Rapid and simple identification of carbapenemase genes, bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye.

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla NDM-1, 9 bla IMP-14a, 2 bla IMP-48, 1 bla IMP-1, 1 bla IMP-4, 1 bla IMP-9, 1 bla IMP-15, 4 bla VIM-2, 1 bla VIM-1, 1 bla IMP-14a & bla VIM-2, 7 bla KPC-2, 3 bla OXA-48 and 3 bla OXA-181) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.

DIAGNOSTIC TEST OF SPUTUM GENEXPERT MTB/RIF FOR SMEAR NEGATIVE PULMONARY TUBERCULOSIS.

The objective of this study was to evaluate the performance of the Gene-Xpert MTB/RIF sputum test for diagnosing pulmonary tuberculosis (TB) among patients sputum acid-fast bacillus (AFB) smear negative results in Thailand, a country with a high prevalence of pulmonary tuberculosis. We studied 151 patients who presented to Srinagarind Hospital, Khon Kaen, Thailand with a 2 week or more history of fever and/or cough and an abnormal chest radiograph between 2010 and 2014; these patients had at least 2 negative sputum AFB smear results. Of these, 76 were diagnosed as having either confirmed or probable pulmonary TB: the 32 confirmed cases were those with a positive sputum culture for Mycobacterium tuberculosis (MTB) and the 44 probable case were those with clinical and radiographic findings consistent with TB and who had a response to anti-TB therapy. Seventy-five cases were diagnosed as not having pulmonary TB. Of the 32 patients with a positive sputum culture for MTB, 26 had a positive GeneXpert MTB/RIF sputum test. Compared to sputum culture for MTB the GeneXpert MTB/ RIF test gave a sensitivity of 83.9% (95% CI: 66.3-94.5) and a specificity of 92.1% (95% CI: 83.6-97), a positive predictive value (PPV) of 81.3% (95% CI: 63.6-92.8) and a negative predictive value (NPV) of 93.3% (95% CI: 85.1-97.8). The GeneXpert MTB/RIF test had a fair sensitivity and specificity for diagnosing smear negative pulmonary TB. It may be useful for diagnosing pulmonary TB in patients with a negative sputum AFB smear. The assay is faster than culture and can detect rifampicin resistant strains of MTB.

Epidemiology and identification of potential fungal pathogens causing invasive fungal infections in a tertiary care hospital in northeast Thailand.

Invasive fungal infections (IFIs) are life threatening and associated with a high mortality rate. Here, we describe the distribution of pathogens, host risk factors, and significance of fungi isolated from patients with IFIs. The study included 861 fungal isolates recovered between 2006 and 2011 from 802 patients at Srinagarind Hospital, Thailand. Based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group 2008 criteria, 28.5% (245/861 isolates) of the fungal isolates were considered to be causative agents of IFIs. The most common fungus was Candida albicans (46%, 396/861 isolates). However, the most common yeast causing IFIs was Cryptococcus neoformans (34.7%, 85/245 isolates), while the most common mould was Penicillium marneffei (10.6%, 26/245 isolates). Cryptococcosis was significantly associated with human immunodeficiency virus infections (P < 0.001). Trend analysis revealed that there was no significant increase in IFI cases (P = 0.34) from 2006 to 2011 or from 2007 to 2011 (P = 0.05), but there was a trend toward significant increases in candidiasis (P = 0.04). The fungal isolates were categorized according to the positive predictive value of their recovery in cultures as being true (>95%), moderate (5%-95%), and rare (<5%) pathogens. This classification system could facilitate the prediction of the likelihood of diseases caused by the isolated fungi.

Evaluation of an immunochromatographic test kit for detecting Mycobacterium tuberculosis complex in sputum samples and on solid and in liquid cultures.

A rapid, cheap and effective method for diagnosing tuberculosis (TB) is essential for TB control. We evaluated the performance of an immunochromatographic assay (ICA) kit (SD Bioline TB Ag MPT64 rapid test) designed for detecting Mycobacterium tuberculosis complex (MTC) in liquid and solid cultures to determine its ability to detect and differentiate MTC directly in sputum samples. We attempted to optimize antigen extraction using several sputum solvents under various conditions. Adding the sputum solvent prior to using the ICA kit gave a sensitivity of 71.7% (43/60) for all acid-fast bacillus (AFB) stain positive specimens and a 100% specificity (20/20) among AFB negative specimens. Without sputum solvent, the ICA kit had 0% sensitivity for detecting MTC in sputum. We also evaluated the ICA test kit for its designed purpose of detecting MTC in 80 solid and liquid culture specimens positive for MTC using the niacin accumulation test or polymerase chain reaction (PCR) (16s-23s ITS). The ICA kit gave 100% sensitivity and specificity. We also evaluated the ICA test kit on 3 reference specimens of MTC, 15 nontuberculous mycobacteria (NTM) species, 7 bacterial species and 5 Candida albicans specimens. The tested ICA kit gave 100% specificity. The tested ICA kit was useful for detecting and differentiating MTC in solid and liquid cultures, but not useful for detecting MTC in sputum samples even treated with sputum solvent. The tested ICA kit should be used only for liquid and solid culture specimens. Therefore, the tested kit is inappropriate for use in evaluating sputum samples.

