Isolation and Genomic Characterization of Schaalia turicensis from a Patient with Gonococcal Urethritis, Thailand.

Schaalia turicensis is facultative anaerobic Gram-positive bacillus that commonly inhabits the oropharynx, gastrointestinal, and genitourinary tract of healthy individuals. This organism has been co-isolated with Neisseria gonorrhoeae from 15-year-old Thai male patient with gonococcal urethritis in Bangkok, Thailand. In this study, we characterized the class 1 integron in S. turicensis isolate using whole-genome sequencing and bioinformatics analysis. Sequencing analysis confirmed the presence of an imperfect class 1 integron located on chromosome and a novel 24.5-kb-long composite transposon, named Tn7083. The transposon Tn7083 carried genes encoding chloramphenicol resistance (cmx), sulfonamide resistance (sul1), and aminoglycoside resistance [aph(6)-Id (strB), aph(3'')-Ib (strA), aph(3')-Ia].

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

HAEMOPHILUS INFLUENZAE FROM PATIENTS AT THE LARGEST UNIVERSITY TERTIARY CARE CENTER, THAILAND 2012 - 2015.

Haemophilus influenzae was isolated from 556 different patients, mostly 10 years or under, at a tertiary referral hospital in Bangkok, Thailand during 2012 - 2015. Peak period of detection was from January to March. Thirty-nine percent of the isolates were ß-lactamase positive. ß-Lactamase-negative ampicillin-resistant H. influenzae (BLNAR) constituted 2% of ß-lactamase-negative cases. H. influenzae was susceptible to ampicillin (58%), amoxicillin/clavulanate (99%), cefotaxime (100%), ceftriaxone (100%), cefuroxime (99%), ciprofloxacin (99%), chloramphenicol (86%), tetracycline (75%), and trimethoprim-sulphamethoxazole (52%). ß-Lactamase-producing isolates (72%) showed high minimal inhibitory concentration (MIC) to ampicillin (128-516 µg/ml) and all BLNAR isolates low ampicillin MIC (2-16 µg/ml). These findings indicate that the level of ampicillin resistance in H. influenzae depended on differences in resistance mechanism.

Role of gyrB Mutations in Pre-extensively and Extensively Drug-Resistant Tuberculosis in Thai Clinical Isolates.

DNA gyrase mutations are a major cause of quinolone resistance in Mycobacterium tuberculosis We therefore conducted the first comprehensive study to determine the diversity of gyrase mutations in pre-extensively drug-resistant (pre-XDR) (n = 71) and extensively drug-resistant (XDR) (n = 30) Thai clinical tuberculosis (TB) isolates. All pre-XDR-TB and XDR-TB isolates carried at least one mutation within the quinolone resistance-determining region of GyrA (G88A [1.1%], A90V [17.4%], S91P [1.1%], or D94A/G/H/N/V/Y [72.7%]) or GyrB (D533A [1.1%], N538D [1.1%], or E540D [2.2%]). MIC and DNA gyrase supercoiling inhibition assays were performed to determine the role of gyrase mutations in quinolone resistance. Compared to the MICs against M. tuberculosis H37Rv, the levels of resistance to all quinolones tested in the isolates that carried GyrA-D94G or GyrB-N538D (8- to 32-fold increase) were significantly higher than those in isolates bearing GyrA-D94A or GyrA-A90V (2- to 8-fold increase) (P < 0.01). Intriguingly, GyrB-E540D led to a dramatic resistance to later-generation quinolones, including moxifloxacin, gatifloxacin, and sparfloxacin (8- to 16-fold increases in MICs and 8.3- to 11.2-fold increases in 50% inhibitory concentrations [IC50s]). However, GyrB-E540D caused low-level resistance to early-generation quinolones, including ofloxacin, levofloxacin, and ciprofloxacin (2- to 4-fold increases in MICs and 1.5- to 2.0-fold increases in IC50s). In the present study, DC-159a was the most active antituberculosis agent and was little affected by the gyrase mutations described above. Our findings suggest that although they are rare, gyrB mutations have a notable role in quinolone resistance, which may provide clues to the molecular basis of estimating quinolone resistance levels for drug and dose selection.

Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates.

One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression.

Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates.

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

Characterization of recombinant flagellin B protein from Leptospira interrogans.

Symptoms of the early phase leptospirosis often are non-specific and can be a major problem in making a diagnosis of febrile illnesses. Rapid diagnosis of leptospirosis is of extreme importance, because antibiotic treatment provides greatest benefit when administered in early stage of the disease. Recombinant flagellin B (FlaB) gene (flaB) of Leptospira interrogans serovar Autumnalis strain Akiyami A was heterologously expressed and purified. The 35 kDa recombinant FlaB was 99% similar to the reference strain in GenBank. Rabbit polyclonal antirecombinant FlaB antibodies recognized using immunoblotting yeilded 35-36 kDa doublet from one saprophytic and eight pathogenic Leptospira serovars. Western blot assay showed that recombinant FlaB could distinguish leptospirosis from non-leptospirosis sera. This recombinant FlaB can be used in serodiagnosis of leptospirosis and identification of Leptospira spp.

Aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from non-cystic fibrosis patients in Thailand.

This study aimed to examine aminoglycosides (AMGs) resistance mechanisms, including the AMG-modifying enzyme genes, mexXY, rplY, nuoG, and galU, in the Pseudomonas aeruginosa non-cystic fibrosis (CF) isolates in Thailand. One hundred P. aeruginosa isolates from non-CF patients were examined for susceptibility to AMGs and for the presence of 10 AMG-modifying enzyme genes. Thirty randomly selected isolates were tested for transcription of mexXY and nuoG and mutations in rplY and galU. All the P. aeruginosa isolates exhibited simultaneous resistance to at least 4 AMGs. High resistance rates to amikacin (92%), gentamicin (95%), streptomycin (99%), and tobramycin (96%) were observed, and all isolates were resistant to kanamycin, neomycin, and spectinomycin. Nine AMG-modifying enzyme genes were detected, including aadA1 (84%), aadB (84%), aadA2 (67%), ant(2″)-Ia (72%), strA-strB (70%), aph(3')-IIb (57%), aac(3')-Ia (40%), and aac(6')-IIa (27%). None of the isolates harbored aac(6')-IIb. Of 30 isolates tested, all but 1 isolate expressed MexXY. Two isolates did not express nuoG. Six isolates carried an amino acid change in RplY, but none of the isolates harbored mutation in galU. The results indicated that the AMG-modifying enzyme genes were widespread among the P. aeruginosa non-CF isolates. The MexXY efflux pump and inactivation for rplY played a role in AMG resistance but disruption of nuoG or galU did not.

Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their blaTEM-135 Gene Variant in Bangkok, Thailand, 2015-2017.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) possessing blaTEM-135 is a serious public health threat. With only a single change in the amino acid sequence, blaTEM-135 could evolve into a TEM-type extended-spectrum beta-lactamase (ESBL), which hydrolyzes extended-spectrum cephalosporins, including ceftriaxone and cefixime. We investigated the molecular epidemiological characteristics, types of plasmids in PPNG isolates, and prevalence of PPNG clinical isolates producing TEM-135 beta-lactamases. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was used to determine the molecular epidemiological characteristics of 99 PPNG isolates collected from 2015 to 2017. A mismatch amplification mutation assay was used to examine the blaTEM-135 gene prevalence. Of the 89 identified NG-MAST sequence types, 65 (73.0%) were novel. Only 17.7% (43/243) of PPNG isolates belonged to 16 genogroups. The most frequent plasmid was African, followed by Rio/Toronto, and Asian. The blaTEM-135 allele was found in Rio/Toronto plasmids. The blaTEM-135 allele was present in 23.2% (23/99) of the PPNG isolates. PPNG isolates expressing TEM-135 beta-lactamase exhibited significantly higher penicillin MIC (minimum inhibitory concentration) values than TEM-1 PPNG isolates. The PPNG isolates showed high genetic diversity and a high proportion of blaTEM-135 alleles. Mutation of the blaTEM-135 allele is worrisome as only one mutation could cause TEM-1 to evolve into an ESBL variant that degrades ceftriaxone. Ongoing surveillance of blaTEM-135 and new PPNG isolates is imperative.

Workflow for Identification of Burkholderia pseudomallei Clinical Isolates in Myanmar.

Burkholderia pseudomallei, the highly infectious and causative organism of melioidosis, was first identified in Myanmar in 1911. B. pseudomallei was identified in Myanmar because of its genetic relatedness to Burkholderia species. In this study, we identified two isolates of Burkholderia cenocepacia, two Acinetobacter baumannii complexes, and 18 clinical isolates of B. pseudomallei using Vitek 2. These isolates were first screened using a latex agglutination test, which showed positive results in 20 of the 22 isolates. All isolates were cultured on Ashdown՚s agar and further tested using molecular methods. Specific PCR for type III secretion system (TTSs) gene clusters indicated 19 B. pseudomallei isolates out of 22 isolates. Furthermore, 16S rRNA and recA gene sequencing were used as the gold standard methods and yielded the same results. RapID NF Plus detected 16 B. pseudomallei out of 22 isolates. Vitek 2 and RapID NF Plus should be considered key tools in the diagnosis of melioidosis and surveillance of B. pseudomallei in Myanmar; however, accurate identification must be confirmed by TTS1 PCR. This study evaluated the presumptive workflow for the investigation of B. pseudomallei infections using different methods and options, in line with the available equipment.

