Identification of methicillin-resistant Staphylococcus aureus carrying an exfoliative toxin A gene encoding phage isolated from a hospitalized patient in Turkey.

From the four known isoforms of the staphylococcal exfoliative toxins (ETs), only ETA and ETB are the major causative agents. General knowledge is that the gene for ETA is located on the chromosome, whereas that for ETB is located on a large plasmid. Yoshizawa and co-workers (2000, Microbiol. Immunol. 44(3): 189-191) isolated, for the first time, a temperate phage (φETA) that carried the structural gene for ETA from an ETA-producing strain of Staphylococcus aureus. In this study, we presented eta gene encoding temperate phages isolated from methicillin-resistant S.aureus (MRSA) isolates obtained from patients in a Turkish hospital. Molecular analysis of the phage genome revealed that the eta gene is located upstream to amidase and holin genes, the same as in the φETA genome. However, partial sequence analysis of amidase and holin genes revealed polymorphic variation. In addition to polymorphic variation, restriction fragment length polymorphism (RFLP) analysis of all of the phage genomes showed that the ETA-containing phage is different from the rest of the phage genomes. The phylogenetic dendrogram of pulsed field gel electrophoresis (PFGE) analysis showed that the ETA-carrying MRSA is quite different from the rest of the MRSA strains. This is the first report showing that a MRSA strain carries an ETA-encoding phage.

[Identification of a novel lytic bacteriophage obtained from clinical MRSA isolates and evaluation of its antibacterial activity]. / Klinik MRSA Izolatlarindan Elde Edilen Yeni Bir Litik Bakteriyofajin Tanimlanmasi ve Antibakteriyel Etkisinin Degerlendirilmesi.

Multidrug-resistant bacteria particularly MRSA is well known as a worldwide problem. Since the rate of development of novel antimicrobial agents has been slowed down during the last years, there have been a need for the exploration of alternative solutions for the treatment of resistant bacterial infections. Treatment of infections by bacteriophages (phages) that specifically kill the infecting pathogen, i.e. by the process known as phage therapy, is considered as a possible approach to treat multidrug resistant bacteria. Phage treatment has also been considered to treat Staphylococcus aureus infections. This study was aimed to evaluate the antibacterial and cytotoxic activities of a new lytic phage obtained from clinical MRSA strains. This lytic phage named as f LizAnk was obtained during the phage infectivity studies performed with 13 lysogenic phages against MRSA strains. The antibacterial activity of the f LizAnk phage was determined in vitro in BHI (Brain Heart Infusion) and LB (Leuria Bertani) broths and the in vivo antibacterial activity against MRSA strains and possible cytotoxic effect against mammalian cells were tested on fibroblastic cell cultures (3T3). This study was conducted using 20 MRSA strains isolated from hospitalized patients. Identification of the isolates was performed by conventional methods and methicillin resistance was detected with oxacillin disk diffusion test and mecA gene detection by PCR. The method described by Kaneko et al. [Biosci Biotechnol Biochem 1997; 61(11): 1960-2] was used with some modifications, for induction and isolation of the phages. In vitro studies indicated that this phage killed the six different MRSA strains (in 107 cfu/ml concentrations) in 8 hours, and this powerful lytic effect was similar in both of the liquid media. In vivo studies were performed by using cell cultures prepared in microplates, and the wells have been inoculated with only phage, phage + MRSA mixture, and only MRSA. The cells were then evaluated microscopically as well as by MTT assay which detected alive cells colorimetrically, at 2nd and 24th hours. In our study, the f LizAnk phage did not cause any toxic effect on fibroblast cell cultures, in addition it was observed that the antibacterial effect of the phage against MRSA has proceeded in the cell culture. In conclusion, since the fLizAnk phage described in this study exhibited strong antibacterial activity against MRSA strains and no cytotoxic effect was detected against mammalian cells, it might be safely used alone or in a phage cocktail to treat skin infection caused by MRSA.

A new, simple, rapid test for detection of DNase activity of microorganisms: DNase Tube test.

The DNase test is a simple, economical method that has traditionally been used as a supplemental test to identify pathogenic Staphylococcus. This test also aids in the differentiation of closely-related genera within the Klebsiella-Enterobacter-Serratia division of Enterobacteriaceae and several other pathogens, including screening of C. diphtheriae. Currently DNase activity of microorganisms was tested using DNase agar plate methods. These tests have some drawbacks including the necessity of the extensive time to see the results of DNase activity of bacteria. In here, we developed a new method which is simple, rapid, inexpensive and applicable to examine DNase activity of any bacteria. In this method, simply, bacteria is added to the broth medium containing DNA and followed for the DNA degradation caused by the DNase of bacteria by running in agarose gel. This method we called DNase Tube test showed DNA degradation as fast as in half an hour depending on the DNase activity of the bacteria.

Detection of herpes simplex virus type 1 in addition to Epstein-Bar virus in tonsils using a new multiplex polymerase chain reaction assay.

HSV-1, HSV-2, CMV, EBV, which are the members of the herpes virus family colonize and establish latent infection in human. Although EBV is a well known virus most involved in recurrent bouts of acute tonsillitis, the role and possibility of HSV-1, HSV-2, and CMV for establishing infection in tonsils are not clear. The purpose of this study is to verify whether the tonsils might harbor the HSV-1, HSV-2, and CMV, in addition to EBV, in chronically hyperplastic nasopharyngeal lymphoid tissue. To accomplish the purpose, we developed a new Multiplex Polymerase Chain Reaction (M-PCR) assay using a single consensus forward primer and virus specific reverse primers for DNA polymerase gene of HSV-1, and 2, EBV, and CMV, and investigated its efficiency for detecting HSV1, HSV2, CMV, and EBV. The sample of 52 patients underwent tonsillectomy or adenectomy because of chronic lymphoid hyperplasia without any evidence of acute infections and were investigated for presence of HSV-1, HSV-2, CMV, and EBV. Of the 54 samples, 11 (20.4%) of them were positive for EBV, 4 of them (7.4%) were positive for HSV-1, and none of the samples were positive for HSV-2 and CMV. To the best of our knowledge, this is the first report that tonsils may be the reservoir for HSV-1 in addition to EBV, and HSV-1 may have a role in recurrent tonsillitis and systemic diseases. The MC-PCR assay presented in this study can provide a rapid, sensitive, and economical method for detection of HSV-1, HSV-2, EBV, and CMV in a single PCR tube.