Prevalence of the CRISPR-cas system and its association with antibiotic resistance in clinical Klebsiella pneumoniae isolates.

BACKGROUND AND OBJECTIVE(S): CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae. MATERIALS AND METHODS: A total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum ß-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: Phenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system's presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX-M1 (3%), blaCTX-M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2'')-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system. CONCLUSION: The presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.

Application of CAR-T cell technology in autoimmune diseases and human immunodeficiency virus infection treatment.

Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy approach that has played a tremendous role in the battle against cancer for years. Since the CAR T lymphocytes are unrestricted-major histocompatibility complex T lymphocytes, they could identify more targets than natural T cells, resulting in practical and widespread functions. The good prospects of CAR T-cell therapy in oncology can be additionally applied to treat other diseases such as autoimmune and infectious diseases. CAR-T cell-derived immunotherapy for autoimmune disorders can be allocated to CAR-Tregs and chimeric autoantibody receptor T cells. Other generations of CARs target human immunodeficiency virus (HIV) proteins. In this review, we summarize CAR-T cell therapies in autoimmune disorders and HIV infection.

Molecular diagnosis, phylogenetic analysis, and antifungal susceptibility profiles of Candida species isolated from neutropenic oncological patients.

BACKGROUND: Neutropenia is the most important cause of life-threatening invasive fungal infections (IFIs). Here, we studied the frequency and antifungal susceptibility profiles of Candida species that colonized or caused infections among neutropenic patients with solid or hematological malignancies. METHODS: A total of 362 clinical samples were collected from 138 patients. After initial isolation using a mix of mycological methods, isolates were screened using chromogenic culture media. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for molecular identification. Positive or suspected cases were confirmed using the reference method of sequencing. Antifungal susceptibility testing for voriconazole and caspofungin was carried out using the microbroth dilution method. An in-silico assay was applied for phylogenetic analysis. RESULTS: Thirty-four Candida strains were isolated. C. albicans (47.06%) and C. glabrata (29.41%) were the most frequent strains. Antifungal treatment reduced the chance of Candida colonization by almost 76% in neutropenic patients (OR: 1.759; 95% CI: 1.349 to 2.390; p value: 0.000). An unusual and non-resistant strain, C. lambica, was reported from the bloodstream of a 56-year-old man with hematologic malignancy (HM). Eight isolates were non-susceptible, and one isolate was resistant to voriconazole. Also, four isolates were non-susceptible to caspofungin. CONCLUSION: We can conclude that there is a cause-and-effect relationship between neutropenia, HM background, and Candida species separated from neutropenic patients, which can lead to possible infections. Further and repetitive studies are recommended using different molecular methods for better prediction and management of fungal infections in neutropenic patients.

Interaction Between Altered Gut Microbiota and Sepsis: A Hypothesis or an Authentic Fact?

Sepsis, as an important public health concern, is one of the leading causes of death in hospitals around the world, accounting for 25% of all deaths. Nowadays, several factors contribute to the development of sepsis. The role of the gut microbiota and the response state of the aberrant immune system is dominant. The effect of the human microbiome on health is undeniable, and gut microbiota is even considered a body organ. It is now clear that the alteration in the normal balance of the microbiota (dysbiosis) is associated with a change in the status of immune system responses. Owing to the strong association between the gut microbiota and its metabolites particularly short-chain fatty acids with many illnesses, the gut microbiota has a unique position in the research of microbiologists and even clinicians. This review aimed to analyze studies' results on the association between microbiota and sepsis, with a substantial understanding of their relationship. As a result, an extensive and comprehensive search was conducted on this issue in existing databases.

The Antibacterial Effect of Ciprofloxacin Loaded Calcium Carbonate (CaCO3) Nanoparticles Against the Common Bacterial Agents of Osteomyelitis.

The present study aimed to investigate the biocompatibility, antibacterial/anti-biofilm effects of ciprofloxacin-loaded calcium carbonate (Cip- loaded CaCO3) nanoparticles against the common organisms responsible for osteomyelitis. The antibacterial and biofilm inhibitory activities were studied by determination of minimum inhibitory concentrations (MICs) and minimum biofilm inhibitory concentrations (MBICs), respectively. Hemolytic effects were determined for studying hemocompatibility. The SDS-PAGE method was used to study the interaction of Cip- loaded CaCO3 with plasma proteins. The effects of Cip- loaded CaCO3 on the cell viability of human bone marrow mesenchymal stem cells (hBM-MSCs) was detected. The Cip- loaded CaCO3 nanoparticles were shown a significant antimicrobial effect at lower concentrations than free ciprofloxacin. No significant hemolytic effect was observed. The Cip- loaded CaCO3 nanoparticles have shown interaction with apolipoprotein A1 (28 kDa) and albumin (66.5 kDa). The viability of hBM-MSCs treated with Cip- loaded CaCO3 was more than 96%. Our results indicated that Cip-loaded CaCO3 nanoparticles had favorable in vitro compatibility with human red blood cells, antimicrobial effects, and low cytotoxicity.