Molecular analyses of TEM genes and their corresponding penicillinase-producing Neisseria gonorrhoeae isolates in Bangkok, Thailand.

Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A ß-lactamase (TEM) gene variant, bla(TEM-135), that may be a precursor in the transitional stage of a traditional bla(TEM-1) gene into an extended-spectrum ß-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the bla(TEM-135) gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the bla(TEM-135) allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a bla(TEM) variant (bla(TEM-135)) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

Class 1 integrons in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from clinical isolates.

Resistance to various antimicrobial agents is an increasing problem in Pseudomonas aeruginosa and Acinetobacter baumannii infections. In this study, the roles of integrons were examined in 101 P. aeruginosa isolates and 176 A. baumannii isolates from patients. The frequencies and characteristics of class 1, 2 and 3 integrons were investigated and the horizontal transfer of integrons was assessed. Among these isolates, class 1 integrons with a resistance gene cassette were detected in 69.3% of P. aeruginosa and 31.8% of A. baumannii isolates, but class 2 and 3 integrons were not found. Five novel gene cassette arrays were identified in P. aeruginosa: aacA7-cmlA, aadB-blaOXA,-o-aadA1, aadB-arr-2-cmlA-blaOXA,-o-aadA1, aadB-cmlA-aadA1 and aadB-cmlA-blaOXA-10-aadA15. The integrons found in A. baumannii isolates in this study were previously reported. Horizontal transfer of some class 1 integrons was detected in both P. aeruginosa (2/70) and A. baumannii (5/57). These data confirm the high prevalence of class 1 integrons with a variety of gene cassette combinations among multidrug-resistant P. aeruginosa and A. baumannii clinical isolates.

Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand.

BACKGROUND: The increasing rate of antimicrobial-resistant Neisseria gonorrhoeae poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance. METHODS: We collected 117 N. gonorrhoeae isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and mtrR (a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 µg/ml) were sequenced using Illumina and Nanopore sequencing platforms. RESULTS: The proportions of N. gonorrhoeae isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the mtrR promoter and an H105Y mutation in the mtrR coding region were found in the N. gonorrhoeae isolate with the highest azithromycin MIC value (1 µg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II penA and had mutations in penB and the mtrR coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid. CONCLUSIONS: The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of N. gonorrhoeae, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.

Widespread presence of dfrA12 and its association with dfrA12-aadA2 cassette in Salmonella enterica isolates from swine.

One hundred and eighty-nine Salmonella isolates from swine were tested for susceptibility to nine antimicrobial agents, presence of dfrA12 and class 1 integrons containing dfrA12-orfF-aadA2 cassette. All isolates were multidrug resistant and exhibited highest resistance prevalence to trimethoprim (93%). Most isolates (89%) were intll-positive and 107 isolates (57%) carried dfrA12, all of which were resistant to trimethoprim. Forty-eight dfrA12-harboring strains (45%) were intl1-positive together with dfrA12-aadA2 gene cassette. Fifteen isolates contained dfrA12 but not intl1 and dfrA12-aadA2 cassette. The results indicated a wide distribution of dfrA12 and its role in dissemination of trimethoprim resistance among Salmonella isolates from fattening pigs.

Spread of Antimicrobial-Resistant Salmonella from Poultry to Humans in Thailand.

Food animal production is important for every country. Several antibiotic agents are used in poultry farming to reduce the economic losses arising from mostly untested infectious diseases. This continued study was performed to determine the prevalence of antibiotic-resistant Salmonella in broiler chickens, poultry farmers, and Salmonella bacteremia patients. A total of 121 Salmonella isolates were collected from the Thai provinces of Khon Kaen (65 isolates), Ratchaburi (43 isolates), and Phayao (13 isolates). Salmonella from chicken showed a high rate of resistance to nalidixic acid and tetracycline. Sixty-four percent of Salmonella isolates carried class 1 integrons (intI1 gene-positive). Among the 121 Salmonella isolates, there were 15 serotypes, with S. Enteritidis being the most common. A clonal relationship between the chicken and human isolates was demonstrated by 3 molecular typing methods: enterobacterial repetitive intergenic consensus polymerase chain reaction; pulsed-field gel electrophoresis; and high-throughput multilocus sequence typing. A spread of the sequence type 11 clone was found between chickens and humans. This study revealed a large-scale Salmonella outbreak in Thailand, a link between resistant bacteria from poultry farms and vertical transmission through the food chain, and horizontal transmission of resistance genes. These results can be used for future surveillance and monitoring.

Complete Genome Sequence of Schaalia turicensis Strain CT001, Isolated from a Patient with Gonococcal Urethritis in Thailand.

