Characterization of novel bacteriophage PSKP16 and its therapeutic potential against ß-lactamase and biofilm producer strain of K2-Hypervirulent Klebsiella pneumoniae pneumonia infection in mice model.

BACKGROUND: Severe infections caused by ß- lactamase producers, hypervirulent Klebsiella pneumoniae (BhvKp) with K2 serotype, highlight emergency need for new therapeutic strategies against this pathogen. We aimed to assess the efficacy of a novel phage, PSKP16, in the treating of pneumonia induced by BhvKp in mice models. METHOD: Genome sequences of PSKP16 were analyzed, and associated information can be found in NCBI. We applied treatment in two ways: by using mice for immediate and delayed treatments. Moreover, acute pneumonia obtained by BhvKp with intranasal method, was characterized in terms of histopathology of pulmonary lesions, biomarkers of inflammation level, leukocytes cells infiltration extent in mice, and was assessed treatment of them with PSKP16 multiplicity of infection (MOI: 10), either individually or in combination with gentamicin. Assessment of the ability of PSKP16 to inhibit BhvKp biofilm was studied. RESULTS: PSKP16 was associated with the Drexlerviridae family, and had a genome size of 46,712 bp, and 67 predicted ORFs. Herein, prompt phage administration's efficacy to decrease bacterial load and improve the survival rate in pneumonia models was faster than the synergism model with delay, but both almost displayed similar endpoints. The distribution of BhvKp strains in the lung was consistent with the histopathological findings, simultaneous inflammation, and level of serum tumor necrosis factor-α (TNF α). The phage treatment presented a lack of severe lesions and alveolar edema, reduction of inflammatory cell infiltration, which not only was it not associated with an over-inflammation but also provided a faster correction of blood cell count abnormalities compared to gentamicin. Phage with a high concentration in in vitro model effectively eliminated biofilms. CONCLUSION: It is essential to raise clinical awareness and management of BhvKp infections, signaled as the next superbug in waiting. The results of our study underscore the importance of PSKP16 as a phage with promising therapeutic potential in treating BhvKp-induced pneumonia.

Evaluation of ethanol and EDTA concentrations in the expression of biofilm-producing smf-1, rpfF genes in XDR clinical isolates of Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is able to cause infections in immunocompromised patients, and the treatment of this opportunistic pathogen is complicated due to its virulence factors, antibiotic resistance, and the ability of the bacteria to produce biofilm. The main goals of this study were to assess the susceptibility of extensively drug-resistant (XDR) isolates to ethanol and EDTA, and evaluating the synergistic effect of these disinfectants, and also survey the effect of exposure to sub-inhibitory concentrations of ethanol and EDTA on the expression of biofilm-producing smf-1, rpfF genes. RESULTS: The results showed that EDTA significantly increased the effectiveness of the ethanol and have a synergistic effect. All of the 10 XDR isolates included in the current study harbored smf-1 and rpfF genes and produced biofilm. After exposure to MIC, sub-MIC, synergism, and sub-synergism of ethanol and EDTA, the expression of smf-1 and rpfF genes was repressed significantly. CONCLUSION: In the current study, it was indicated that the expression of biofilm-producing genes was repressed when bacteria are exposed to different concentrations of ethanol and EDTA. Future studies should include more complex microbial communities residing in the hospitals, and more disinfectants use in hospitals. Expression of other virulence genes in different conditions is suggested.

Phenotype and genetic determination of resistance to common disinfectants among biofilm-producing and non-producing Pseudomonas aeruginosa strains from clinical specimens in Iran.

BACKGROUND: Pseudomonas aeruginosa is a common pathogen in Hospitalized patients, and its various resistance mechanisms contribute to patient morbidity and mortality. The main aims of the present study were to assess the susceptibility of biofilm-producing and non-producing P. aeruginosa isolates to the five commonly used Hospital disinfectants, to evaluate the synergistic effect of selected disinfectants and Ethylene-diamine-tetra acetic acid (EDTA), and the effect of exposure to sub-inhibitory concentrations of Sodium hypochlorite on antimicrobial susceptibility test. RESULTS: The results showed that sodium hypochlorite 5% and Ethanol 70% were the most and least effective disinfectants against P. aeruginosa, respectively. The addition of EDTA significantly increased the effectiveness of the selected disinfectants. The changes in the antibiotic-resistance profiles after exposure to sub-inhibitory concentrations of disinfectants were observed for different classes of antibiotics (Carbapenems, Aminoglycosides, Cephalosporins, Fluoroquinolones). As well as near the all isolates harbored efflux pump genes and 117 (97.5%) of isolates produced biofilm. CONCLUSION: In the current study, the mixture of disinfectant and EDTA were the most suitable selection to disinfect Hospital surfaces and instruments. Also, it was clear that exposure to sub-inhibitory concentrations of Sodium hypochlorite results in resistance to some antibiotics in P. aeruginosa species. Strong and intermediate biofilm formers belonged to MDR/XDR strains. Future studies should include more complex microbial communities residing in the Hospitals, and more disinfectants use in Hospitals.