Postbiotics versus probiotics in early-onset colorectal cancer.

Probiotics and postbiotics mechanisms of action and applications in early-onset colorectal cancer (EOCRC) prevention and treatment have significant importance but are a matter of debate and controversy. Therefore, in this review, we aimed to define the probiotics concept, advantages and limitations in comparison to postbiotics, and proposed mechanisms of anti-tumor action in EOCRC prevention and treatment of postbiotics. Biotics (probiotics, prebiotics, and postbiotics) could confer the health benefit by affecting the host gut microbiota directly and indirectly. The main mechanisms of action of probiotics in exerting anticancer features include immune system regulation, inhibition of cancer cell propagation, gut dysbiosis restoration, anticancer agents' production, gut barrier function renovation, and cancer-promoting agents' reduction. Postbiotics are suggested to have different mechanisms of action to restore eubiosis against EOCRC, including modulation of gut microbiota composition, gut microbial metabolites regulation, and intestinal barrier function improvement via different features such as immunomodulatory, anti-inflammatory, antioxidant, and anti-proliferative properties. A better understanding of postbiotics challenges and mechanism of action in therapeutic applications will allow us to sketch accurate trials in order to use postbiotics as bio-therapeutics in EOCRC.

Carbapenem resistance in Bacteroides fragilis: A review of molecular mechanisms.

Carbapenems are an applicable subclass of ß-lactam drugs in the antibiotic therapy of anaerobic infections, especially for poly-microbial cases, due to their broad antimicrobial spectrum on aerobic and anaerobic bacteria. Bacteroides fragilis is the most commonly recovered anaerobic bacteria in the clinical laboratories from mono- and poly-microbial infections. B. fragilis is relatively non-susceptible to different antibiotics, including ß-lactams, tetracyclines, fluoroquinolones, and macrolides. Carbapenems are among the most effective drugs against B. fragilis strains with high-level resistance to different antibiotics. Increased antibiotic resistance of B. fragilis strains has been reported following the overuse of an antimicrobial agent. Earlier contact with carbapenems is linked with increased resistance to them that limits the options for treatment of B. fragilis caused infections, especially in cases caused by multidrug-resistant strains. Several molecular mechanisms of resistance to carbapenems have been described for different carbapenem-resistant bacteria. Understanding the mechanisms of resistance to antimicrobial agents is necessary for selecting alternative antimicrobial agents and the application of control strategies. In the present study, we reviewed the mechanisms contributing to resistance to carbapenems in B. fragilis strains.

An Alliance of Carbapenem-Resistant Klebsiella pneumoniae with Precise Capsular Serotypes and Clinical Determinants: A Disquietude in Hospital Setting.

Carbapenemase-resistant Klebsiella pneumoniae (CRKP) is a genuine burden for physicians and researchers. We aimed at carbapenemase resistance and its relation with capsular serotyping in K. pneumoniae and studied some clinical determinants, which may influence the clinical infections. Initially, 61 K. pneumoniae isolates obtained from various clinical specimens were confirmed at the molecular level and then antimicrobial susceptibility test was performed followed by capsular serotyping performed by multiplex PCR. All isolates were subjected to the detection of carbapenemase genes including bla KPC, bla NDM-1, bla OXA-48, bla VIM, and bla IMP. Clinical and demographic data of all patients were reviewed including age, gender, underlying diseases, and the treatment obtained. Multidrug-resistance was a predominant feature in 77% K. pneumoniae strains. Presence of extended-spectrum beta-lactamase was detected phenotypically in 59% K. pneumoniae strains. Carbapenem resistance was noticed phenotypically in 24.6% isolates. bla OXA-48 and bla NDM-1 were the most frequent carbapenemase genes. bla NDM-1 positive isolates correlated with gentamicin, amikacin, imipenem, and meropenem resistance (p < 0.05). The nosocomial isolates mostly harbored bla OXA-48 gene (p < 0.02). Amongst all the K. pneumoniae isolates, 59% isolates could be typed and serotype K54 had the highest prevalence followed by K20 and K5. Correlation between the carbapenemase genes, serotype and type of infection showed that bla OXA-48 positive strains had a significant association with K20 serotype and urinary tract infections (p=0.2) while, K20 serotype and bla KPC positive strains were significantly associated with wound infections (K20, p=0.3 and bla KPC, and p=0.4). Mucoid phenotype was not found related to presence of specific carbapenemase genes or serotypes except serotype K20 (p < 0.001). Patients with monotherapy had treatment failure in comparison to the combination therapy for bla KPC-associated infections. In conclusion, the present investigation exhibited the significant association between K20 serotype with bla OXA-48. The predominance of K54 reveals the possibility of endemicity in our hospital setting. K. pneumoniae isolated from wound specimens significantly harbors K20 serotype and bla KPC gene. Comprehensive clinical information and the distribution of antibiotic resistance genes, and serotypes may play important roles in the treatment process.

