Somporn Srifuengfung

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

ABSTRACT 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 β-lactamase (TEM) gene variant, blaTEM-135, that may be a precursor in the transitional stage of a traditional blaTEM-1 gene into an extended-spectrum β-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 blaTEM-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 blaTEM-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 blaTEM variant (blaTEM-135) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

Prevalence and antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis isolated from patients in Bangkok, Thailand

Haemophilus influenzae serotype b (Hib) is significant because it causes invasive diseases, e.g. meningitis, pneumonia and bacteraemia. Hib vaccine is available in Thailand, however it is not on the Expanded Program of Immunization in Thailand owing to its high cost. Hib vaccine has limited use in Thailand, mostly in private hospitals. There are eight biotypes of H. influenzae identified by biochemical tests [1]. Production of b-lactamase is the most common reason for b-lactam resistance. Two different types of b-lactamase (TEM-1 and ROB-1 types) have been described in H. influenzae, but TEM-1 is predominant (93.7%) globally [2]. The blaTEM-1 gene is usually located on large (40 kb) chromosomally integrated conjugative elements and less commonly on small plasmids (at least three distinct plasmids with sizes from 4304 bp to 5646 bp have been characterised) [3]. In addition to blactamase, another b-lactam resistance mechanism is mutation in the ftsI gene encoding penicillin-binding protein 3 (PBP3), leading to altered PBP that has low affinity for ampicillin. Isolates with this mechanism of resistance are referred to as b-lactamasenegative ampicillin-resistant (BLNAR). Moraxella catarrhalis is also an important pathogen. The aim of this study was to determine the prevalence of different biotypes and serotypes, b-lactamase production, b-lactamase genes, ftsI gene mutation in H. influenzae and the blaBRO gene in M. catarrhalis as well as antimicrobial susceptibility. A total of 638 H. influenzae and 297 M. catarrhalis non-duplicate isolates were collected from different patients at Siriraj Hospital (Bangkok, Thailand) during January 2007 to December 2011. For H. influenzae, 57.7% of patients were male and 42.3% were female (M:F sex ratio 1.36:1). For M. catarrhalis, 54.5% of patients were male and 45.5% were female (M:F sex ratio 1.20:1). The patient age distribution ranged from 3 months to 85 years. Clinical specimens of H. influenzae were from various sites (Table 1), but specimens of M. catarrhalis were from the respiratory tract and eye. Sputum was acceptable for culture if it contained >25 polymorphonuclear cells and <25 epithelial cells per low-power field. All isolates were either from infections or from colonisation because in some cases they were isolated with other bacteria. Biotypes were determined by indole, urease and ornithine decarboxylase tests. Serotypes b and non-b were determined by agglutination using latex particles coated with serotype b antiserum (Bio-Rad, Hercules, CA). b-Lactamase production was determined by nitrocefin test (Becton Dickinson, Cockeysville, MD). Study of the blaTEM and blaROB-1 genes in b-lactamaseproducing H. influenzae, the ftsI gene in BLNAR H. influenzae and

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 β-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) β-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.

Gonococcal Antimicrobial Susceptibility and Prevalence of blaTEM-1, blaTEM-135 Genes in 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 and drug susceptibility of Salmonella isolated from patients in Bangkok, Thailand

Salmonella is a public health problem in many parts of the world, especially in developing countries. Globally, non-typhoidal Salmonella causes 93.8 million diarrhoeal illnesses and 155 000 deaths each year [1]. In 2010, Salmonella caused 3.4 million invasive infections and 681 000 deaths. The most widely reported serotypes are Salmonella enterica serotype Enteritidis and S. enterica serotype Typhimurium [1]. There are >2500 serovars and >50 serogroups of Salmonella [2]. The drugs of choice for treatment are extended-spectrum cephalosporins in children and fluoroquinolones in adults. We aimed to determine the serogroups, serotypes and antimicrobial susceptibility of Salmonella isolated from patients in Bangkok Thailand. A total of 728 non-duplicate Salmonella isolates were collected from different patients during January 2011 to April 2013. This study was conducted at Siriraj Hospital (Bangkok, Thailand), Thailand’s largest university-based tertiary care centre with 2600 beds and 100 000 annual admissions. Multiple isolates from different sites in the same patient were included only once. At the time of positive Salmonella isolation, 35.7% of isolates were from outpatients and 64.3% were from inpatients. The mean standard deviation age of patients was 31.34 27.89 years (range 3 days to 95 years), with a male to female ratio of 1.1:1 (51.8% males and 48.2% females). The occurrence of salmonellosis in different age groups was as follows: 60 years, 20.1%; <1 year, 17.6%; and 1–9 years, 17.5%, accounting for 55.2% of all patients. Serogroups were determined by slide agglutination using Salmonella polyvalent A–I antisera reagent and specific Salmonella antisera serogroups, including A, B, C, C1, C2, D, Vi, E, F, I, G and H (Oxoid Ltd., Basingstoke, UK). Serovars were determined for 146 available blood isolates by slide agglutination using specific Salmonella H-antisera and the Sven Gard method (S&A Reagents Lab Ltd., Bangkok, Thailand). Testing was conducted by the Kauffman–White scheme [2]. Antimicrobial susceptibility testing was performed by the disk diffusion method [3]. Escherichia coli ATCC 25922 was used as a control strain. Salmonella were mostly isolated from faeces (506; 69.5%) and blood (176; 24.2%), accounting for 93.7% of all isolates (Table 1). Salmonella serogroup B (33.4%) was predominant and was mostly found in faeces. In antimicrobial susceptibility testing, serogroup C1 had the lowest sensitivity both to ceftriaxone (14.9%) and cefotaxime (15.2%). For serotyping, the 146 available blood isolates were distributed into 27 serotypes. Salmonella Choleraesuis (34.2%) was the most common serotype, and S. Enteritidis (31.5%) ranked second, accounting for 65.8% of all isolates. Salmonella Typhi was not found. Salmonella Choleraesuis demonstrated higher drug resistance than S. Enteritidis. For