The effect of antimicrobial activity of Teucrium Polium on Oral Streptococcus Mutans: a randomized cross-over clinical trial study.

BACKGROUND: The purpose of this study is to determine the effect of a mouthwash containing Teucriumpolium herb on Streptococcus mutans in mouth. METHODS: This study was a randomized, crossover, double-blind clinical trial, where we selected 22 volunteers (dental students) randomly and we divided them into two groups. The study had two phases. In each phase, one group acted as the intervention group, while the other one was the control group. Both the intervention and control groups were given the mouthwash with and without Teucriumpolium, respectively. S. mutans of saliva were measured before and after each phase to compare the effects of the mouthwashes. A three-week washout period was considered between the two phases. An independent two-sample t-test was utilized to compare the mean of S. mutans colonies. Additionally, we used a standard AB/BA crossover model to find the results of the treatment and the impact of carryover on the residual's biological effects. The significance level was considered 0.05 in this experiment. RESULTS: There is no significant difference observed between the two groups in the number of S. mutans before using the mouthwashes. When the mouthwash containing Teucriumpolium was used, there was a significant decrease in the number of S. mutans colonies in both phases' extract (P = 0.002). CONCLUSION: The results of this study indicate the mouthwash containing aqueous extract of Teucrium polium can majorly reduce the colonization of S. mutans in human saliva. TRIAL REGISTRATION: Ethical issues approved by the Ethics Committee of the Rafsanjan University of Medical Sciences with the approval number of 937/9/31, IRCT code Number of IRCT2013121815842N1 and it was approved on 06/16/2014. The study was conducted in the period of September to November 2014.

Evaluation of the therapeutic effect of a novel bacteriophage in the healing process of infected wounds with Klebsiella pneumoniae in mice.

OBJECTIVE: Bacterial wound infections have recently become a threat to public health. The emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae highlights the need for a new treatment method. The effectiveness of bacteriophages has been observed for several infections in animal models and human trials. In this study, we assessed the effectiveness of bacteriophages in the treatment of wound infections associated with MDR and biofilm-producing K. pneumoniae and compared its effectiveness with that of gentamicin. METHODS: A lytic phage against MDR K. pneumoniae was isolated and identified. The effectiveness of phages in the treatment of wound infection in mice was investigated and its effectiveness was compared with gentamicin. RESULTS: The results showed that the isolated phage belonged to the Drexlerviridae family. This phage acts like gentamicin and effectively eliminates bacteria from wounds. In addition, mice in the phage therapy group were in better physical condition. CONCLUSION: Our results demonstrated the success of phage therapy in the treatment of mice wounds infected with K. pneumoniae. These results indicate the feasibility of topical phage therapy for the safe treatment of wound infections.

Isolation of new Klebsiella pneumoniae phage PSKP16.

Background and Objectives: Klebsiella pneumoniae is a clinically relevant opportunistic pathogen belonging to the Enterobacteriaceae family. It is in the top three bacteria associated with antimicrobial resistance deaths globally, and one of the most dangerous bacteria causing nosocomial infections. Phage therapy offers a potential option for the treatment of drug-resistant bacterial infections. Materials and Methods: Phage PSKP16 was isolated against K. pneumoniae, capsular type K2 (isolated from a wound infection). PSKP16 is a new lytic phage with a Siphovirus-like morphology. Results: PSKP16 is a linear double stranded DNA phage with a GC content of 50% and genome size of 46,712 bp, for which we predicted 67 ORFs. PSKP16 belongs to the genus Webervirus and shows high evolutionary proximity to Klebsiella phages JY917, Sushi, and B1. Conclusion: Phage isolation is fast, cheap and efficient, but it requires time and characterization (which adds expense) to ensure that the isolated phages do not pose a health risk, which is essential to safely use phage therapy to treat life-threatening bacterial infections.

Isolation, Characterization and in vitro Evaluation of Specific Bacteriophages Targeting Extensive Drug Resistance Strains of Pseudomonas aeruginosa Isolated from Septic Burn Wounds.

