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].

Genomic analysis of Enterococcus faecium strain RAOG174 associated with acute chorioamnionitis carried antibiotic resistance gene: is it time for precise microbiological identification for appropriate antibiotic use?

BACKGROUND: Preterm labor syndrome is associated with high perinatal morbidity and mortality, and intra-amniotic infection is a cause of preterm labor. The standard identification of causative microorganisms is based on the use of biochemical phenotypes, together with broth dilution-based antibiotic susceptibility from organisms grown in culture. However, such methods could not provide an accurate epidemiological aspect and a genetic basis of antimicrobial resistance leading to an inappropriate antibiotic administration. Hybrid genome assembly is a combination of short- and long-read sequencing, which provides better genomic resolution and completeness for genotypic identification and characterization. Herein, we performed a hybrid whole genome assembly sequencing of a pathogen associated with acute histologic chorioamnionitis in women presenting with PPROM. RESULTS: We identified Enterococcus faecium, namely E. faecium strain RAOG174, with several antibiotic resistance genes, including vancomycin and aminoglycoside. Virulence-associated genes and potential bacteriophage were also identified in this genome. CONCLUSION: We report herein the first study demonstrating the use of hybrid genome assembly and genomic analysis to identify E. faecium ST17 as a pathogen associated with acute histologic chorioamnionitis. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. The occurrence of E. faecium ST17 raised the awareness of the colonization of clinically relevant E. faecium and the carrying of antibiotic resistance. This finding has brought the advantages of genomic approach in the identification of the bacterial species and antibiotic resistance gene for E. faecium for appropriate antibiotic use to improve maternal and neonatal care.

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.

Rapid diagnosis of intra-amniotic infection using nanopore-based sequencing.

OBJECTIVES: Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. METHODS: Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. RESULTS: Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5-9 h from DNA extraction. CONCLUSIONS: This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

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.

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.