Tanittha Chatsuwan

Accuracies of Leuconostoc phenotypic identification: a comparison of API systems and conventional phenotypic assays

BackgroundCommercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria.MethodsClinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-β hemolysis in our institute during 1997–2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCRs, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005–2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays.ResultsOut of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed.ConclusionThe current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available.

Serotype coverage of pneumococcal conjugate vaccine and drug susceptibility of Streptococcus pneumoniae isolated from invasive or non-invasive diseases in central Thailand, 2006-2009.

The serotype of 172 S. pneumoniae isolates obtained from normally sterile sites from January 2006 to February 2009 in Thai patients was evaluated. The most common serotypes were 6B, 23F, 14, 19F, and 19A in patients <5 year-old, and 6B, 19A, 23F, 4, 9V in patients >65-year old. Seven-valent pneumococcal conjugated vaccine (PCV-7) covered 70.3%, 43.6%, and 43.5% of patients <5, 5-64 and > or = 65 years of age, respectively, while PCV-13 covered 81.2%, 59.7%, and 60.9%, respectively. PCV-9, PCV-10, PCV-11 had very similar coverage as PCV-7. The antibiotic susceptibility rates of the isolates from sterile sites were 88.7-95.7% for penicillin, 90.6-98.4% for cefotaxime, 92.2-100% for ofloxacin and 100% for ciprofloxacin. PCV-7 covered 83% and 100%, respectively, of penicillin and cefotaxime non-susceptible isolates in patients <5-year old.

Drug-resistant nontyphoidal Salmonella bacteremia, Thailand.

To the Editor: Despite improved public health, serious infections with nontyphoidal Salmonella enterica remain a major clinical and public health concern in Thailand and worldwide (1,2). Life-threatening Salmonella infections resistant to fluoroquinolones, extended-spectrum cephalosporins, or both, have been increasingly reported (3). Use of antimicrobial drugs for disease prevention and growth promotion in food animals has been implicated in this increase in drug resistance (4). Because of extensive global travel, such increases affect the medical community domestically and internationally (5). We report a pilot survey of drug resistance in Salmonella spp. in Thailand.

Comparison of methods for identifying causative bacterial microorganisms in presumed acute endophthalmitis: conventional culture, blood culture, and PCR

BackgroundIdentification of bacterial pathogens in endophthalmitis is important to inform antibiotic selection and treatment decisions. Hemoculture bottles and polymerase chain reaction (PCR) analysis have been proposed to offer good detection sensitivity. This study compared the sensitivity and accuracy of a blood culture system, a PCR approach, and conventional culture methods for identification of causative bacteria in cases of acute endophthalmitis.MethodsTwenty-nine patients with a diagnosis of presumed acute bacterial endophthalmitis who underwent vitreous specimen collection at King Chulalongkorn Memorial Hospital were enrolled in this study. Forty-one specimens were collected. Each specimen was divided into three parts, and each part was analyzed using one of three microbial identification techniques: conventional plate culture, blood culture, and polymerase chain reaction and sequencing. The results of the three methods were then compared.ResultsBacteria were identified in 15 of the 41 specimens (36.5%). Five (12.2%) specimens were positive by conventional culture methods, 11 (26.8%) were positive by hemoculture, and 11 (26.8%) were positive by PCR. Cohen’s kappa analysis revealed p-values for conventional methods vs. hemoculture, conventional methods vs. PCR, and hemoculture vs. PCR of 0.057, 0.33, and 0.009, respectively. Higher detection rates of Enterococcus faecalis were observed for hemoculture and PCR than for conventional methods.ConclusionsBlood culture bottles and PCR detection may facilitate bacterial identification in cases of presumed acute endophthalmitis. These techniques should be used in addition to conventional plate culture methods because they provide a greater degree of sensitivity than conventional plate culture alone for the detection of specific microorganisms such as E. faecalis.Trial registrationThai Clinical Trial Register No. TCTR20110000024.

Changing trends in serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae causing invasive diseases in Central Thailand, 2009-2012.

