Antisense peptide nucleic acids againstftsZ andefaA genes inhibit growth and biofilm formation of Enterococcusfaecalis.

Enterococcus faecalis is one of the important causes of nosocomial infections. Nowadays, increasing prevalence of antibiotic-resistant bacteria and slow progress in recognizing new antimicrobial agents has limited the efficiency of conventional antibiotics, which cause to find novel strategies to overcome bacteria. Therefore, in this study, we aimed to assess the role of efaA gene in the biofilm formation and the role of ftsZ gene in the controlling of bacterial growth by the anti-sense PNAs(Peptide Nucleic Acid).E. faecalis ATCC® 29212™was used for the study of PNAs designed to targeting the start codon section of the ftsZ andefaA genes. PNA attachment to RNA was confirmed by blotting. Electroporation technique was used for the intracellular transfer of anti-ftsZ PNAs. The spot-plating method was used to the assessment of alteration in bacterial growth. Biofilm formation assay and real-time PCR were used for detection of biofilm inhibitory effect of cell penetrating peptide (CPP) conjugated to anti-efaA PNAs.ByftsZ PNAs treatment, no growth was seen from the strain in agar by a spot plating method and the inhibition zone of anti-ftsZ PNAs was not seen. PNAs against the efaA gene decreased by 95% the expression of the efaA gene and biofilm formation. In addition, the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay showed no toxicity on MCF7 cells for both of anti-ftsZand anti-efaA PNAs.This study used new genetic and molecular tools to inhibit pathogenicity and infection by E. faecalis. In this study, we suggested that efaA gene plays a critical role in the biofilm formation and anti-efaA PNAs could decrease the formation of biofilm, as well as, anti-ftsZ PNAs could eliminate bacterial growth.

AdeB efflux pump gene knockdown by mRNA mediated peptide nucleic acid in multidrug resistance Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii isolates cause critical problems in health-care environments. AdeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically essential antibiotics in A. baumannii, such as ciprofloxacin. This study aimed to target adeB gene with antisense peptide nucleic acid (PNA) and investigate its effect on resistance to antibiotics. NCBI database was used to design appropriate PNA to target adeB gene, by connecting PNA to mRNA, the translation of mRNA can be prevented. Three clinical isolates and A. baumannii ATCC 17978 were treated with the designed PNA by electroporation and competence procedure. Minimum Inhibitory concentration (MIC) of ciprofloxacin, colistin, and tetracycline were determined by microbroth dilution method. In addition, the expression level of adeB gene was measured by quantitative real-time PCR (qRT-PCR). Isolates used in this study had mutations in gyrA and parC genes corresponding to resistance to ciprofloxacin. MIC of resistance to ciprofloxacin after treatment with PNA was reduced from 32 µg/ml to16 µg/ml in A. baumannii ATCC 17978 isolate. Susceptibility level of tetracycline, in the 2 clinical isolates was decreased from 64 µg/ml to 32 µg/ml and in the other isolate was reduced from 128 µg/ml to 64 µg/ml. The expression level of adeB gene was decreased in A. baumannii ATCC 17978 (P > 0.01) but not in clinical isolate (P = 0.107). Findings of the present study indicate overexpression of adeB efflux pump has extra effect on resistance to antibiotics in isolates with a defined mechanism of resistance. Antisense technology is a feasible technique to suppress the function of these genes, which may be further exploited to control multidrug-resistant isolates.

Peptide nucleic acid-mediated re-sensitization of colistin resistance Escherichia coli KP81 harboring mcr-1 plasmid.

Escherichia coli is a gram-negative bacterium and it causes a variety of diseases in humans. It causes a wide range of clinical infections in humans; urinary tract infections is the most prevalent infection caused by uropathogenic Escherichia coli. In recent years, the observation of antibiotic-resistant genes such as resistance to colistin, makes the Escherichia coli resistant to antibiotics like colistin (polymyxin E), because of that the use of new therapies like peptide nucleic acid (PNA) has attracted the consideration of scientists. The aim of this study is the assessment of the inhibitory role of PNA against mcr-1 gene and reduction of mcr-1 gene expression and MIC in colistin resistant E. coli by PNA. NCBI database was used to design PNA. Our study was carried out on E. coli KP81 bacteria containing the mcr-1 gene. Microbroth dilution (MIC) method was used to survey phenotypic sensitivity and determine the sensitivity of the bacteria to the colistin antibiotic. E. coli KP81 isolates were further investigated by polymerase chain reaction to assess the presence of mcr-1 genes and target genes were quantified by real-time PCR assay using specific primers. The MIC result after treatment with specific PNA showed that the resistance to colistin reduced about three fold and the resistance level dropped from 32 µg/ml to 4 µg/ml. The expression analysis of mcr-1 gene in E. coli KP81 isolate indicates the PNA, 95% reduced the expression of the mcr-1 gene. Our observations showed that by inhibiting the expression of mcr-1, sensitivity to colistin can be defeated. Using higher concentrations of PNA and an in vivo study can reveal more clinical application of this method.

