Genetic diversity and antimicrobial resistance pattern of Salmonella enterica serovar Enteritidis clinical isolates in Thailand.

OBJECTIVE: To trace the history of antimicrobial resistance in Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) circulating in Thailand, we characterised clinical isolates obtained during 2004-2007. METHODS: Antimicrobial resistance profiles, multi-locus variable number tandem repeat analysis (MLVA) types and 3 representative virulence determinants (spvA, sodCI and sopE) were established from SE isolates (n = 192) collected from stool and blood of patients throughout Thailand during the period 2004-2007. RESULTS: Resistance was found in SE against 10 out of 11 antimicrobials studied. The highest resistance ratios were observed for nalidixic acid (83.2%), ciprofloxacin (51.1%) and ampicillin (50.5%), and 25.5% were multidrug resistant. Based on five polymorphic tandem repeat loci analysis, MLVA identified 20 distinct types with three closely related predominant types. A significant increase of AMP resistance from 2004 to 2006 was strongly correlated with that of a MLVA type, 5-5-11-7-3. CONCLUSION: The usage of antimicrobials in human medicine or farm settings might act as selective pressures and cause the spread of resistant strains. Hence, a strict policy on antimicrobial usage needs to be implemented to achieve the control of resistant SE in Thailand.

EMERGENCY ROOM: AN UNRECOGNIZED SOURCE OF EXTENDED-SPECTRUM ß-LACTAMASE PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae are the leading causes of hospital-associated infections, but community-acquired cases are increasingly being reported. This study determined the prevalence of ESBL-producing E. coli and K. pneumoniae carriers, their bla genes and risk factors of 452 patients admitted to the emergency room (ER) of Ramathibodi Hospital, Mahidol University, Bangkok, Thailand between April and August 2011. Prevalence of ESBL-producing E. coli and K. pneumoniae from rectal swabs was 16.5% and 1.0%, respectively. Factors associated with ESBL-producing carriers were a previous history of hospital admission (p = 0.001) and visits to health care facilities (p = 0.002) during the previous 3 months. All ESBL-producing isolates were susceptible to imipenem, meropenem and ertapenem. The majority (78%) of ESBL-producing E. coli isolates showed very high resistance to cefotaxime and ceftriaxone (MIC50 and MIC90 > 256 µg/ml). ESBL-producing E. coli harbored chromosomal blaTEM (96%), blaCTX-M (70%) and blaSHV (1%), while 8%, 73% and 3%, respectively, were located on plasmid. The prevalence of these genes in ESBL-producing K. pneumoniae was 75%, 50% and 25%, respectively on chromosome; and 100%, 25% and 50%, respectively on plasmid. Nucleotide sequence analysis revealed that these bla genes were of the type blaTEM-1' blaTEM-116' blaCTX-M-15' blaCTX-M-161' blaSHV-12, blaSHV-28 and blaSHV-148. Detailed epidemiologic and clinical characteristics of ER patients with history of prior hospital visits should be carried out to identify the ESBL-producing organisms they have acquired in order to institute appropriate treatment for these patients as well as control measures against further dissemination of these life-threatening organisms.

Detection and genetic characterization of norovirus in environmental water samples in Thailand.

The aim of this study was to detect and characterize noroviruses (NoVs) in environmental water samples. One hundred and fourteen water samples were collected from a river and irrigation canals in central Thailand during 2006-2007. NoVs were detected by RT-nested PCR in 13% of the samples. The river samples (22%) contained NoVs at a higher frequency than the irrigation canal samples (4%). Among the 15 NoV-positive samples, 9 harbored genogroup (G) I, 2 samples with GII, and 4 samples with mixed GI and GII. DNA sequencing of PCR amplicons and phylogenetic analysis of partial capsid gene revealed that 5 samples were of genotype GI-2, 1 sample was GI-6, and 1 sample was a mix of GI-2 and GII-unclassified genotypes. NoVs in water samples quantified using quantitative RT-PCR were in the range of 4.91 x 10(2) -1.26 x 10(3) copies/ml for NoV GI and 3.51 x 10(3) copies/ml for NoV GII. This is the first study demonstrating the presence of NoV variants in water samples collected from a river and the adjacent canals of Thailand.

Dissemination of class I integron in Acinetobacter baumliannii isolated from ventilator-associated pneumonia patients and their environment.

