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.

Detection of outer membrane porin protein, an imipenem influx channel, in Pseudomonas aeruginosa clinical isolates.

Decreased permeability to imipenem is the most frequent mechanism of imipenem resistance in Pseudomonas aeruginosa. We have determined the presence of OprD porin protein, an imipenem influx channel, in 70 carbapenem-resistant P. aeruginosa clinical isolates by Western blot analysis using rabbit anti-OprD polyclonal antibody. Ninety-eight percent (54 of 55 isolates) of imipenem-and meropenem-resistant P. aeruginosa clinical isolates were negative for OprD porin production. A small group of isolates resistant to imipenem but susceptible to meropenem (2 isolates) produced OprD protein but at a level 3-5 times lower than the wild type P. aeruginosa ATCC27853 strains. This study indicates that the loss of OprD porin protein was the main mechanism for imipenem resistance in P. aeruginosa clinical isolates. Determination of the status of OprD level in P. aeruginosa may help in the better selection of appropriate carbapenem antibiotics.

Antibacterial activity of carbapenem-based combinations againts multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Multidrug-resistant (MDR) Acinetobacter baumannii are increasingly encountered and frequently susceptible only to colistin with their MIC values close to resistance breakpoint. Antibacterial activity of two carbapenem-based combinations were explored in order to overcome the bacterial resistance. MATERIAL AND METHOD: Thirty clinical isolates of MDRA. baumannii were employed to assess in vitro antibacterial activity of two carbapenem-based regimens. Imipenem combined with colistin and meropenem combined with colistin and sulbactam were the first and second regimens, respectively. All isolates were resistant to imipenem (MIC range: 8-128 microg/ml) and meropenem (MIC range: 64-256 microg/ml) but still susceptible to colistin (MIC range: 0.5-2 microg/ml). The MIC range of sulbactam was 4-64 microg/ml. None of the isolates produced metallo-beta-lactamase. RESULTS: Synergistic antibacterial effect of imipenem combined with colistin was observed against 100 percent of A. baumannii isolates by the checkerboard microdilution panel method. In a subsequent time kill study, the most active concentration of this regimen was the combination of imipenem at the fixed concentration of 32 microg/ml and colistin at the 1/4 of the MIC values of each isolate that exerted significantly higher bactericidal activity than imipenem at 32 microg/ml alone and colistin alone at the 1/4 of the MIC values. The scanning electron micrographs demonstrated major cell morphological change and cell wall destruction after 2-hour exposure to this combination. The triple combinations of meropenem, sulbactam and colistin showed synergy against 96.7 percent of MDR A. baumannii while double combinations of either meropenem and sulbactam, meropenem and colistin, and sulbactam and colistin showed synergy effects of 70%, 73.3% and 53.3%, respectively The time kill study using ten isolates also showed better killing effect by the triple combination than any of the double combinations. CONCLUSION: Antibacterial activity against MDR A. baumannii of imipenem plus colistin was superior over any single of the two agents. The addition of sulbactam to meropenem and colistin may further improve their antibacterial activity. The double or triple carbapenem-based combinations offer promising alternatives in the treatment of infections due to MDR A. baumannii.

Prevalence of extended-spectrum beta-lactamase and class 1 integron integrase gene intl1 in Escherichia coli from Thai patients and healthy adults.

Among 120 Escherichia coli isolates from Thai patients, 37 and 9 isolates were extended-spectrum beta-lactamase (ESBL) and suspected ESBL producers respectively while 5 E. coli isolates from 120 Thai healthy adults were suspected ESBL producers. Integrase (intl1) gene was detected in 99% of the clinical and 87% of the non-clinical isolates. Among 37 ESBL producers, percent recovery of bla(TEM), bla(CTX-M), bla(SHV) and bla(VEB) was 78%, 78%, 8% and 8%, respectively. Twenty-five isolates of ESBL producers carried both bla(TEM) and bla(CTX-M), 2 isolates carried 3 genes (bla(TEM), blac(CTX-M), and bla(SHV)) and 3 showed no detectable bla gene. Among the 14 suspected ESBL producers, intl1 and bla(TEM) were detected in 13 isolates. ESBL producers from clinical samples were resistant to most of the tested antimicrobial agents compared to non-ESBL producers and isolates from healthy adults with about half of the latter susceptible to all tested antimicrobial agents. Only one clinical isolate was resistant to imipenem. Susceptibility to trimethoprim/sulfamethoxazole among the clinical isolates in ESBL producer group (27%) and non-producer group (33%) were comparable, whereas the percent susceptibility of the non-clinical isolates was about twice that of the clinical isolates.