BACKGROUND: Antibiotic resistant bacteria and various infections caused by them especially extensive drug resistance (XDR) strains and worrying statistics of mortality due to these strains and also the lack of a clear vision for development and production of new effective antibiotics have made the necessity of using alternative therapies more apparent. MATERIALS AND METHODS: In this study, specific phages affecting the Pseudomonas aeruginosa XDR strain were extracted from hospital wastewater and their laboratory characteristics along with lysis effect on 40 XDR strains of P. aeruginosa were investigated. RESULTS: The results indicated that three isolated phages (PaB1, PaBa2 and PaBa3) belonged to the Myoviridae and Pododoviridae families and were specific to Pseudomonas aeruginosa strains. More than 98% of phages absorbed their host in less than 10 minutes (Adsorption time <10 min) and completed their lytic cycle after 40 minutes (latent time = 40 min). Burst size of PaBa1, PaBa2 and PaBa3 was 240, 250 and 220 pfu/cell, respectively. PaBa1 lysed 62.5% of the XDR strains with the highest efficiency. The three Phage cocktail was effective against 67.5% of the studied strains. CONCLUSION: The results of this study indicate the significant potential of these phages for therapeutic use and prophylaxis of infections caused by this bacterium.

Antibacterial effect of nano-chlorhexidine on Enterococcus faecalis biofilm in root canal system: An in vitro study.

Background: A new method to improve the properties of the materials is nano-encapsulation, which improves the biological properties, antibacterial activity along with reduction of toxicity. Due to the spread of nano-knowledge, the present study was performed to evaluate the antibacterial effect of nano-chlorhexidine (CHX) on Enterococcus faecalis biofilm in the root canal system. Materials and Methods: In this in vitro experimental study, 55 matured single-root mandibular premolars were decoronated and the canals were prepared by single length method up to #F3 ProTaper Universal system. Five teeth were selected as negative control. Then, the teeth were randomly divided into three experimental groups (n = 15) and a positive control group (n = 5). The experimental groups were irrigated with 2% nano- CHX gel, 2% CHX solution, and 5.25% sodium hypochlorite (NaOCl), respectively. Finally, the number of colonies was counted. Kruskal-Wallis test was used to compare the number of colonies among groups. The level of significance was set at P < 0.05. Results: The mean number of colonies in the groups of nano-CHX, NaOCl, CHX, and positive control were obtained as 17.73 ± 18.69, 35.53 ± 36.42, 38.8 ± 31.8, and 96.8 ± 22.52, respectively. There was a significant decrease in the number of colonies in all the experimental groups compared to the control group (P < 0.05). However, difference in the number of colonies among these three groups was not significant (P > 0.05). Conclusion: The use of nano-CHX in removing E. faecalis biofilm from root canal is as effective as the use of CHX and NaOCl.

Antifungal Efficacy of a Permanent Silicon Soft Liner Containing Silver Nanoparticles.

Objectives: This study aimed to evaluate the in vitro antifungal efficacy of addition of silver nanoparticles (SNPs) to Mucopren® silicone soft liner material. Materials and Methods: Twenty disc samples (8 × 2 mm) of Mucopren® silicone soft liner containing 0wt% (control), 0.5wt%, 1wt%, 2wt%, and 3wt% SNPs were fabricated. Samples were powdered and added to 150 mL of Sabouraud dextrose agar culture medium and placed on separate culture dish plates. Each plate was inoculated with 106 colony forming units per milliliter (CFUs/mL) of Candida albicans (PTCC 5027) according to the CLSI protocol, and incubated at 37℃. The colony count was verified at 24 h, and the antifungal effect of the samples was evaluated according to the percentage of viable cells in the 2 subgroups with/without thermocycling. Data were analyzed using SPSS version 20 via ANOVA and t-test (P<0.05). Results: All experimental groups showed higher antifungal activity than the control group, and this effect was dose-dependent (P<0.05). The lowest colony count was recorded in the 3wt% group. Thermocycling had no significant effect on the antifungal efficacy, except in 0.5wt% concentration of SNPs (P=0.013). Conclusion: Addition of SNPs to Mucopren soft liner conferred antifungal effects. Further mechanical stability and toxicity studies are still required.

Use of a new multiplex quantitative polymerase chain reaction based assay for simultaneous detection of Neisseria meningitidis, Escherichia coli K1 , Streptococcus agalactiae, and Streptococcus pneumoniae.

