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Background@#With limited information available, the association among urinary tract infections, urease-producing bacteria and the presence of magnesium ammonium phosphate (MAP) urolithiasis in canines in Thailand requires more study. @*Objectives@#This study aimed to investigate the association between demographic characteristics of canines and the presence of MAP urolithiasis in canines, and to evaluate antimicrobial susceptibility patterns of bacteria isolated from canine uroliths. @*Methods@#A total of 56 canines admitted for treatment with surgical removal of uroliths were recruited. Demographic characteristics and clinical chemistry data were recorded.Bacteria isolated from the removed uroliths were identified. Chemical compositions of the uroliths were analyzed by Fourier transform infrared spectrometer. Potential risk factors were determined with univariable and multivariable logistic regression analyses. @*Results@#Of 56 canine urolithiasis, bacteria were isolated from uroliths of 38 canines (27 MAP and 11 non-MAP) but not from uroliths of 18 canines (5 MAP and 13 non-MAP). The most common bacteria found in nidus of MAP uroliths was Staphylococcus pseudintermedius (approximately 51%). An antimicrobial resistance was frequently found in Staphylococci isolates (42.86%). Multivariate logistic regression analysis showed that the predictors of MAP urolith in canine urolithiasis were being female (p = 0.044; adjusted odds ratio [OR], 10.22; 95% confidence interval [CI], 1.06– 98.24) and the positive urolith culture (p = 0.012; adjusted OR, 8.60; 95% CI, 1.60–46.30). @*Conclusions@#Our results indicate that S. pseudintermedius (a urease-producing bacterium) is the major causative bacteria of MAP uroliths. A positive urolith culture and being female are risk factors of MAP urolithiasis in canines.
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To evaluate antibacterial activity and the bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (A. zerumbet) rhizomes. Methods: Eight reference microbial strains including two Gram-positive bacteria [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)] and six Gram-negative bacteria [Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATTC 700603), Proteus mirabilis (DMST 8212), Salmonella enterica subsp. enterica serovar Vellore. (ATCC 15611), Shigella flexneri (ATCC 12022) and Pseudomonas aeruginosa (ATCC 27853)], were used to test antimicrobial susceptibility by the broth microdilution method. Bioactive compounds were analyzed by using HPLC. Results: The minimum inhibitory concentration values of A. zerumbet extract were 8 mg/mL for Staphylococcus aureus, Escherichia coli and Shigella flexneri and 16 mg/mL for Enterococcus faecalis and the other four Gram-negative bacilli. HPLC chromatograms revealed that the A. zerumbet extract contained hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. Conclusions: The constituents of A. zerumbet rhizomes could be a potential source of antibacterial compounds, warranting further study of A. zerumbet extract.
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Objective: To compare biofilm formation in trimethoprim/sulfamethoxazole (SXT)- susceptible Escherichia coli (E. coli) (SSEC) and SXT-resistant E. coli (SREC) isolated from patients with urinary tract infections, and study the motile ability and physical characteristics of the isolates. Methods: A total of 74 E. coli isolates were tested for antimicrobial susceptibility with the disc diffusion assay. Based on the SXT-susceptibility test, the E. coli isolates were divided into SSEC (N = 30) and SREC(N = 44) groups. All E. coli isolates were examined for motile ability by using a motility test medium, and for checking biofilm formation a scanning electronmicroscope was used. Bacterial colony size was measured with a vernier caliper and bacterial cell length was measured under a light microscope. The bacterial growth rate was studied by plotting the cell growth (absorbance) versus the incubation time. Results: The frequencies of non-motility and biofilm formation in the SREC group were significantly higher than that in the SSEC group (P < 0.01). The SREC bacterial cell length was shorter than that in the SSEC group [(1.35 ± 0.05) vs. (1.53 ± 0.05) mm, P < 0.05)], whereas the bacterial colony size and mid-log phase of the growth curve were not significantly different. Conclusions: The present study indicated that biofilm formation and phenotypic change of uropathogenic E. coli can be attributed to the mechanism of E. coli SXT resistance.