Treatment outcomes of new and previously-treated smear positive pulmonary tuberculosis at Srinagarind Hospital, a tertiary care center in northeast Thailand.

OBJECTIVE: To study treatment outcomes of new and previously-treated smear positive pulmonary TB according to current WHO guideline 2010 and factors related to treatment success at Srinagarind Hospital. MATERIAL AND METHOD: Adult patients who had smear-positive pulmonary TB treated at Srinagarind Hospital between January 2005 and December 2010 were enrolled in the present study. RESULTS: Over a 6-years period, 322 patients (272 new and 50 previously-treated cases) were diagnosed smear positive pulmonary TB. The mean age was 48.85 (SD 17.9) years and the male to female ratio was 1.8:1. The mean duration of symptoms in the previously-treated group was longer than the new cases (2.39 vs. 1.99 months, p = 0.38). Symptoms, underlying diseases, HIV status, and organ involvement between these two groups were not different. Two-thirds (72.1% of patients) had cough, 35.4% had fever and 20.5% weight loss. Twenty-three percent of cases had underlying DM and 7.8% were HIV positive. Disseminated TB was found in 18.9% of cases. The mean duration of treatment in new cases was 6.88 months vs. 11.20 months in previously-treated cases. The common regimens for new cases included 2IRZE/4IR (72.8%) and 2IRE/7IR (19.1%) vs. 2IRZE/4IR (62%), 2IRE/7IR (12%), and other regimens for MDR patients (6%) among the previously-treated cases. However in previously-treated group IRZES/IRZE/IRE (p = 0.001), second-line drugs (p = 0.002), and MDR regimens (p < 0.001) were statistically more common treatments than in the new cases group. About 60% of cases were treated at TB clinic. The success rate among new cases who had completed treatment at Srinagarind Hospital was higher than for previously-treated ones (94.8% vs. 86.4%; p = 0.04). Among previously-treated cases (n = 50); 24 were defaulters, 19 were relapses, and seven were failures. For the defaulted and relapsed cases, patients usually received the IRZE/IR or IRZES/IRZE/IRE regimens. On the other hand, for failure cases, patients usually received the second-line drugs or MDR regimens. The overall success rate in defaulted cases was 87.6%, vs. 68.4% who relapsed and 57.1% who failed (p = 0.067). For new cases, the isolations found DR-TB 3.6% and MDR-TB 0%. For previously-treated cases, the isolations found DR-TB 16.67% and MDR-TB 6.25%. The only one factor related to successful outcomes was treatment at TB clinic (adjusted OR 2.01, 95% CI 1.18-3.43). CONCLUSION: Previously-treated pulmonary TB had less success rate than new cases. Culture and susceptibility for previously-treated group were recommended before starting treatment. Treatment at TB clinic improved treatment outcomes.

Evaluation of seegene anyplex MTB/NTM real-time detection assay for diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is a common central nervous system infectious disease. Polymerase chain reaction (PCR) assay is a useful method for the rapid diagnosis of TBM. The Seegene Anyplex MTB/NTM real-time detection assay has good sensitivity and specificity for detection of tuberculosis in respiratory specimens, though, data regarding other specimens are lacking. This study aims to define the diagnostic role of Seegene Anyplex MTB/NTM real-time detection assay in TBM in adults. METHODS: This was a retrospective study of 367 adults with symptomatic community acquired meningitis between December 2013 and December 2019. Cerebrospinal fluid (CSF) had been sent for conventional diagnosis, including culture to identify Mycobacterium tuberculosis, and Seegene Anyplex MTB/NTM real-time detection assay. Other diagnostic examinations were performed as necessary. RESULTS: Of the 367 patients in the study, 37 were diagnosed with TBM (14 with definite TBM and 23 with probable TBM). Between the total TBM cases (n = 37) and non-TBM cases (n = 330), clinical sensitivity was 32.4% and specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 93.0%. Between the definite TBM cases (n = 14) and non-TBM cases (n = 330), clinical sensitivity was 50.0% and specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 97.9%. CONCLUSION: Due to lack of sensitivity, we suggest Seegeen Anyplex MTB/NTM real-time detection assay should not be used to rule out TBM but is useful for definite diagnosis.