Contribution of Arginine Catabolic Mobile Element and Copper and Mercury Resistance Element in Methicillin-Resistant Staphylococcus aureus: A Vantage Point.

Different clones of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are dominating geographically. One of the significant, hypervirulent, CA-MRSA and a significant health concern clones is USA3000, found worldwide regionally with varying frequencies. The clone harbors several mobile genetic elements (MGEs) including, arginine catabolic mobile element (ACME) and copper and mercury resistance genes (COMER), accomplished by horizontal gene transfer from S. epidermidis. Evidence suggests that ACME and COMER have a more prominent role in enhancing biofilm capacity and ultimately persistent infections. This review highlights the comprehensive view on ACME and COMER structure, their distribution, and the mechanism of action along with pathogenetic features of USA3000 encompassing their role in biofilm formation, adhesion, quorum sensing, resistance to antibiotics, chemotaxis, and nutrient uptake. We also provided an insight into the role of ACME and COMER genes in the survival of bacterium. Our results shed light on the emergence of two independent clones possessing ACME (North American) and COMER (South American) elements which later disseminated to other regions. ACME and COMER both are adjacent to staphylococcal cassette chromosome mec type IV (SCCmec IV). The acquisition of mecA, followed by COMER or ACME has been shown as a significant factor in the rise and fall of MRSA strains and their complex ability to adapt to hostile environments. The presence of ACME increases fitness, thereby allowing bacteria to colonize the skin and mucous membrane while COMER contributes to genetic stability by knocking over the copper-mediated killing in macrophages. Evidence suggests that ACME and COMER have a more prominent role in enhancing biofilm capacity and ultimately persistent infections. Interestingly, ACME strains have been shown to possess the ability to counteract skin acidity, thereby allowing increased skin colonization. A profound understanding of MGEs in S. aureus plays an important role in the prevention of epidemic clones.

Induction of proteome changes involved in biofilm formation of Enterococcus faecalis in response to gentamicin.

BACKGROUND: Enterococcus faecalis is a significant cause of nosocomial infections and other diseases, including endocarditis, bacteremia, and urinary tract infections. This microorganism forms biofilms to overcome difficult environmental conditions, such as lack of oxygen, lack of water, and the presence of antimicrobials. These biofilms make diseases difficult by changing their proteome contents, protecting the bacterium, and increasing their pathogenicity. This study aimed to evaluate gentamicin's effect on proteome changes and biofilm formation in E. faecalis. METHOD: Twenty-five clinical isolates and one standard isolate were selected for the experiments. A label-free/gel-free proteomic and microtiter plate techniques were used to study proteome changes and biofilm formation, respectively. RESULTS: Gentamicin significantly increased the biofilm formation in 62% of isolates and the rest of the isolates; no significant change was observed. The abundance of lactate utilization protein C, ribosomal RNA small subunit methyltransferase H, and protein translocase subunit SecA were increased. However, the abundances of proteins effective in cell division and metabolism, such as replication initiation protein and segregation and condensation protein A, were decreased. CONCLUSION: The present study's findings exhibited that antibiotics might have adverse effects on treatment and increase microorganisms' pathogenicity. It was observed in gentamicin as induction of biofilm formation through different mechanisms, particularly changes in the expression of specific proteins in E. faecalis.

Comparison of superantigens and attachment factors genes of Staphylococcus aureus in clinical isolates and nasal colonizers in the same patients.