Bioequivalence, antibacterial activity and therapeutic outcome of a generic meropenem (Mapenem).

BACKGROUND: The authors aimed to compare the bioequivalence and antibacterial activity of a generic meropenem with the original meropenem and studied its preliminary therapeutic outcome. MATERIAL AND METHOD: A randomized, open-label, crossover study was employed to assess the bioequivalence and antibacterial activity. Twenty-six healthy males were recruited at Siriraj Hospital, Thailand and randomized to firstly receive either a single intravenous 30-minute infusion of a generic (Mapenem) or original meropenem (Meronem) and vice versa for the second period. The washout period was one week. Ten milliliters of blood samples were collected before meropenem infusion and at 0, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, 360, 470 and 480 minutes after the beginning of the drug infusion. Blood samples were coded and separated into plasma and serum samples. Plasma samples were used to determine drug concentrations by HPLC-UV detector and the data were analyzed for Cmax, AUC0-t and AUC0-inf. Serum samples were assayed in triplicate for measuring generic and original meropenems' inhibitory activities of a meropenem-susceptible E. coli ATCC 25922 in the same agar plate. An open-label design was used to preliminarily study of the therapeutic outcome and adverse effects of the generic meropenem in 30 patients. RESULTS: All enrolled twenty-six volunteers completed the whole study. The statistical analysis of 90% confidence interval of Cmax, A UC0-t, and AUC0-inf of the generic and original meropenems were 87.7 to 101.7%, 96.3 to 102.4% and 96.3 to 102.3%, respectively. The results were within the standard range of bioequivalence acceptance criteria (80-125%) and the powers of the test were greater than 80%. Using E. coli ATCC 25922 in the blind assay of serum inhibition activity, the inhibitory zone sizes (mm) of the generic compared to original meropenems were not statistically different with respect to every time points of blood collections (p < 0.05). Correlation of mean values of serum meropenem levels and the widths of inhibitory zone sizes of the same samples collected at the same intervals showed good linear relationship with r = 0.891; R2 = 0.794 (p < 0.01) for the generic meropenem and r = 0.885; R2 = 0.784 (p < 0.01) for the original meropenem. The therapeutic result with the generic meropenem for various indications was successful or improved in 24 cases from 30 cases (80%) and the bacterial cure rate was 23 in 30 clinical isolates (76.7%). Adverse reactions probably related to the study medication were rash and elevated liver enzymes in 1 and 3 patients, respectively, and all resolved spontaneously. CONCLUSION: In the present study, the generic meropenem exhibited indifferent bioequivalence and antibacterial activity compared to the original meropenem. There was also a good correlation between serum levels and inhibitory zone sizes produced by the same serum samples in every periods of blood collection. Clinical efficacy of the generic meropenem was shown to be satisfactory without notable severe adverse reaction.

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.

Discriminatory powers of molecular typing techniques for methicillin-resistant Staphylococcus aureus in a university hospital, Thailand.

Discriminatory powers of various molecular techniques were evaluated for typing of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Siriraj Hospital, Bangkok, Thailand. Thirty MRSA isolates were randomly selected in this study. They were characterized by pulsed-field gel electrophoresis, Clal-mecA and Clal-Tn554 polymorphisms, ribotyping, and PCR-based methods including SCCmec typing, spa and coa gene polymorphism, and repeat units in hypervariable region downstream of mecA. Individual molecular typing technique distinguished those MRSA isolates into 2 to 5 types. Eleven genetic backgrounds of MRSA isolates were elucidated by combination of typing methods with trimethoprim/sulfamethoxazole (TMP/SXT) susceptibility. Combination of all typing methods including TMP/SXT susceptibility yielded a discriminatory index of 0.94. Combination of PCR-based methods and TMP/SXT susceptibility, with the discriminatory index of 0.89, is a practical typing approach suitable for rapid epidemiological investigation of MRSA isolates in a hospital setting.

