand during the period November 2013-February 2015. We screened 339 A. baumannii, nonrepetitive clinical isolates to determine drug susceptibility. Among all isolates, we found that 7.9% was nondrug-resistant A. baumannii (NR-AB). Carbapenem-resistant A. baumannii (CR-AB) strains accounted for 84.9% of the total isolates, with extensively drug-resistant A. baumannii (XDR-AB) accounting for 7.9% of the total isolates. We further investigated class D carbapenemase genes using multiplex-PCR amplification and class B metallo-ß-lactamase genes, including blaIMP, blaVIM, and blaNDM-1 genes, using PCR and sequencing methods. We found that 300 (88.5%) isolates carried acquired class D carbapenemase genes, including blaOXA-23 (82.6%), blaOXA-24 (0.3%), and blaOXA-58 (6.5%). The genes blaIMP and blaVIM were not detected in any isolates. The blaNDM-1 was detected in 31 isolates from two hospitals (9.1%). All of the blaNDM-1-positive A. baumannii (NDM-AB) had ISAba125 sequences upstream of the blaNDM-1 gene. A coexistence of three resistance genes-blaOXA-23-blaOXA-58-blaNDM-1-was found in one isolate. A repetitive element palindromic-PCR (REP-PCR) revealed that all A. baumannii isolates were genetically diverse and could be divided into 33 genotypes. Only three genotypes were found to be predominant in all hospitals. Data from our study indicate the widespread emergence of multiple resistance determinants in A. baumannii isolates in Thailand, suggesting the need for more stringent infection control measures.

Biofilm formation of methicillin-resistant coagulase negative staphylococci (MR-CoNS) isolated from community and hospital environments.

Methicillin-resistant coagulase negative staphylococci (MR-CoNS) are the major cause of infectious diseases because of their potential ability to form biofilm and colonize the community or hospital environments. This study was designed to investigate the biofilm producing ability, and the presence of mecA, icaAD, bap and fnbA genes in MR-CoNS isolates. The MR-CoNS used in this study were isolated from various samples of community environment and five wards of hospital environments, using mannitol salt agar (MSA) supplemented with 4 µg/ml of oxacillin. The specie level of Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus warneri was identified by specific primers of groESL (S. haemolyticus), rdr (S. epidermidis) and nuc (S. hominis and S. warneri). The remainder isolates were identified by tuf gene sequencing. Biofilm production was determined using Congo red agar (CRA) and Microtiter plate (MTP) assay. The mecA and biofilm associated genes (icaAD, fnbA and bap) were detected using PCR method. From the 558 samples from community and hospital environments, 292 MR-CoNS were isolated (41 from community environments, and 251 from hospital environments). S. haemolyticus (41.1%) and S. epidermidis (30.1%) were the predominant species in this study. Biofilm production was detected in 265 (90.7%) isolates by CRA, and 260 (88.6%) isolates were detected by MTP assay. The staphylococci isolates derived from hospital environments were more associated with biofilm production than the community-derived isolates. Overall, the icaAD and bap genes were detected in 74 (29.5%) and 14 (5.6%) of all isolates from hospital environments. When tested by MTP, the icaAD gene from hospital environment isolates was associated with biofilm biomass. No association was found between bap gene and biofilm formation. The MR-CoNS isolates obtained from community environments did not harbor the icaAD and bap genes. Conversely, fnbA gene presented in MR-CoNS isolated from both community and hospital environments. The high prevalence of biofilm producing MR-CoNS strains demonstrated in this study indicates the persisting ability in environments, and is useful in developing prevention strategies countering the spread of MR-CoNS.

Enhanced Antibacterial Activity of Acinetobacter baumannii Bacteriophage ØABP-01 Endolysin (LysABP-01) in Combination with Colistin.