Molecular Epidemiology Survey of Staphylococcus aureus Panton-Valentine Leukocidin-positive Isolated from Sanandaj, Iran.

BACKGROUND: Staphylococcus aureus strains that are Panton-Valentine leukocidin (PVL) positive cause severe skin and soft tissue infections as well as necrotizing pneumonia. The presence of PVL gene is a marker for methicillin-resistant S. aureus; therefore, survey on prevalence and phylogenetic distribution of PVL is of great importance for public health. The aim of this research was molecular epidemiology survey of S. aureus PVL positive, isolated from two tertiary hospitals of Sanandaj. MATERIALS AND METHODS: A total of 264 staphylococci isolates were collected from clinical specimens, hospital personnel and hospital environment of two tertiary hospitals of Sanandaj, in 2012 (Toohid and Besat). Bacterial cultures and biochemical tests were performed for S. aureus detection. Then, polymerase chain reaction (PCR) and repetitive sequence-based PCR (rep-PCR) were used for the determination of prevalence and molecular epidemiology of S. aureus PVL, respectively. Data were analyzed using the Fisher's exact test (P < 0.05). RESULTS: From 264 staphylococci isolates, 88 (33.33%) were detected as S. aureus. Furthermore, 20 out of 88 (22.72%) strains of S. aureus were PVL positive according to PCR results. Rep-PCR showed six main clusters of S. aureus samples. PVL had similar clonality between different samples. No significant relationship was observed between PVL positive S. aureus and rep-PCR patterns (P = 0.98). CONCLUSION: These results showed that a clone of S. aureus PVL positive has spread between the community and hospital settings. Therefore, appropriate measures are required to prevent the spread of staphylococci and other bacteria in hospitals.

Peptide nucleic acids (PNAs): currently potential bactericidal agents.

In recent years, the emergence of ESBL-producing and multi-drug resistant bacteria have been increased and designing novel components is necessary for confrontation these bacteria. Peptide nucleic acids (PNAs) are one of the synthetic components that bind to single strand DNA and RNA. Applications of these components are wide while, and one of the important applications of these components is inhibition of gene expression and knock downing the target gene follow as inhibition of bacterial growth. For PNA targeting gene, peptide-PNAs (PPNA) activity cannot be occurred without sequence homology, at the same time, it has been affected by sequence-based specific target and dose-dependent-based manner. Choosing the conserved sequence in different bacterial genus can provide broad-spectrum antimicrobial activity. In this review article, we studied several research papers and extract PNA targeting genes that cause gene knock down and inhibition of bacterial growth. Some novel opportunities for advancement and the design ultra-narrow-spectrum antimicrobial drugs against multi-drug can be accessible by utilizing PNA against necessary genes of pathogens. These results open novel vision for therapeutic intervention. Future researches are required to evaluate the safety, toxicity and pharmacokinetics properties of PPNAs in order to be utilized in clinical treatment.

Actinomycosis in Iran: Short Narrative Review Article.

Actinomycosis is an indolent, slowly progressive infection caused by anaerobic or microaerophilic bacteria, primarily of genus Actinomyces, which colonize the mouth, colon and vagina. Mucosal disruption may lead to infection virtually at any sites in the body. The aim of this study was to underline different features of actinomycosis and to represent total data about etiologic agents, clinical, diagnostic and therapeutic approaches these infections. From a total of 38 case reports or series, ninety one cases were obtained by using of relevant articles reported as recorded cases in Iran (1972 to 2012). Analyzed data represented 21 cases of oral-servicofacial (23.1%), 7 cases of thoracic (7.7%), 17 cases of abdominal (18.7%), 21 cases of disseminated forms (23.1%) and 25 cases of others (27.5%). Findings indicated more common of these infections in men (61.5%). Actinomyces naeslundii (21 cases) was found as the most common causative agents in comparison with A. Israeli (15 cases), A. viscosus (3 cases) and A. bovis (1 case). The most patients had been successfully treated with penicillin although some cases needed surgery along with antibiotic therapy. Since some clinical features of actinomycosis are similar to malignancies, so the differential diagnosis of invasive forms must be considered. This report emphasizes on the importance of differential diagnosis of actinomycosis from similar diseases by clinicians.