To describe the trends in serotype distribution and antimicrobial susceptibility of S. pneumoniae causing invasive pneumococcal diseases (IPD) we tested 238 pneumococci isolates from normally sterile sites between 2009 and 2012 and compared these findings with previous data collected within our network. Serotyping was performed for 15 serotypes contained in the 7-,10-, 13-, and experimental 15-valent pneumococcal conjugate vaccines (PCV). The most common serotypes found were 6B (13.9%), 19A (12.6%), 14 (8.0%), 18C (5.9%), and 6A (3.8%); and 39.9% were non-PCV15 serotypes. One of 81 patients with available data had breakthrough infection with vaccine serotype (19F). There was a significant increase of serotype 19A among children ≤5 years (5.6% in 2000–2009 vs 18.3% in 2009–2012, P = 0.003). The all-age serotype coverage was 36.4%, 41.5%, 59.3%, and 59.7% for PCV7, PCV10, PCV13, and PCV 15, respectively. The corresponding coverage in children ≤5 years were 46.4%, 48.8%, 73.2%, and 73.2% respectively. High susceptibilities to penicillin (89.7%), cefotaxime (95.7%), cefditoren (90.2% by Spanish breakpoints), ofloxacin (97.9%), and levofloxacin (100%), but low to cefdinir (50.0%), cefditoren (45.1% by US-FDA breakpoints), macrolides (<50%), clindamycin (67.7%), tetracycline (41.4%), and trimethoprim-sulfamethoxazole (32.4%) were observed. Serotype 19A was less susceptible to penicillin (80.0 vs 91.2%, P = 0.046), cefditoren (66.7 vs 95.5% by Spanish breakpoints, P = 0.004), and tetracycline (9.1 vs 45.5%, P = 0.024) than non-19A isolates. These data emphasize the need for continued surveillance to monitor changes in serotypes as well as antimicrobial susceptibilities in order to guide strategies for prevention and treatment.

In Vitro Activities of Carbapenems in Combination with Amikacin, Colistin or Fosfomycin against Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

Carbapenem-resistant Acinetobacter baumannii clinical isolates (n=23) were investigated for carbapenem resistance mechanisms and in vitro activities of carbapenems in combination with amikacin, colistin, or fosfomycin. Major carbapenem resistance mechanism was OXA-23 production. The vast majority of these isolates were OXA-23-producing A. baumannii ST195 and ST542, followed by novel STs, ST1417, and ST1423. The interuption of carO by a novel insertion sequence, ISAba40, was found in two isolates. The combinations of imipenem and fosfomycin, meropenem and amikacin, imipenem and amikacin, and imipenem and colistin were synergistic against carbapenem-resistant A. baumannii by 65.2%, 46.2%, 30.8%, and 17.4%, respectively. Surprisingly, the combination of imipenem and fosfomycin was the most effective in this study against A. baumannii, which is intrinsically resistant to fosfomycin. Imipenem and fosfomycin inhibit cell wall synthesis; therefore, fosfomycin may be an adjuvant and enhance the inhibition of cell wall synthesis of carbapenem-resistant A. baumannii when combined with imipenem.

Serotype distribution and antibiotic susceptibility of invasive Streptococcus pneumoniae isolates in patients aged 50 years or older in Thailand

As the 13-valent pneumococcal conjugate vaccine (PCV13) has been approved for use in adults aged 50 y and older, we evaluated vaccine-serotype coverage rate in Thai adult patients with invasive pneumococcal infections before the vaccine was widely used. Of the 157 S. pneumoniae isolates from normal sterile sites during January 2005 to September 2012, 150 (95%) from blood, mean patients’ age 69.6 (range 50–89) years, the overall serotype coverage by PCV13 was 58%. The vaccine covered 50%, 56%, 59%, and 68% of the invasive isolates from patients aged 50–59, 60–69, 70–79, and ≥80 y, respectively. The most common vaccine serotypes were 6B (17%), 19A (9%), 18C (5%), and 23F (4%). The susceptibility rates of penicillin and ceftriaxone were 95% and 96% for nonmeningitis criteria; and 46% and 92% for meningitis criteria, respectively. The susceptibilities to other antibiotics were: chloramphenicol 76%, clindamycin 80%, erythromycin 57%, levofloxacin 100%, ofloxacin 94%, tetracycline 39%, trimethoprim/sulfamethoxazole 37%, linezolid 99%, and vancomycin 100%, respectively. These data served as a reference for monitoring of vaccine serotype coverage with future increased vaccine utilization.

Mechanisms of carbapenem resistance in Acinetobacter pittii and Acinetobacter nosocomialis isolates from Thailand