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Methicillin-resistant Staphylococcus aureus, a well-known nosocomial pathogen in tertiary healthcare facilities, can cause severe life-threatening symptoms. Nowadays, prevention and control of outbreaks related to hospital-acquired infections need molecular information to distinguish and definitely define a real etiology. For the last decade, molecular techniques have been developed and applied to an epidemiological study of infectious diseases. Among them, polymerase chain reaction-based typing techniques are most feasible to be used as molecular tools in clinical microbiology laboratory in Thailand. In this study, PCR-based typing methods, including SCCmec typing, variable numbers of tandem repeats typing of hypervariable region downstream of mecA (HVR) locus and spa gene, were applied in order to determine genetic background, and major endemic clones in Srinagarind Hospital, Khon Kaen. A total of 247 MRSA isolated from 124 patients of Srinagarind Hospital during July 2007 through December 2008 were characterized by the PCR-based typing methods described above. Five SCCmec types were identified as type-III (60.7%), type-IIIA (30.8%), type-II SCCmec (6%), type-III DCS (1.7%), and type-I variant with class C mec complex (0.9%), respectively. HVR and spa typing differentiated MRSA into 5 and 10 groups, respectively. Combination of all genetic markers could identify two major clones, III-15-7 (43.6%), and IIIA-7-7 (22.2%). Medical wards and medical intensive care unit were considered as endemic areas of these two clones. Information in this study may be applied to infection control measure and lead to development of suitable PCR-typing techniques for MRSA in clinical laboratory.
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Haemophilus influenzae is a part of normal upper respiratory flora of human, which can cause a wide variety of infections. Other members of genus Haemophilus rarely cause human infection but are frequently isolated from clinical specimens, such as sputum. The pathogenicity between H. influenzae and other Haemophilus species is different therefore a reliable method for identification of H. influenzae is essential. The aim of this study was to compare the identification methods for Haemophilus by four phenotypic tests with that by a PCR-based method. A total of 101 Haemophilus isolates were identified by biochemical tests and the XV requirement test by using XV paper strip technique, porphyrin test and Staphylococcus streak technique. The PCR-based method was performed using specific primers for 16SrDNA, p6 genes of H. influenzae and sodA gene of H. parainfluenzae. Using the XV paper strip technique, porphyrin test and biochemical tests, 88 and 13 isolates were identified as H. influenzae and H. parainfluenzae respectively, whereas 54 H. influenzae and 47 H. parainfluenzae were identified by using Staphylococcus streak technique (66.4 % agreement with that of the three tests). The PCR-based method revealed that 83 H. influenzae and 12 H. parainfluenzae were identified, whereas 6 isolates could not be categorized into both species. This study showed that identification of Haemophilus by the XV paper strip technique, porphyrin test and biochemical test gave 93.1 % agreement with that of the PCR method, whereas the Staphylococcus streak technique gave only 71.3 % agreement.
