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].

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.

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.

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.

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.

Antibacterial oral sprays from kaffir lime (Citrus hystrix DC.) fruit peel oil and leaf oil and their activities against respiratory tract pathogens.

BACKGROUND AND AIM: Kaffir lime fruit peel oil and Kaffir lime leaf oil have been reported for their activities against respiratory tract pathogens. The purpose of the study was to develop clear oral sprays to be used as a first-defense oral spray. EXPERIMENTAL PROCEDURE: Clear antibacterial oral sprays were prepared and analyzed for their respective active major compounds, using GC-MS. The sprays were tested against a Gr. A streptococcal clinical isolate and 3 standard respiratory tract pathogens, using Broth microdilution method. A 4-month stability test was carried out as well. RESULTS AND CONCLUSION: Six clear oral sprays, three formulae composed of Kaffir lime fruit peel oil (6, 10, 13%v/v KLO) and the other three formulae containing Kaffir lime leaf oil (4, 8, 12%v/v KLLO), were developed. The active compounds in KLO were α-terpineol and terpinene-4-ol whereas that in KLLO was citronellal. All oral sprays exhibited antibacterial activity against one Group A streptococcal clinical isolate and three respiratory pathogenic pathogens, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247, among which the strongest activity was against H. influenzae ATCC 49247. The antibacterial activity of all oral sprays remained unchanged in an accelerated stability test, at 4, 30, and 45 °C under 75% relative humidity, throughout the 4-month storage.

Complete Genome Sequences of Three Neisseria gonorrhoeae Isolates from Thailand with Multidrug Resistance and Multilocus Sequence Type 1903.

Multilocus sequence typing (MLST) sequence type 1903 (ST1903) is the most common ST of ceftriaxone-resistant Neisseria gonorrhoeae Here, we report three completed genome sequences of MLST ST1903 N. gonorrhoeae isolates collected from patients at Faculty of Medicine Siriraj Hospital, a university hospital in Bangkok, Thailand, in 2009 to 2011.

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.

Antimicrobial Resistance in Commensal Escherichia coli Isolated from Pigs and Pork Derived from Farms Either Routinely Using or Not Using In-Feed Antimicrobials.

The aims of this study were (i) to evaluate whether routine in-feed antimicrobial use in pigs or not resulted in differences in antimicrobial resistance (AMR) E. coli at different pig producing stages, and (ii) to determine whether resistant strains were presented in pig meat postslaughter. A total of 300 commensal E. coli isolates were obtained and examined for antibiograms, AMR genes, plasmid replicons, and molecular types. The isolates were from two farms either using (A) or not using in-feed antimicrobials (NA), sampled four times during the production cycle and once postslaughter. E. coli resistant to aminoglycosides containing aadA1, aadA2, and aadB and extended-spectrum beta-lactamase-producing (ESBLP) E. coli containing blaCTX-M-1 were significantly increased in the nursery and growing periods in farm A compared to farm NA. IncI1-Iγ and IncHI2 were common in the nursery period and were shown to transfer blaCTX-M genes by conjugation. ST10 was the most common type only found in live pigs. ST604, ST877, ST1209, and ST2798 ESBLP were found only in live pigs, whereas ST72, ST302, and ST402 ESBLP were found in pig meat.

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.

Gonococcal Antimicrobial Susceptibility and the Prevalence of blaTEM-1 and blaTEM-135 Genes in Neisseria gonorrhoeae Isolates from Thailand.

We studied the antimicrobial susceptibility and prevalence of the blaTEM-1 and blaTEM-135 genes in Neisseria gonorrhoeae isolates obtained in Thailand. The isolates were tested using the disk diffusion method, and 100% of 370 isolates were found susceptible to cefixime, ceftriaxone, cefotaxime, ceftazidime, cefepime, spectinomycin, and azithromycin. Some of the isolates were resistant to penicillin (85.7%), ciprofloxacin (88.0%), ofloxacin (97.4%), or tetracycline (89.1%). Penicillinase-producing N. gonorrhoeae accounted for 83.8% of isolates, with 70.0% of these further identified as penicillinase-producing plus tetracycline resistant N. gonorrhoeae. Penicillin, tetracycline, and ciprofloxacin are not recommended for treatment because of the high prevalence (89.7%) of multidrug resistant gonococci. A study of genes controlling enzyme of beta-lactamase production (blaTEM-1 and blaTEM-135) was performed using mismatch amplification mutation assay PCR method and DNA sequencing. Beta-lactamase positive N. gonorrhoeae carried blaTEM-1 (69.6%) and blaTEM-135 (30.4%), indicating that there is a significant increase and spread of blaTEM-135 among gonococci in Thailand.

Prevalence of the M Protein Gene in Group C and Group G Streptococci Isolated from Patients in Thailand.

We surveyed group C and group G ß-hemolytic streptococci for emm and emmL (emm -like) genes which encode the M protein, as well as determined their antimicrobial susceptibilities. A total of 97 isolates 79 GCS/GGS isolates and 18 isolates from other groups were tested for the M protein gene by PCR. Focusing on invasive infections with group A (GAS), group C (GCS), and group G (GGS) ß-hemolytic streptococci isolated from blood, the M protein gene was found in 90.0%, 84.6%, and 78.3% of isolates, respectively. The hypervariable N terminal region of the emm was sequenced from 62 isolates, and 26 types of the emm gene were identified. Based on these results, type emm222.2 may be endemic to Thailand. The results of antimicrobial susceptibility testing of groups C, G, and non-groups A to G isolates indicated high susceptibility (range 82-100%) to penicillin, cefotaxime, chloramphenicol, clindamycin, erythromycin, linezolid, ofloxacin, and vancomycin, whereas the isolates showed low susceptibility (range 0-15.6%) to tetracycline.

Plasmid metagenomics reveals multiple antibiotic resistance gene classes among the gut microbiomes of hospitalised patients.

Antibiotic resistance genes are rapidly spread between pathogens and the normal flora, with plasmids playing an important role in their circulation. This study aimed to investigate antibiotic resistance plasmids in the gut microbiome of hospitalised patients. Stool samples were collected from seven inpatients at Siriraj Hospital (Bangkok, Thailand) and were compared with a sample from a healthy volunteer. Plasmids from the gut microbiomes extracted from the stool samples were subjected to high-throughput DNA sequencing (GS Junior). Newbler-assembled DNA reads were categorised into known and unknown sequences (using >80% alignment length as the cut-off), and ResFinder was used to classify the antibiotic resistance gene pools. Plasmid replicon modules were used for plasmid typing. Forty-six genes conferring resistance to several classes of antibiotics were identified in the stool samples. Several antibiotic resistance genes were shared by the patients; interestingly, most were reported previously in food animals and healthy humans. Four antibiotic resistance genes were found in the healthy subject. One gene (aph3-III) was identified in the patients and the healthy subject and was related to that in cattle. Uncommon genes of hospital origin such as blaTEM-124-like and fosA, which confer resistance to extended-spectrum ß-lactams and fosfomycin, respectively, were identified. The resistance genes did not match the patients' drug treatments. In conclusion, several plasmid types were identified in the gut microbiome; however, it was difficult to link these to the antibiotic resistance genes identified. That the antibiotic resistance genes came from hospital and community environments is worrying.

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.

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.