ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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ABSTRACT
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ABSTRACT
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.
ABSTRACT
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.