Causative agents and resistance among hospital-acquired and ventilator-associated pneumonia patients at Srinagarind Hospital, northeastern Thailand.

Abstract. Hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) have an impact on health care costs and mortality. The aim of this study was to identify the causative agents, antibiotics prescribed, cost of treatment and drug resistance trends among HAP and VAP patients at a tertiary-care hospital in northeastern Thailand during 2008 and 2009. The incidences of HAP in 2008 and 2009 were 0.7/1,000 and 0.55/1,000 hospital days, respectively. The incidences of VAP in 2008 and 2009 were 13.6/1,000 and 12.6/1,000 ventilator days, respectively. About 70% of HAP were caused by Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae; and 70% of VAP were caused by A. baumannii, P. aeruginosa, and K. pneumoniae. The ranking in the causative agents of HAP and VAP was not different, but more antimicrobial resistant organisms were seen in 2009. More than half of the costs of nosocomial infection treatment in 2008 and 2009 were the costs for HAP and VAP, 16.8 and 17.5 million Baht, respectively. Fewer A. baumannii and P. aeruginosa isolates were sensitive to carbapenems. Only one-fifth of A. baumannii isolates were sensitive to cefoperazone/sulbactam. The only two antimicrobial agents with consistently good activity against A. baumannii were tigecycline (approximately 85%) and colistin (approximately 99%). Fifty-seven point six percent of P. aeruginosa isolates were sensitive to ceftazidime, 72.4% were sensitive to piperacillin/tazobactam, 95.9% were sensitive to netilmycin and 99.2% were sensitive to colistin. Forty-seven percent of K. pneumoniae isolates were extended spectrum beta-lactamase and sensitive to carbapenems. Methicillin-resistant S. aureus was the cause of 6-7% of HAP/VAP cases in our study.

Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand.

The aims of this study were to develop multiplex PCR for simultaneous detection of five superantigenic toxin genes (sea, seb, sec, sed and tst-1) in Staphylococcus aureus isolated from 149 clinical samples and nasal swabs from 201 healthy subjects in Thailand, and to compare prevalence and expression of those genes between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The sensitivity of multiplex PCR was 10(3) CFU/ml (60 CFU/PCR reaction) for DNA templates extracted by both boiling and extraction methods. S. aureus strains from patients (65%) harbored more superantigenic toxin genes than healthy subjects (54%). MRSA (80%) isolated from patients harbored more superantigenic toxin genes than MSSA (52%). Sea was the most frequently found gene in S. aureus strains from patients and carriers. MRSAisolates harbored sea and produced SEA more frequently than MSSA isolates (p <0.05) and MRSA isolates (59%) from blood samples consisted of a higher number of superantigenic toxin producers than MSSA (9%) (p < 0.05). More S. aureus strains isolated from patients with severe septicemia contained superantigenic toxin genes (94%) and produced toxins (82%) than those from non-severe patients (64% and 57%, respectively). The multiplex PCR method described here offers a reliable tool for simultaneous detection of various staphylococcal toxin genes.

Risk factors and clinical outcomes of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli septicemia at Srinagarind University Hospital, Thailand.

Escherichia coli producing extended spectrum beta-lactamase (ESBL) has emerged as a worldwide, public health problem. The aims of this study were to determine the incidence of ESBL-producing E. coli septicemia and evaluate the factors associated with the infection and the clinical outcomes. We reviewed 145 cases of E. coli septicemia among adult patients admitted to Srinagarind University Hospital in northeastern Thailand between 2005 and 2006. The incidence of ESBL-producing E. coli septicemia was 9.9 cases per 10,000 hospital admissions. The factors significantly associated with ESBL-producing E. coli septicemia were: 1) hospital acquisition [odds ratio (OR) 6.46; 95% confidence interval (CI) 2.01-20.79], 2) previous use of a fluoroquinolone, (OR 19.14; 95% CI 5.82-62.96), and 3) use of a central venous catheter (OR, 8.59; 95% CI, 1.11-66.27). Seventy-two hours after receiving empiric treatment, a significantly greater proportion of patients with ESBL-producing E. coli septicemia had a worse clinical outcome than those with non-ESBL producing E. coli septicemia (p = 0.01). The overall mortality rate was significantly higher among the ESBL-producing E. coli septicemia group than the non-ESBL producing E. coli septicemia group (29% vs 11.5%, respectively, p = 0.02). A high APACHE II score, ESBL-producing E. coli septicemia, primary septicemia, and having a non-urinary tract infecting as a source of septicemia were significantly independent factors related to mortality among patients with E. coli septicemia. ESBL-producing E. coli septicemia is an important cause of nosocomial infection and is related to higher mortality risk, especially among those with primary septicemia and secondary septicemia due to a non-urinary tract infection.