Purpose. The emergence of carbapenem resistance in non‐baumannii Acinetobacter has increased in clinical settings worldwide. We investigated the prevalence and mechanisms of carbapenem resistance in A. pittii and A. nosocomialis Thai isolates. Methodology. Acinetobacter calcoaceticus‐Acinetobacter baumannii (Acb) complex isolates were identified by gyrB mulitplex PCR. Carbapenem susceptibilities were studied by the agar dilution method and carbapenemase genes were detected by multiplex PCR. Reductions of the outer membrane proteins (OMPs) were evaluated by SDS‐PAGE. Overexpressions of efflux pumps were detected by using efflux pump inhibitors and RT‐PCR. Results. Of the 346 Acb isolates, 22 and 19 were A. pittii and A. nosocomialis, respectively. The carbapenem resistance rates were 22.7 % in A. pittii and 26.3 % in A. nosocomialis. Three carbapenem‐resistant A. pittii carried blaOXA‐23. One carbapenem‐resistant A. pittii harboured blaOXA‐58, while another isolate co‐harboured blaOXA‐58 and blaIMP‐14a. blaOXA‐58 was also found in three carbapenem‐susceptible A. pittii. Five carbapenem‐resistant A. nosocomialis carried blaOXA‐23. Eighteen A. pittii isolates carried blaOXA‐213‐like. Reduced OMPs were found in carbapenem‐resistant and ‐susceptible A. pittii carrying blaOXA‐58, but were not detected in carbapenem‐resistant A. nosocomialis isolates. Overexpression of adeE was found in carbapenem‐resistant A. pittii. No efflux pump genes were present in carbapenem‐resistant A. nosocomialis. Conclusion. The major mechanisms of carbapenem resistance in A. pittii and A. nosocomialis were the production of OXA‐23 and OXA‐58. Overexpression of adeE played a role in carbapenem resistance in A. pittii. Since blaOXA‐58 was found in carbapenem‐susceptible A. pittii, using carbapenems in the treatment of A. pittii infection should be considered.

High prevalence of ceftriaxone resistance among invasive Salmonella enterica serotype Choleraesuis isolates in Thailand: The emergence and increase of CTX-M-55 in ciprofloxacin-resistant S. Choleraesuis isolates

S. Choleraesuis is a highly invasive zoonotic pathogen that causes a serious systemic infection in humans. The emergence and increase of resistance to ceftriaxone and ciprofloxacin among S. Choleraesuis has become a serious therapeutic problem. The present study demonstrated high frequency of antimicrobial resistance in Salmonella Choleraesuis among 414 nontyphoidal Salmonella isolates from bacteremic patients in Thailand. High rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistances were observed in S. Choleraesuis isolates. The dissemination of the self-transferable blaCTX-M-14-carrying IncFIIs, IncFII, and IncI1 plasmids and blaCMY-2-carrying IncA/C plasmid along with the clonal spread of blaCMY-2-harbouring S. Choleraesuis isolates contributed to the high frequency of resistance to extended-spectrum cephalosporins (ESCs; third- and fourth-generation cephalosporins) during 2005-2007. We reported the first occurrence of ceftazidime-hydrolysing CTX-M-55 in S. Choleraesuis isolates which dramatically increased and became the most abundant CTX-M variant among ESC-resistant S. Choleraesuis isolates during 2012-2016. The spread of clone pulsotype B3 was due to the dissemination of IncA/C plasmids carrying both blaCTX-M-55 and qnrS1 among ciprofloxacin-resistant S. Choleraesuis isolates harbouring D87G in GyrA. These isolates were apparently responsible for the high rates of co-resistance to ESCs and ciprofloxacin (51.3%) during 2012-2016. This study emphasizes the importance to have an action plan to control the dissemination of antimicrobial resistance in S. Choleraesuis since this poses a threat to global health due to travel and trade in animal food products.

Peritoneal Dialysis-Related Peritonitis Due to Melioidosis: A Potentially Devastating Condition

♦ Background: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in Southeast Asia and Northern Australia. Although a wide range of clinical manifestations from this organism are known, peritonitis associated with peritoneal dialysis (PD) has rarely been reported. ♦ Patients and Methods: Peritoneal dialysis patients from all regions in Thailand were eligible for the study if they had peritonitis and either peritoneal fluid or effluent culture positive for B. pseudomallei. Patient data obtained included baseline characteristics, laboratory investigations, treatments, and clinical outcomes. When possible, PD fluid and removed Tenckhoff (TK) catheters were submitted for analyses of minimal inhibitory concentration (MIC) and microbial biofilm, respectively. ♦ Results: Twenty-six patients were identified who were positive for peritoneal B. pseudomallei infection. The recorded mean age was 50 ± 15 (24 – 75) years, and the majority (58%) were female. Most of the cases were farmers living in Northeastern and Northern Thailand. Almost half of the cases had diabetes. Infections were reported commonly during the monsoon season and winter. The clinical presentations of peritonitis were similar to the manifestations from other microorganisms. Nine patients (41%) died (7 from sepsis), 6 fully recovered, and 7 switched to permanent hemodialysis. The mortality was potentially associated with sepsis (p = 0.007), infection during the monsoon season (p = 0.017), high initial dialysate neutrophils (p = 0.045), and high hematocrit (p = 0.045). Although no antibiotic resistance to ceftazidime and carbapenems was detected, approximately 50% of patients died with this treatment. Microbial biofilms were identified on the luminal surface of 4 out of 5 TK catheters, but the removal of the catheter did not alter the outcomes. ♦ Conclusion: Peritoneal dialysis-related peritonitis due to melioidosis is uncommon but highly fatal. Increased awareness, early diagnosis, and optimal management are mandatory.