BACKGROUND AND OBJECTIVES: Neisseria meningitidis, Escherichia coli K1, Streptococcus agalactiae, and Streptococcus pneumoniae cause 90% of bacterial meningitis. Almost all infected people die or have irreversible neurological complications. Therefore, it is essential to have a diagnostic kit with the ability to quickly detect these fatal infections. MATERIALS AND METHODS: The project involved 212 patients from whom cerebrospinal fluid samples were obtained. After total genome extraction and performing multiplex quantitative polymerase chain reaction (qPCR), the presence or absence of each infectious factor was determined by comparing with standard strains. RESULTS: The specificity, sensitivity, positive predictive value, and negative predictive value calculated were 100%, 92.9%, 50%, and 100%, respectively. So, due to the high specificity and sensitivity of the designed primers, they can be used instead of bacterial culture that takes at least 24 to 48 hours. CONCLUSION: The remarkable benefit of this method is associated with the speed (up to 3 hours) at which the procedure could be completed. It is also worth noting that this method can reduce the personnel unintentional errors which may occur in the laboratory. On the other hand, as this method simultaneously identifies four common factors that cause bacterial meningitis, it could be used as an auxiliary method diagnostic technique in laboratories particularly in cases of emergency medicine.

Inhibitory effect of probiotic yeast Saccharomyces cerevisiae on biofilm formation and expression of α-hemolysin and enterotoxin A genes of Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus, as an opportunistic pathogen, is the cause of a variety of diseases from mild skin infections to severe invasive infections and food poisoning. Increasing antibiotic resistance in S. aureus isolates has become a major threat to public health. The use of compounds produced by probiotics can be a solution to this problem. Thus, the purpose of this study was to investigate the effect of Saccharomyces cerevisiae on some virulence factors (biofilm, α-hemolysin, and enterotoxin A) of S. aureus. MATERIALS AND METHODS: Supernatant and lysate extracts were prepared from S. cerevisiae S3 culture. Sub-MIC concentrations of both extracts were separately applied to S. aureus ATCC 29213 (methicillin-sensitive S. aureus; MSSA) and S. aureus ATCC 33591 (methicillin-resistant S. aureus; MRSA) strains. Biofilm formation of these strains was measured by microtiter plate assay and expression level of α-hemolysin and enterotoxin A genes (hla and sea, respectively) using real-time PCR technique. RESULTS: The supernatant extract has reduced both biofilm formation and expression of sea and hla genes, while lysate extract had only anti-biofilm effects. The MRSA strain showed more susceptibility to yeast extracts than MSSA strain in all tests. CONCLUSION: The present study exhibited favorable antagonistic effects of S. cerevisiae S3, as a probiotic yeast, on MSSA and MRSA strains. Based on the findings of this study, the compounds produced by this yeast can be used to control S. aureus infections; however, further similar studies should be conducted to confirm the findings of the present study.

Polymerase Chain Reaction-Based Assays for the Diagnosis of Active and Relapsed Cases of Human Brucellosis.

This study aimed to compare polymerase chain reaction (PCR) with serum agglutination test (SAT) in the diagnosis of patients before and 6 months after treatment. Peripheral blood specimens from 50 patients with brucellosis (case group) and 30 subjects without brucellosis (control group) were selected and entered into the study. The diagnosis of brucellosis was established using SAT ≥ 1:160 and 2-mercaptoethanol (2-ME) ≥ 1:80 with clinical signs and symptoms compatible with brucellosis. For each case, both before treatment and 6 months after completion of therapy, SAT, 2-ME, and PCR were performed. Subjects in the control group were assessed by the same tests at the initial visit. In the case group, 50 patients (36 males, 14 females) with the mean age of 43.6 ± 14.5 years were evaluated. The mean age of the control group was 40.6 ± 14 years. Among the 50 patients whose nested PCR assays were initially positive, 43 (86%) were negative 6 months after completing treatment. Relapse occurred in five (10%) patients within 6 months after treatment and all were PCR positive. None of the patients in the control group was PCR positive. Results show that PCR seems to be highly sensitive and specific, and therefore is a useful method for both the initial diagnosis and detection of relapse or chronic brucellosis.

Evaluation of Different Primers for Detection of Brucella by Using PCR Method.

INTRODUCTION: Brucellosis is a worldwide zoonosis and a significant cause of loss of health in humans and animals. Traditionally, classic diagnosis is carried out by isolation of Brucella, which is time-consuming, technically challenging and potentially dangerous. The aim of this study was to expand a molecular test that would be used for the develop detection of Brucella in a single reaction with high sensitivity and specificity, by targeting IS711element. METHODS: This study was carried out from 2015 to 2016 at the Ayatolla Rohani hospital in Babol, Iran. The present study was designed to develop PCR assay, based on IS711 gene for rapid diagnosis of Brucella spp. and immediate detection of Brucella, with high sensitivity and specificity. Four pairs of oligo-nucleotide primers with sizes of 547, 403, 291 and 127bp respectively, were planned to exclusively amplify the targeted genes of Brucella species. RESULTS: Our results show that, five PCR primers set up, would be helpful in amplifying the DNAs from the genus Brucella with high specificity and sensitivity so it can be 12 fg, for Brucella species to provide a valuable tool for diagnosis. CONCLUSION: This method can be more useful than serological and biochemical tests and in addition, this reduces the number of required tests more rapidly and economically.