Multidrug resistant Acinetobacter baumannii has become the most common cause of health care-associated infections at Maharaj Nakhon Si Thammarat Hospital, Thailand. The objective of the study was to detect integrons using PCR-based method from 96 A. baumannii isolates from ventilator-associated pneumonia (VAP) patients and their environment. Antibiotic susceptibility was determined using a disk diffusion technique. Forty-six isolates exhibited integrase genes, with only class I and class II integron detected in 43 and 3 A. baumannii isolates, respectively. Twenty-seven of 52 clinical and 19 of 44 environmental isolates were integron-positive. Detection rate of integron-positive A. baumannii isolated from VAP patients increased from 25% to 83% over the 4 month study period. The majority (91%) of integron-positive A. baumannii showed resistance to 6 or more of 11 antibiotics tested and 72% of class I integron-positive isolates were imipenem-resistant. Thus, class I integron-positive A. baumannii had spread among the VAP patients and into hospital environment, the latter acting as reservoirs of potential pathogens possessing drug resistance genes.

Clonal spread of carbapenem resistant Acinetobacter baumannii in the patients and their environment at BMA Medical College and Vajira Hospital.

OBJECTIVE: To determine the clonal spread of carbapenem-resistant Acinetobacter baumannii (CRAB) in the patients and their environment at BMA Medical College and Vajira Hospital. MATERIAL AND METHOD: A prospective study on CRAB isolated from the clinical specimens of 30 patients and 300 from their environmental samples were carried out from September 1-15, 2008. The CRAB isolates were genotyped using PCR-based typing method. RESULTS: Twenty-six (86.7%) and 20 (66.7%) cases of 30 patients had their environment contaminated with A. baumannii and CRAB, respectively Environmental contamination rates of A. baumannii and CRAB were 18.0% (54/300) and 13.0% (39/300), respectively. The most contaminated sites with CRAB were bedside cupboards (26.7%), followed by bedrails and bed sheets (20%), BP cuffs (16.7%), over bed tables and nurse station counters (13.3% each) and push carts (10%). Four molecular types were classified among 65 CRAB isolates. Molecular type 1 was the most prevalent (90.7%) and found in all kinds of environmental samples except patient record folder and computer keyboard/mouse. About 37% of the patients had at least one of their environmental samples contaminated with CRAB clonally related with their own types. CONCLUSION: Clonal spread of CRAB was demonstrated to emphasize the important of hand hygiene, contact precaution and patient's environmental decontamination in controlling the spread of CRAB in the hospital.

Epidemiological characteristics of Acinetobacter baumannii infections at Phramongkutklao Hospital.

OBJECTIVE: To describe epidemiological characteristics of Acinetobacter baumannii infections and identify molecular patterns of A. baumannii isolated from the patients admitted in Phramongkutklao Hospital. MATERIAL AND METHOD: A retrospective study on previously isolated A. baumannii from the clinical specimens submitted to the microbiology laboratory of Phramongkutklao Hospital from January to March 2008 were carried out together with molecular typing using PCR-based method. Clinical data were obtained from IC surveillance and patients' records. RESULTS: 114 A. baumannii were isolated from 80 patients. A. baumannii was a cause of healthcare-associated infection (90%, 72 of 80 cases), colonization (7.5%), and community-acquired infection (2.5%) with mortality rate of 50%. Majority of the patients from which A. baumannii were isolated were male (58.8%), age over 60 years (56.3%), diagnosed with lower respiratory diseases (26.3%), had A. baumannii ventilator-associated pneumonia (66.7%), and admitted in medical department (57.5%) with median length of hospital stay 35 days. PDR- and MDR- A. baumannii were accounted for 67.5% and 21.1%, respectively. All isolates showed sensitive to tigecycline and colistin. Using PCR-based typing was able to distinguish 6 molecular types among 114 A. baumannii isolates. Molecular type 2 was the most common type (47.4%) and widely spread in 14 wards. Spread of clonally related isolates was found in 14 cases admitted in 8 medical wards and ICUs. CONCLUSION: Multiple clones of PDR- and MDR- A. baumannii were widely spread in the hospital. Clonally related A. baumannii infected 14 cases in 8 wards.

Bed rails and endotracheal tube connectors as possible sources for spreading Acinetobacter baumannii in ventilator-associated pneumonia patients.