Endolysins are lytic enzymes produced by bacteriophages with their ability to degrade the cell wall of bacterial hosts. Endolysin (LysABP-01) from Acinetobacter baumannii bacteriophage ØABP-01 was cloned, overexpressed and characterized. Endolysin LysABP-01 has a globular structure consisting of lysozyme-like (N-acetyl-ß-D-muramidase) catalytic domain. It contains 185 amino acids which correspond to a 21.1 kDa protein. The lytic activity of the recombinant endolysin protein was determined by a plate lysis assay for its ability to lyse the autoclaved cell (crude cell wall) of the different bacterial species. LysABP-01 can degrade the crude cell wall of A. baumannii strains, Escherichia coli and Pseudomonas aeruginosa but not of Staphylococcus aureus. The antibacterial activity of LysABP-01 and its synergism with various antibiotics were tested. The results exhibited elevated antibacterial activity in a combination of the sub-MIC LysABP-01 and colistin. The checkerboard assay for measuring antibiotic synergy of LysABP-01 and colistin was performed. This combination was synergistic against various drug-resistant strains of A. baumannii (FIC index < 0.5). In summary, our study highlights the ability of LysABP-01 endolysin to hydrolyze the A. baumannii cell wall and its synergistic interaction with colistin.

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water.

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.

Distribution among Thai children of methicillin-resistant Staphylococcus aureus lacking cna, fnbA and icaAD.

Staphylococcus aureus is a bacterium causing infections in both community and hospitals. S. aureus nasal colonization increases the risk of acquiring S. aureus infection. In this study, the prevalence of S. aureus nasal colonization was determined in Thai children, showing that nasal colonization was found in 78 of 217 subjects and the methicillin-resistant S. aureus (MRSA) carriage rate was present in 5 of 217 children. Among 78 S. aureus isolates, resistance to penicillin, erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole, oxacillin and cefoxitin was found in 99%, 8%, 1%, 4%, 1%, 8% and 6% of the isolates, respectively. The presence of 3 adhesin genes, investigated by PCR, revealed that among 78 isolates, fnbA, icaAD and cna was detected in 73, 72 and 48 isolates, respectively but not in the 5 MRSA isolates. Ninety-five percent and 31% of S. aureus isolates were able to form strong and weak biofilms, respectively. Four MRSA isolates that lacked icaAD were non-biofilm-forming strains. These results revealed a high prevalence of S. aureus nasal colonization in healthy Thai children with 2% being MRSA. Effective strategies to prevent S. aureus transmission and infection are therefore needed in the Thai community.

McsA and the roles of metal-binding motif in Staphylococcus aureus.

McsA is a key modulator of stress response in Staphylococcus aureus that contains four CXXC potential metal-binding motifs at the N-terminal. Staphylococcus aureus ctsR operon encodes ctsR, clpC, and putative mcsA and mcsB genes. The expression of the ctsR operon in S. aureus was shown to be induced in response to various types of heavy metals such as copper and cadmium. McsA was cloned and overexpressed, and purified product was tested for metal-binding activity. The protein bound to Cu(II), Zn(II), Co(II), and Cd(II). No binding with any heavy metal except copper was found when we performed site-directed mutagenesis of Cys residues of three CXXC motifs of McsA. These data suggest that two conserved cysteine ligands provided by one CXXC motif are required to bind copper ions. In addition, using a bacterial two-hybrid system, McsA was found to be able to bind to McsB and CtsR of S. aureus and the CXXC motif was needed for the binding. This indicates that the Cys residues in the CXXC motif are involved in metal binding and protein interaction.

Prevalence of methicillin-resistant Staphylococcus aureus among university students in Thailand.

We studied the prevalence of methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) nasal colonization among healthy young Thai adults. MSSA nasal colonization was found in 30 of 200 subjects (15%). The prevalence of MRSAnasal carriage was 1% (2 of 200) detected by cefoxitin/oxacillin disk diffusion and oxacillin salt screening methods. These carriers were associated with health care risk factors. The two MRSA isolates were mecA positive, SCCmec type II. All S. aureus isolates were tested for antibiotic resistance. Their resistance rates to penicillin, erythromycin, clindamycin, oxacillin and cefoxitin were 96.7, 26.7, 26.7, 6.7 and 6.7%, respectively. All MSSA and MRSA isolates were susceptible to gentamicin, chloramphenicol, trimethoprim/sulfamethoxazole, rifampicin, linezolid, fusidic acid, mupirocin, ciprofloxacin and vancomycin. The results of this first study of MRSA nasal colonization among healthy young Thai adults suggests MRSA is present in the Thai community.