First experience of Candida non-albicans isolates with high antibiotic resistance pattern caused oropharyngeal candidiasis among cancer patients.

BACKGROUND AND AIM: In cancer patients, Candida species can cause a variety of diseases particularly oropharyngeal candidiasis which is a common infection. In this study, an attempt has been made to determine susceptibility pattern of four antifungal agents against the Candida species isolated from cancer patients with oropharyngeal candidiasis. MATERIALS AND METHODS: Samples were taken from 50 cancer patients with oropharyngeal candidiasis by the physician, and isolation and identification of Candida spp. was done based on standard procedures. Antifungal resistance pattern was carried out according to CLSI guidelines, and 18s ribosomal RNA among Candida spp. was identified using multiplex polymerase chain reaction. RESULTS: Of the 50 patients, 18 (36%) were females and 32 (64%) were males; mean age was 38.4 years. Leukemia and lymphoma were the most frequent cancer types in the studied group, accounting for 17 (34%) and 12 (24%), respectively. A total of 29 Candida spp. were isolated from 29 cancer patients, of which 17 were C. albicans and 12 were C. non-albicans. All the Candida spp. were confirmed having 18s ribosomal RNA. Among all the Candida spp., C. non-albicans showed higher resistance pattern to amphotericin B (MIC 07 µg/ml) and ketoconazole (MIC = 05 µg/ml). CONCLUSION: In conclusion, oropharyngeal Candidiasis is a serious infection among cancer patients. The isolated Candida spp. were resistant to common antifungal agents, which may lead to longer hospital stay, more expensive/toxic drugs and higher mortality. Therefore, interval surveillance is necessary in developing institutional guidelines.

Comparison of Genital Chlamydia trachomatis Infection Incidence Between Women With Infertility and Healthy Women in Iran Using PCR and Immunofluorescence Methods.

BACKGROUND: For a long time, infertility has been one of the most sequels in medical sciences with microbial agents as one group of its causes. The possible etiological role of Chlamydia trachomatis in infertility was suggested years ago, but it has not yet been proved completely. To decrease the severe involvements of C. trachomatis infections, screening by efficient diagnostic methods are necessary. OBJECTIVES: In this study we attempted to determine the incidence of C. trachomatis in infertile women and compared this with healthy women. MATERIALS AND METHODS: This case-control study was performed on 150 infertile women with unknown causes and without physiological deficiency for infertility. The control group consisted of 200 fertile safe and impregnated women. Presence of C. trachomatis in the two groups was examined by direct and indirect immunofluorescence tests and PCR. RESULTS: C. trachomatis was detected by direct immunofluorescence method in 23 (15.3%) infertile women compared and 7 (3.5%) healthy controls. Using indirect immunofluorescence tests, a positive test titer of 1:16 as well as the above results were detected in 34 (22.6%) of the infertile cases and 9 (4.5%) of the controls. C. trachomatis was detected by PCR method in 48 (32%) infertile women and 13 (8.7%) among the controls. CONCLUSIONS: The results of our study suggest that there is a significant association between C. trachomatis infection and female infertility.

Simultaneous Detection of Integrase and Antibiotic Resistance Genes within SXT Constin in Vibrio cholerae O1 El Tor Strains Isolated from Iran Using Multiplex-PCR.

OBJECTIVES: Amongst the various antibiotic resistant elements in Vibrio. cholerae, SXT constin (SXT-C) is important. We were going to design a quick method for determination of antibiotic resistance gene pattern in SXT-C. MATERIALS AND METHODS: Ninety four V. cholerae O1 El Tor isolates were used in this study. Antibiotic susceptibility testing, multiplex PCR amplification of SXT-C containing dfrA1, sulII, strB and the int in a multiplex PCR were done. RESULTS: Results of our study showed that 92 (97.8%) out of 94 isolates were positive for all above genes. Multiplex PCR results correlated with the antibiotic susceptibility data obtained by using disc diffusion assay. CONCLUSION: Using this multiplex PCR, it would be easily possible to demonstrate the presence of antibiotic resistance in SXT-C which, in turn, allows for a suitable treatment in cholera patients causing reduction in the mortality and morbidity rate of the infected individuals.