Identification of medically important Candida species by polymerase chain reaction-restriction fragment length polymorphism analysis of the rDNA ITS1 and ITS2 regions.

AIM: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP) method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR) products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. MATERIALS AND METHODS: One hundred and fifty candidal strains isolated from various clinical samples were studies/ included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. RESULTS: The majority of Candida isolates were isolated from urine (78.6%) while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. CONCLUSION: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

Molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae at a Turkish centre: Is the increase of resistance a threat for Europe?

OBJECTIVES: In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) have become an important threat to hospitalised patients. This study aimed to identify the genetic mechanisms of carbapenem resistance in CRKP isolated from patients in a Turkish centre. METHODS: During 2013-2014, a total of 98 K. pneumoniae isolated from patients at Çukurova University Balcali Hospital (Adana, Turkey) determined phenotypically as resistant to carbapenems were screened genotypically for the presence of carbapenemase enzymes by multiplex PCR. RESULTS: Of the 98 patients for whom genetic investigation was made, 93 (94.9%) were adults, 56 (57.1%) were male and 81 (82.7%) were diagnosed as infected. The mean and median age were 51.8±20.5years and 55 years (range 1-89 years), respectively. The nosocomial infection rate was 87.8% (86/98). The mortality rate was 41.8% (41/98). Fifty-eight patients (59.2%) were admitted to intensive care units. Of the 12 non-nosocomial infections, 5 (41.7%) originated from the inpatient clinic of the urology department. The mean and median hospital length of stay (LOS) were 20.7±20.8days and 17days (range 0-90 days), respectively. The most common carbapenemase gene detected was blaOXA-48 (74.5%), followed by blaVIM (45.9%) and blaSME (37.8%). The blaNDM gene was detected in 20 isolates (20.4%). The most effective antibiotics were tigecycline and colistin, with susceptibility rates of 87.5% and 74.3%, respectively. CONCLUSIONS: Multiple resistance mechanisms were present in CRKP isolates in Turkey. Most of the isolates harboured blaOXA-48, blaVIM and blaSME genes; meanwhile, the rate of 20.4% for blaNDM is alarming.

COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS.

Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

NDM-1 and rmtC-Producing Klebsiella pneumoniae Isolates in Turkey.

BACKGROUND: The resistance of aminoglycosides in strains that produce beta-lactamase can be developed through the multidrug resistant encoding genes carried by common plasmids. Recently, the association between 16S rRNA methyltransferase resistance and beta-lactamase enzymes carried by the same plasmids has drawn increased attention from researchers, particularly the association in aminoglycoside-resistant strains with a minimum inhibitory concentration (MIC) of ≥ 256 µg/mL. OBJECTIVES: We aimed to investigate the co-existence of 16S rRNA methyltransferase and beta-lactamase genes in multidrug resistant (MDR) Klebsiella pneumoniae strains isolated from clinical samples. METHODS: We determined the molecular mechanisms of aminoglycoside resistance and its relationship with resistance to carbapenem and beta-lactam group antibiotics in 40 extended-spectrum beta-lactamase (ESBL)-positive carbapenem- and aminoglycoside-resistant K. pneumoniae strains. Multidrug resistant K. pneumoniae was isolated from various clinical samples in the faculty of medicine of Cukurova University, Turkey. First, the resistance of aminoglycoside and beta-lactam antibiotics was phenotypically investigated using the Kirby-Bauer disk diffusion test, double disk synergy test, and modified Hodge test. The MIC values of aminoglycoside were determined using the agar dilution method. Polymerase chain reaction was performed to detect the carbapenemases, ESBL, and 16S rRNA methyltransferase genes. The results were confirmed by a sequence analysis. RESULTS: Twenty K. pneumoniae strains showed resistance to amikacin, and 40 were resistant to gentamicin. The MIC value was found to be > 512 µg/mL in five amikacin-resistant strains and > 128 µg/mL in 10 gentamicin-resistant isolates. The rmtC gene, a type of 16S rRNA methyltransferase, was amplified in four isolates (MIC amikacin: > 512 µg/mL, gentamicin: > 128 µg/mL). Of these four isolates, three had the blaNDM-1 gene and all contained at least one ESBL gene. CONCLUSIONS: This study demonstrated the co-existence of rmtC and blaNDM-1 genes for the first time in Turkey. The spread of this resistant type should be monitored and limited through molecular surveillance.