Molecular analysis and antimicrobial resistance of Vibrio cholerae O1 in northeastern Thailand.

A total of 84 clinical Vibrio cholerae O1 isolates were collected from Khon Kaen (KK), Udon Thani (UT), Loei (LI), and Nong Khai (NK), northeastern Thailand during cholera outbreaks in 2007 and 2008. The majority of V. cholerae O1 strains carried nearly all the virulence-associated genes (ctxA, zot, and ace) except for four isolates and one isolate from UT and NK, respectively, which carried only tcpA, ompU, hlyA and toxR. None of the V. cholerae O1 strains carried sto. Pulsed field gel-electrophoresis (PFGE) profiling of 16 randomly chosen isolates showed the same PFGE pattern, except for one NK isolate, which was sensitive to all seven antibiotics used in the antimicrobial susceptibility tests. The tests revealed that multi-drug resistance to tetracycline and co-trimoxazole were present in KK strains (92%), followed by LI (75%) and UT (52%) strains. All strains were sensitive to norfloxacin but intermediate resistance to ciprofloxacin was found in a single strain from KK and LI. Differences in antimicrobial resistance among V. cholerae strains with the same PFGE pattern reflect differences in the antimicrobial agents used in each area of northeastern Thailand.

High rate of reinfection and possible transmission of Mycobacterium avium complex in Northeast Thailand.

The Mycobacterium avium complex (MAC) includes two main species of non-tuberculous mycobacteria (NTM), M. avium and Mycobacterium intracellulare. These can cause serious disease, especially in immunocompromised patients. Little information is available concerning genetic diversity of NTM. We used multilocus sequence typing (MLST) based on a highly discriminative gene set to analyze MAC serially isolated from patients to determine the rate of MAC reinfection. Genomic DNA was sequenced from 49 MAC isolates (15 cases comprised of 11 true infections and 4 instances of colonization). More than half of the MAC isolates tested were found to be multidrug resistant. The discriminatory power was assessed of 24 house-keeping genes (fusA, atpD, pheT, glnA, topA, secA, argH, glpK, murC, cya, pta, rrl, rrs, hsp65, rpoB, 16S-23S rRNA ITS, recF, lipT, pepB, gnd, aspB, groEL, sodA and est) previously used for genotyping of MAC and other NTM. Seven genes (fusA, secA, rpoB, hsp65, 16S rRNA, 23S rRNA, 16S-23S rRNA ITS) had a discriminatory power index higher than 0.9 and were included in the optimized set that we used. This set was significantly better for genotyping and diagnosis of MAC than previously used 4-gene, 5-gene and 9-gene sets. MLST using our 7-gene set indicated that the rate of reinfection was 54.55% (6/11 cases). Persistent infections (n = 5 cases, 45.45%) were found. A changing of clone in the same patient was found in 1/4 (25%) of the colonization cases. Two small clusters of possible MAC transmission between humans were found. Our study demonstrated that the high frequency of apparent treatment failure of MAC might be artefactual, as a consequence of a high rate of MAC reinfection in Thai population. Our useful highly discriminative gene set for MAC species and clonal strain analysis could be further applied for the diagnosis and patient management.

Drug susceptibility patterns of Mycobacterium tuberculosis and clinical outcomes of drug-resistant tuberculosis at Srinagarind Hospital, a tertiary care center in northeastern Thailand.