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ABSTRACT
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Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported in Pseudomonas aeruginosa. Class B carbapenemases or metallo-b-lactamases (MBLs) are the most clinical concern. Currently, there is no recommendation available from the Clinical Laboratory Standards Institute (CLSI) for MBL detection. In this study, we attempted to evaluate the performance for phenotypic detection of MBLs in imipenem-nonsusceptible clinical isolates of P. aeruginosa from Srinagarind Hospital. Five MBL-positive control strains, each producing IMP-1, IMP-4, IMP-9, VIM-1, or VIM-2 enzyme, and 74 clinical isolates of P. aeruginosa were screened for the presence of MBLs by combined-disk test (CDT) and double-disk synergy test (DDST) on a single agar plate using imipenem (10 µg IPM) and meropenem (10 µg MEM) disks as substrates and 292 µg of EDTA as an MBL inhibitor. Multiplex PCR for detection of MBL genes was used as a gold standard. Genotypic confirmation revealed that 9 of the 74 clinical isolates (12.2%) carried MBL genes, blaVIM (6 isolates) and blaIMP (3 isolates). Comparison of the MBL phenotypic tests to the multiplex PCR revealed that the CDT using IPM+EDTA/IPM correctly differentiated all MBL-producing isolates (sensitivity of 100%) but gave three false-positive isolates (specificity of 95.4%), whereas that with MEM+EDTA/MEM showed sensitivity and specificity of 92.9% and 92.3% respectively. In addition, the DDST using either IPM or MEM with EDTA gave the same result for all isolates with sensitivity and specificity of 92.9% and 96.9% respectively. These methods are simple and accurate for detection of MBLs in imipenem-nonsusceptible P. aeruginosa isolates from this hospital. Early detection of MBL producers and strict infection control will contribute to prevent further spread of these resistant strains.
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Burkholderia pseudomallei (B. pseudomallei) is a major causative agent of melioidosis in endemic areas such as Southeast Asia and Northern Australia. Rapid diagnosis is required for appropriate treatments in septicemic melioidosis. In this study, we aimed to evaluate the specific polyclonal antibody (pAb) to B. pseudomallei for potentially use in the development of diagnostic assays and pathogenesis studies. In determination the cross reaction of the pAb to other bacteria by indirect ELISA, the pAb to B. pseudomallei has no cross-reactivity with other bacteria. In contrast, the pAb reacted with 24 clinical isolates of B. pseudomallei-extracted antigens. By ELISA, the pAb to B. pseudomallei could be used to detect secreted antigens in 3 hour culture supernatants of 1.5 x 104 cfu/ml B. pseudomallei. In addition, opsonization of the bacteria with the pAbs could enhance bacterial internalization by U937-derived macrophages when compared with bacterial culture alone. The mechanism of these observations, however, needs to be further investigated. In conclusion, we suggest that the pAbs to B. pseudomallei can be potentially used for development of diagnosis method and pathogenesis study.
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Staphylococcus aureus, a well-known human pathogen, causes a variety of infections in human, e.g. superficial skin infection through life-threatening infection. S. aureus is able to produce many enzymes, including exotoxins which lead to tissue inflammation and injury. Moreover, it also plays an importance role in clinical practice by exhibiting resistance phenotype to methicillin. Many virulence determinants are located on mobile genetic elements (MGEs), such as bacteriophages, pathogenicity islands (PAI), and genomic islands, and existed variably in the bacterial population. Then, virulence genes can be used as genetic markers for clinical manipulations, and nosocomial control measurement. The objective of this study was to determine virulence genes associated with those MGEs in S. aureus samples both in methicillin-susceptible S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA). A total of 100 MSSA and MRSA isolates (50 of each) were randomly selected from clinical samples of patients at Srinagarind Hospital during November, 2006 through June, 2007. All isolates were determined for the presence of eta, lukDE, lukSF-PV, tst-1, sak, sea, sec, sel, and sep genes by PCR. In case of MRSA, staphylococcal cassette chromosme mec (SCCmec) types were also determined in order to study the association among the determinants and their allotypes. The results showed that most of S. aureus samples harbored at least one virulence gene, and most of them carried lukDE (90 %), and sak (88 %). High potential virulence genes, eta and lukSF-PV, were detected in 2 and 10 isolates of MSSA only. However, sea gene was detected more frequent in MRSA than MSSA (P \< 0.05). While sec gene was significantly recognized less in MRSA than MSSA isolates (P \< 0.05). The other staphylococcal enterotoxins such as sec, sel and sep were detected in small samples of S. aureus, and none was found to harbor tst-1 gene. The molecular information associated to virulence genes on MGEs may be useful in clinical practice and hospital epidemiology in Srinagarind Hospital, and other tertiary care facilities.