This study aimed to determine molecular patterns of Acinetobacter baumannii using a PCR-based technique with REP-1, REP-2 and M13 primers to distinguish the patients' strains and the environmental strains (condensate, endotracheal tube connector, bed rail and nurses hands). There were 67 cases of ventilator-associated pneumonia (VAP) among 600 patients using mechanical ventilators in 10 wards from March to July 2006. The incidence of VAP was 11.2% or 8.9/1,000 ventilator days with a 54.5% fatality rate. Among 19 of 22 A. baumannii VAP patients, 68.4% (13/19) had their environmental samples contaminated with A. baumannii and the most common contaminated sites were bed rails and endotracheal tube connectors (36.8% each). Multidrug resistant (MDR) A. baumannii were involved in 77.3% of A. baumannii VAP. Molecular typing of 96 A. baumannii isolates was able to differentiate A. baumannii isolates into 7 types. Type 2 was the most common and found in 77.3% (17/22) of A. baumannii VAP patients admitted in 6 of 7 wards. Identical fingerprints were found in clinical isolates and their bed rails, endotracheal tube connectors and condensates of 5 patients. The results demonstrate that multiple clones of MDR A. baumannii were widely spread in the hospital. Bed rails and contaminated endotracheal tube connectors could be potential sources of A. baumannii spread.

The differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection.

BACKGROUND: Dengue haemorrhagic fever is an important public health problem and mainly occurs in children less than 15 years of age. Recently, the incidence of the disease have increased in adults but data on clinical and laboratory presentations of those affected are limited. OBJECTIVES: To assess and compare clinical manifestations and laboratory findings of dengue virus infected children and adults in Thailand. STUDY DESIGN: A 1-year study was conducted from September 2003 to August 2004 for dengue virus infected patients admitted to Phetchabun Provincial Hospital, Thailand. Physical signs, symptoms, and laboratory features were recorded. All dengue patients were confirmed using immunochromatographic test on convalescent sera. RESULTS: Based on serology-confirmed dengue virus infection, there was 286 dengue patients including 15 (5.3%) dengue fever and 271 (94.7%) dengue haemorrhagic fever (DHF). Among DHF cases, clinical classifications were DHF I, 40.9%; DHF II, 43%; and DHF III or dengue shock syndrome (DSS), 10.8%. Of all dengue patients, 231 cases (80.8%) were children aged less than 15 years and 55 cases (19.2%) were adults. The highest proportion of child cases was DHF I (42.9%), whereas that of adults was DHF II (51%). Some clinical manifestations were more common in adult patients, such as petechiae, melena, headache, retro-orbital pain, joint pain, myalgia, nausea and vomiting (p-value<0.05). Signs found commonly in children were epistaxis, oliguria, and liver enlargement (p-value<0.05). Haemoconcentration, thrombocytopenia, increased alanine aminotransferase, and longer prothrombin time were found to be significantly higher in adults than in children (p-value<0.05). CONCLUSIONS: Some clinical presentations of dengue disease and laboratory findings in adults are different from those in children. Therefore, adults as well as pediatric cases of DHF need appropriate and prompt case management to reduce the mortality rate of DHF.

Risk factors for multi-drug resistant Acinetobacter baumannii nosocomial infection.

OBJECTIVE: To assess factors associated with multi-drug resistant Acinetobacter baumannii (MDR-AB) nosocomial infection. MATERIAL AND METHOD: This hospital-based case-control study was conducted in patients admitted to Siriraj Hospital, Bangkok, Thailand between January 1, 2005 and December 31, 2005. The study population consisted of 155 cases with MDR-AB nosocomial infection and 310 controls without nosocomial infection. The cases were matched with controls by age and ward of admission with a ratio of 1:2. RESULTS: The average age of the present study population was 63.5 +/- 18.7 years among cases and 62.9 +/- 18.2 years among controls. The mean of length of stay in hospital among cases was 4.9 +/- 1.4 weeks and controls 1.8 +/- 1.0 weeks. The most common site of MDR-AB nosocomial infection was lower respiratory tract (74.8%). The antimicrobial susceptibility of MDR-AB was 3.9% to cetriaxone and 42.1% to cefoperazone/sulbactam. Multiple logistic regression analysis showed the following associated factors with MDR-AB nosocomial infection: duration of admission prior to MDR-AB nosocomial infection > 1 week (OR = 2.06; 95% CI 1.09-3.89), indwelling urinary catheter > 1 week (OR = 8.24; 95% CI 3.81-17.82), mechanical ventilation > 1 week (OR = 5.73; 95% CI 2.96-11.10), central venous line > 1 week (OR = 3.29; 95% CI 1.48-7.31), nasogastric intubation > 1 week (OR = 6.22; 95% CI 3.24-11.93), prior administration of 3rd-4th generation cephalosporins (OR = 1.80; 95% CI 1.04-3.13), metrodazole (OR = 2.59; 95% CI 1.21-5.56), and piperacillin-tazobactam (OR = 4.68; 95% CI 1.93-11.32). CONCLUSION: A case-control study in medical and surgical patients in Siriraj Hospital in 2005 revealed risk factors for AB nosocomial infection. Prolonged admission of more than 2 weeks, use of devices, and prior treatment with certain antimicrobials were found to be significant risk factors for the infection. To reduce the infection, strict infection control measures must be applied to the patients with these risk factors. Education to medical personnel and enforcement of infection control practices are all needed to reduce antimicrobial resistant bacterial nosocomial infection.