Drug-resistant tuberculosis is a major public health problem. The aim of this study was to assess the local susceptibility patterns of Mycobacterium tuberculosis and clinical outcomes of drug resistant tuberculosis (DR-TB) at Srinagarind Hospital, a tertiary care center in northeastern Thailand. Between January 2004 and December 2008, 1,052 patients had culture-proven M. tuberculosis infections at Srinagarind Hospital. M. tuberculosis was resistant to isoniazid (2.3%), rifampicin (2.8%), ethambutol (3.8%), streptomycin (2.1%), kanamycin (0.7%) and ofloxacin (1.9%). The occurrences of multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) were 1.2% and 0.38%, respectively. Of the 65 DR-TB patients, complete medical records were found for 55. The male to female ratio was 2.2:1. The mean age was 50 years. Thirteen patients had MDR-TB. The duration of symptoms in the MDR-TB group was longer than the non-MDR-TB group, 11.6 months vs 2.6 months, respectively. Half of MDR-TB and one-third of non-MDR-TB patients had a previous history of being treated for tuberculosis. Nearly 20% of cases were HIV positive. Mono-drug resistance was initially treated with standard first-line drugs (CAT 1). The clinical course was more likely to be worse during the maintenance phase if there was resistance to rifampicin. Whenever there was resistance to two, three or four drugs, the antituberculosis drugs were prescribed based on susceptibility patterns. Only 30% of patients with MDR-TB and XDR-TB responded to treatment. Culture and sensitivity testing for M. tuberculosis cases is recommended in patients at high risk for DR-TB, such as patients previously treated for tuberculosis and those HIV positive.

The first vancomycin-intermediate Staphylococcus aureus strains isolated from patients in Thailand.

We screened 533 and 361 methicillin (meticillin)-resistant Staphylococcus aureus strains isolated in a university hospital in 2002 and 2003 and in 2006 and 2007, respectively, and identified 4 (0.8%) of the strains in the first group and 8 (2.2%) of the strains in second group as heterogeneous vancomycin-resistant S. aureus (heterogeneous VISA) strains and 3 (0.8%) of the strains in the second group as VISA strains. This is the first report of VISA strains isolated from patients in Thailand.

Post operative penicillin-non-susceptible Streptococcus pneumoniae meningitis and septic shock in a child.

The authors describe a one-year-old girl with a fronto-ethmoidal encephalomeningocele who developed wound infection, purulent meningitis and septic shock 5 hours after operation. The patient was treated with intravenous ceftazidime and vancomycin empirically. The cerebrospinal fluid (CSF) and eye discharge grew Streptococcus pneumoniae (S. pneumoniae). The minimal inhibitory concentration (MIC) by E-test of penicillin and cefotaxime were 1.0 and 0.38 ug/ml respectively so the antibiotics were switched to cefotaxime 300 mg/kg/day. She recovered completely after appropriate treatment. Penicillin-non-susceptible S. pneumoniae should be considered as one of the causes of post-operative serious infection of the face and neck in the era of increasing prevalence of penicillin-resistant S. pneumoniae.

Analysis of drug-susceptibility patterns and gene sequences associated with clarithromycin and amikacin resistance in serial Mycobacterium abscessus isolates from clinical specimens from Northeast Thailand.

Mycobacterium abscessus is an important infectious agent highly associated with drug resistance and treatment failure. We investigated the drug resistance situation of M. abscessus in Northeast Thailand and the possible genetic basis for this. Sixty-eight M. abscessus clinical isolates were obtained from 26 patients at Srinagarind Hospital during 2012-2016. Drug susceptibility tests and sequencing of erm(41), rrl and rrs genes were performed. Mycobacterium abscessus was resistant to 11/15 antibiotics (nearly 100% resistance in each case). Partial susceptibility to four antibiotics was found (amikacin, tigecycline, clarithromycin and linezolid). Non-massiliense subspecies were significantly associated with clarithromycin resistance (p<0.0001) whereas massiliense subspecies were associated with tigecycline resistance (p = 0.028). Inducible clarithromycin resistance was seen in 22/68 (32.35%) isolates: 21 of these isolates (95.45%) belonged to non-massiliense subspecies and resistance was explicable by the T28C mutation in erm(41). Inducible clarithromycin resistance was found in one isolate of the massiliense subspecies. Acquired clarithromycin resistance explicable by the A2271G/C mutation of rrl was seen in only 7/16 (43.75%) of strains. Inducible and acquired resistance mechanisms can be interchangeable during the course of infection. Rrs mutations were not associated with amikacin resistance in our study. Antibiotic resistance in subspecies of M. abscessus was reported from Northeast Thailand. Known resistance-associated mutations cannot explain all of the resistance patterns observed.

Molecular Characterization of Carbapenemase-Nonproducing Clinical Isolates of Escherichia coli (from a Thai University Hospital) with Reduced Carbapenem Susceptibility.

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 ß-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 µg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 µg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 µg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 µg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.

Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes.

Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250.IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future.