Schaalia turicensis, a Gram-positive bacillus, is a potential pathogen in genital infections. Here, we report the complete genome sequence of S. turicensis strain CT001, which was coisolated with Neisseria gonorrhoeae. Comprehensive analysis revealed the presence of a composite transposon carrying an imperfect class 1 integron in S. turicensis.

Complete Genome Sequence of Neisseria gonorrhoeae Multilocus Sequence Type ST7363 Isolated from Thailand.

A Neisseria gonorrhoeae multilocus sequence type (MLST) ST7363 strain was isolated from a patient at the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, in 2010 and completely sequenced. This strain is susceptible to ceftriaxone and cefixime. A complete circular chromosome and circular plasmids were assembled from combined Oxford Nanopore Technologies (ONT) and Illumina sequencing.

Detection of outer membrane porin protein, an imipenem influx channel, in Pseudomonas aeruginosa clinical isolates.

Decreased permeability to imipenem is the most frequent mechanism of imipenem resistance in Pseudomonas aeruginosa. We have determined the presence of OprD porin protein, an imipenem influx channel, in 70 carbapenem-resistant P. aeruginosa clinical isolates by Western blot analysis using rabbit anti-OprD polyclonal antibody. Ninety-eight percent (54 of 55 isolates) of imipenem-and meropenem-resistant P. aeruginosa clinical isolates were negative for OprD porin production. A small group of isolates resistant to imipenem but susceptible to meropenem (2 isolates) produced OprD protein but at a level 3-5 times lower than the wild type P. aeruginosa ATCC27853 strains. This study indicates that the loss of OprD porin protein was the main mechanism for imipenem resistance in P. aeruginosa clinical isolates. Determination of the status of OprD level in P. aeruginosa may help in the better selection of appropriate carbapenem antibiotics.

High prevalence of bla(OXA)-23 in oligoclonal carbapenem-resistant Acinetobacter baumannii from Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Acinetobacter baumannii has emerged in health care settings as a pandrug-resistant pathogen. Carbapenems are ineffective for treatment of this pathogen. Here we explored the molecular epidemiology and mechanism of carbapenem resistance in clinical isolates of carbapenem-resistant A. baumannii (CRAB). Antibiotic susceptibility by disk diffusion test was performed using imipenem and meropenem disk on 200 different clinical CRAB isolates. All isolates were resistant and gave inhibition zones of both antibiotic disks < or = 13 mm. Polymerase chain reaction (PCR) was carried out on 37 randomly selected isolates to amplify the common carbapenem hydrolyzing beta-lactamase genes (bla(OXA23)-like, bla(OXA-24/40)-like, bla(OXA-58), bla(IMP), and bla(VLM)). Clones were resolved by PCR-randomly amplified polymorphic DNA (PCR-RAPD) and plasmid profiling. PCR amplification and DNA sequencing revealed the existence of bla(OXA-23) downstream of the insertion element, ISAba1, in all 37 isolates tested. This segment was present in the carbapenem-resistant genomic resistant island AbaR4. These isolates were resolved into three RAPD types (Type I, 20 isolates; Type II, 16 isolates; and type III, 1 isolate) and 10 plasmid profiles. The CRAB isolates investigated here were oligoclonal and carbapenem resistance was conferred by the presence of bla(OXA-23). The presence of this beta-lactamase gene in many clonal isolates indicated its wide spread.

The sequence of pbp2b from penicillin-resistant Streptococcus pneumoniae in Thailand.

OBJECTIVE: Penicillin resistance in Streptococcus pneumoniae isolates results from altered penicillin-binding proteins (PBPs), especially PBP2, which has a reduced affinity to penicillin. This study evaluated drug resistance and the gene sequence of the conserved motif pbp2b of penicillin-resistant isolates in Thailand. MATERIAL AND METHOD: Penicillin-resistant pneumococci with minimum inhibitory concentrations (MIC) for penicillin > or = microg/ ml and penicillin-susceptible strains were identified from clinical specimens. The pbp2b genes were amplified by polymerase chain reaction (PCR), and the purified PCR product was cloned into E. coli. The recombinant plasmid clones containing pbp2b were sequenced and evaluated for mutations corresponding to penicillin and cefotaxime resistance. RESULTS: Penicillin-susceptible S. pneumoniae isolates were susceptible to 12 other antibiotics tested (range 95-100%) while penicillin-nonsusceptible isolates were resistant to most antibiotics except amoxicillin/clavulanate and levofloxacin. Sequence analysis of pbp2b showed a substitution of A for T451 next to the region of the SSN triad in all six resistant isolates tested and mutations clustered around the KTG triad in two isolates. Using the ClustalW alignment program, Thai isolates differed from those of European countries, but were more similar to those from Japan than Korea. CONCLUSION: Penicillin or cefotaxime resistance in S. pneumoniae in Thailand was due to affinity reduction of PBP2b, similar to changes found in other Asian isolates.