A novel bacteriocin from Enterococcus faecalis 478 exhibits a potent activity against vancomycin-resistant enterococci.

The emergence of multidrug-resistant enterococci (MDRE) and particularly vancomycin-resistant enterococci (VRE) is considered a serious health problem worldwide, causing the need for new antimicrobials. The aim of this study was to discover and characterize bacteriocin against clinical isolates of MDRE and VRE. Over 10,000 bacterial isolates from water, environment and clinical samples were screened. E. faecalis strain 478 isolated from human feces produced the highest antibacterial activity against several MDRE and VRE strains. The optimum condition for bacteriocin production was cultivation in MRS broth at 37°C, pH 5-6 for 16 hours. The bacteriocin-like substance produced from E. faecalis strain EF478 was stable at 60°C for at least 1 hour and retained its antimicrobial activity after storage at -20°C for 1 year, at 4°C for 6 months, and at 25°C for 2 months. A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) analysis showed that the amino acid sequences of the bacteriocin-like substance was similar to serine protease of E. faecalis, gi|488296663 (NCBI database), which has never been reported as a bacteriocin. This study reported a novel bacteriocin with high antibacterial activity against VRE and MDRE.

New facet of non-O1/non-O139 Vibrio cholerae hemolysin A: a competitive factor in the ecological niche.

Different serogroups of Vibrio cholerae may inhabit the same ecological niche. However, serogroup O1/O139 strains are rarely isolated from their ecological sources. Quite plausibly, the non-O1/non-O139 vibrios and other bacterial species suppress growth of O1/O139 strains that share the same niche. Our bacterial inhibition assay data indicated that certain non-O1/non-O139 strains used a contact-dependent type VI secretion system (T6SS) to suppress growth of the O1 El Tor, N16961 pandemic strain. Comparative proteomics of the O1 and the suppressive non-O1/non-O139 strains co-cultured in a simulated natural aquatic microcosm showed that SecB and HlyD were upregulated in the latter. The HlyD-related effective factor was subsequently found to be hemolysin A (HlyA). However, not all hlyA-positive non-O1/non-O139 strains mediated growth suppression of the N16961 V. cholerae; only strains harboring intact cluster I HlyA could exert this activity. The key feature of the HlyA is located in the ricin-like lectin domain (ß-trefoil) that plays an important role in target cell binding. In conclusion, the results of this study indicated that non-O1/non-O139 V. cholerae suppressed the growth of the O1 pandemic strain by using contact-dependent T6SS as well as by secreting the O1-detrimental hemolysin A during their co-persistence in the aquatic habitat.

Quinolone Resistance Determinants of Clinical Salmonella Enteritidis in Thailand.