Endemic Serratia marcescens in a neonatal intensive care unit.

OBJECTIVE: To study the endemicity of Serratia marcescens in a neonatal intensive care unit (N.I.C.U). MATERIAL AND METHOD: During the first 4 months of 2001, neonates in the N.I.C.U. in a teaching hospital were screened for S. marcescens by serial throat swabs and collections of other appropriate clinical specimens. Environmental cultures were also done in the same period. Isolated S. marcescens were tested for antimicrobial susceptibility and for genotyping by pulsed field gel electrophoresis. RESULTS: During the period, 104 neonates were studied. S. marcescens were isolated in 34.6% of the cases. Environmental cultures were positive for S. marcescens in 1.4%. There were 10 patterns of antibiogram of the 190 strains isolated. All strains belonged to pulsotype A. CONCLUSION: The study confirmed that S. marcescens was endemic in the N.I.C.U. and belonged to one genotype.

Detection of Clostridium difficile toxin A and B genes from stool samples of Thai diarrheal patients by polymerase chain reaction technique.

The prevalence of Clostridium difficile isolated from stools of Thai adult patients with suspected antibiotic-associated diarrhea (AAD) was 18.64 per cent. The recovery rate of toxin genes (tcdA and tcdB) by polymerase chain reaction (PCR) from stool samples yielded almost the same compared to the recovery rate of the toxin detection by enzyme immunoassay (EIA), which were 44.9 per cent and 46.7 per cent, respectively. Correlation of toxin gene detection by PCR and toxin detection by EIA was 90.6 per cent. All but one stool sample, the tcdA gene was detected together with the tcdB gene. Both genes were always detected together from tox gene-positive strains. Although, there were some discrepancy results for certain samples, the direct PCR-based-detection of C. difficile tox genes in stool samples seems to be the appropriate method for the diagnosis of C. difficile diarrhea. The PCR assay should be a recommended technique to be used routinely in laboratories. Further optimization of the technique to increase the sensitivity of the PCR assays is still needed. However, a quantitative isolation of the organism from stools of suspected antibiotic-associated diarrhea (AAD) or antibiotic-associated colitis (AAC) patients may give some evidence for clinicians in hospitals who cannot perform PCR-based or EIA-based techniques, since 48.6 per cent of the isolates were demonstrated as toxigenic strains.

Occurrence of extended-spectrum beta-lactamase in clinical isolates of Klebsiella pneumoniae in a University Hospital, Thailand.

The occurrence and antimicrobial susceptibility of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients attending Siriraj Hospital in Bangkok from August 2000 to January 2001 were determined. ESBL-producing isolates were screened with four different methods: disk diffusion according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines, Etest ESBL (CT/CTL and TZ/TZL), Oxoid combination discs and MIC Etest strip. Antimicrobial susceptibility testing were determined by a microdilution automatic method (VITEX system, bioMerieux). Of 22,178 clinical specimens, 400 (1.8%) K. pneumoniae were isolated Of 26% (104/400) of these isolates were suspected to be ESBL-producing. Rates of detection of ESBL-producing K. pneumoniae were 18.67%, 30% and 23.78% for blood, sputum and urine samples, respectively. Susceptibility testing has revealed that all 70 tested isolates including 53 isolates from blood and sputum and 17 isolates from urine samples were susceptible to imipenem (MIC< or =4 mg/L). None of the tested isolates were susceptible to cephalosporins, cephamycin and aztreonam. Rate of susceptibility to ciprofloxacin, levofloxacin, gentamicin and tobramycin were 60%, 64%, 28% and 9%, respectively, for isolates from blood and sputum; 71%, 71%, 18% and 6% for urinary isolates. The present findings revealed a high occurrence rate of multi-drug resistance ESBL-producing K. pneumoniae in patients attending the university hospital. Imipenem was highly active against ESBL-producing K. pneumoniae.