Salmonella Enteritidis has emerged as a global concern regarding quinolone resistance and invasive potential. Although quinolone-resistant S. Enteritidis has been observed with high frequency in Thailand, information on the mechanism of resistance acquisition is limited. To elucidate the mechanism, a total of 158 clinical isolates of nalidixic acid (NAL)-resistant S. Enteritidis were collected throughout Thailand, and the quinolone resistance determinants were investigated in the context of resistance levels to NAL, norfloxacin (NOR), and ciprofloxacin (CIP). The analysis of point mutations in type II topoisomerase genes and the detection of plasmid-mediated quinolone resistance genes showed that all but two harbored a gyrA mutation, the qnrS1 gene, or both. The most commonly affected codon in mutant gyrA was 87, followed by 83. Double codon mutation in gyrA was found in an isolate with high-level resistance to NAL, NOR, and CIP. A new mutation causing serine to isoleucine substitution at codon 83 was identified in eight isolates. In addition to eighteen qnrS1-carrying isolates showing nontypical quinolone resistance, one carrying both the qnrS1 gene and a gyrA mutation also showed a high level of resistance. Genotyping by multilocus variable number of tandem repeat analysis suggested a possible clonal expansion of NAL-resistant strains nationwide. Our data suggested that NAL-resistant isolates with single quinolone resistance determinant may potentially become fluoroquinolone resistant by acquiring secondary determinants. Restricted therapeutic and farming usage of quinolones is strongly recommended to prevent the emergence of fluoroquinolone-resistant isolates.

An efficient virus concentration method and RT-nested PCR for detection of rotaviruses in environmental water samples.

Water samples were concentrated by the modified adsorption-elution technique followed by speedVac reconcentration of the filter eluates. Reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) was used to detect rotavirus RNA in concentrates of the water. The detection limit of the rotavirus determined by RT-nested PCR alone was about 1.67 plaque forming units (PFU) per RT-PCR assay and that by RT-nested PCR combined with concentration from 1l seeded tap water sample was 1.46 plaque forming units per assay. Water samples were collected from various sources, concentrated, and determined rotavirus RNA. Of 120 water samples, rotavirus RNA was detected in 20 samples (16.7%); 2/10 (20%) of the river samples, 8/30 (26.7%) of the canal samples, and 10/40 (25%) of the sewage samples but was not found in any tap water samples (0/40). Only three water samples were positive for rotavirus antigen determined using an enzyme-linked immunosorbent assay (ELISA). Alignment analysis of the sequenced PCR product (346-bp fragment) was performed in eight rotavirus-positive samples using the rotavirus sequence deposited in the GenBank. All samples gave the correct VP7 sequence. Results of analysis showed two samples similar to human rotavirus (97-98%), five similar to rotavirus G9 sequence (94-99%), and one sample similar to animal rotavirus (97%). PCR inhibitors were not observed in any concentrated water samples. In all 20 (of 120) samples where rotaviruses were found, fecal coliforms including Escherichia coli were also found, but of the samples testing negative for rotaviruses, 76 were fecal coliforms positive and 69 were E. coli positive. The combination of the virus concentration method and RT-nested PCR described below made it possible to effectively detect rotaviruses in environmental water samples.

Characterization of antibiotic resistance in Escherichia coli isolated from shrimps and their environment.

Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet (A), tet (B), tet (C), tet (D), tet (E), and tet (G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet (A) (69.1%; 38 of 55) was the most common followed by tet (B) (56.4%; 31 of 55) and tet (C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-orfF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.

Possible horizontal transmission of Salmonella via reusable egg trays in Thailand.

Salmonella contamination of eggshells, egg contents, reusable egg trays, and various environmental samples was assessed. Although the overall Salmonella contamination rate from egg farms was low (3.2%), over a quarter (26.7%) of egg trays from farms and more than one third (36.7%) of trays from the market were contaminated. Salmonella strains isolated from reusable egg trays were analyzed by serotyping, antimicrobial susceptibility test and XbaI pulsed-field gel electrophoresis (PFGE) typing. Five serovars (S. Braenderup, S. Emek, S. Weltevreden, S. Stanley, and S. Derby) were isolated, and half of the strains assessed were found to be resistant to one or more of the six antimicrobial agents examined. The overall resistance rates to nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline, and ampicillin were 40.7%, 36.0%, 26.7% and 3.5%, respectively. The PFGE types were matched against sample location and drug resistance. S. Braenderup PFGE type A2 (susceptible to all tested drugs) was isolated from all sample sites; PFGE type A2 (resistant to nalidixic acid) was isolated from Farm C and the market. S. Braenderup PFGE type A1 (resistant to four drugs) was isolated from Farms A and C. S. Weltevreden PFGE type C3 (susceptible to all tested drugs) was isolated from Farms A and B and type C4 (susceptible to all tested drugs) was isolated from Farm A and the market. The distribution of the related genotypes and resistance patterns of Salmonella in egg farms and the market indicate drug-resistant strains of Salmonella may be spread on reusable egg trays.

Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction.

The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.