BACKGROUND: Staphylococcus aureus (S. aureus) is a bacterial pathogen can cause a wide range of nosocomial infections. Nasal colonization by S.aureus plays important role both in the epidemiology and pathogenesis of infection. OBJECTIVES: The purpose of this study was to investigate the association of clinical isolates and nasal colonizers of S. aureus in the same patients by molecular methods, and their antibiotic susceptibility pattern. METHODS: A total of 181 S. aureus isolates were collected from 100 patients admitted that including 100 clinical isolates and 81 nasal swabs from the same patients (19 cases were found as noncarriers). Superantigens and adhesion genes were identified by PCR. Molecular typing of the isolates was performed by repetitive element polymerase chain reaction (Rep-PCR). Antimicrobial susceptibility pattern of the isolates was conducted by disk diffusion. MIC of the isolates to vancomycin was determined by microbroth dilution. The ability of S. aureus isolates to form biofilm was determined by microtiter plate assay. RESULTS: The most frequent adhesion gene in both clinical isolates and nasal colonizer was clfA with 93% and 76%, respectively. Staphylococcal enterotoxin A (SEA) was the most commonly superantigen (68%) in both nasal colonizers (71.6%) and clinical isolates (65%). The highest resistance rate was to erythromycin (45.3%) with 36% and 56.8% in clinical and nasal colonizer isolates, respectively. All S. aureus isolates were susceptible to linezolid and vancomycin. Multiple drug resistance (MDR) was detected in 36% (n = 65) of the isolates. Biofilm formation was identified in 160 (88.4%) isolates with 87% and 90% in clinical isolates and nasal colonizers, respectively. Repetitive element polymerase chain reaction (Rep-PCR) typing divided 181 S. aureus isolates into six clusters. Twelve isolates from clinical isolates and nasal carriers were closely related. CONCLUSION: There is a high concordance rate between colonizing and clinical isolates of S. aureus in terms of adhesion factors and superantigen genes. It is suggested that nasal decolonization could be effective in the preventing of S. aureus infections.

Role of CRISPR-Cas system on antibiotic resistance patterns of Enterococcus faecalis.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.

Evaluation of Human Respiratory Syncytial Virus and Human Parainfluenza Virus Type 3 among Hospitalized Children in Northwest of Iran.

BACKGROUND: Acute respiratory tract infections (ARTIs) are the leading cause of illnesses in children. Human respiratory syncytial virus (HRSV) and human parainfluenza viruses (HPIVs) are among the most common etiologic agents associated with viral respiratory tract infections in children worldwide. Nevertheless, limited information is available on the spread of infections of these two viruses in northwest Iran. OBJECTIVE: The purpose of the current study is to evaluate the frequency of RSV and HPIV-3 and clinical features among Iranian children with confirmed respiratory infections between April 2019 and March 2020. METHODS: 100 nasopharyngeal swabs were collected from hospitalized patients (under 5 years old) with ARTI from Tabriz Children's Hospital. Detection of respiratory viruses was performed using the nested RT-PCR method. RESULTS: Respiratory syncytial virus and HPIV-3 were recognized in 18% (18/100) and 2% (2/100) of children, respectively. Ten (55.6%) of the RSV-positive samples were male, while 8 (44.4%) were female. HPIV-3 was found only among 2 male patients (100%). Most patients (61.1%) with RSV infection were less than 12 months old. Additionally, samples that were positive for HPIV-3 were less than 12 months old. RSV infections had occurred mainly during the winter season. CONCLUSIONS: This study confirms that RSV can be one of the important respiratory pathogens in children in northwestern Iran. However, according to this study, HPIV-3 has a lower prevalence among children in this area than RSV. Therefore, implementing a routine diagnosis for respiratory pathogens can improve the management of respiratory infections in children.

Mucosa-Associated Escherichia coli in Colorectal Cancer Patients and Control Subjects: Variations in the Prevalence and Attributing Features.

Accumulating evidence indicates that specific strains of mucosa-associated Escherichia coli (E. coli) can influence the development of colorectal carcinoma. This study aimed to investigate the prevalence and characterization of mucosa-associated E. coli obtained from the colorectal cancer (CRC) patients and control group. At two referral university-affiliated hospitals in northwest Iran, 100 patients, 50 with CRC and 50 without, were studied over the course of a year. Fresh biopsy specimens were used to identify mucosa-associated E. coli isolates after dithiothreitol mucolysis. To classify the E. coli strains, ten colonies per sample were typed using enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR). The strains were classified into phylogroups using the quadruplex PCR method. The PCR method was used to examine for the presence of cyclomodulin, bfp, stx1, stx2, and eae-encoding genes. The strains were tested for biofilm formation using the microtiter plate assay. CRC patients had more mucosa-associated E. coli than the control group (p < 0.05). Enteropathogenic Escherichia coli (EPEC) was also found in 23% of CRC strains and 7.1% of control strains (p < 0.05). Phylogroup A was predominant in control group specimens, while E. coli isolates from CRC patients belonged most frequently to phylogroups D and B2. Furthermore, the frequency of cyclomodulin-encoding genes in the CRC patients was significantly higher than the control group. Around 36.9% of E. coli strains from CRC samples were able to form biofilms, compared to 16.6% E. coli strains from the control group (p < 0.05). Noticeably, cyclomodulin-positive strains were more likely to form biofilm in comparison to cyclomodulin-negative strains (p < 0.05). In conclusion, mucosa-associated E. coli especially cyclomodulin-positive isolates from B2 and D phylogroups possessing biofilm-producing capacity colonize the gut mucosa of CRC patients.

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.

Comparative analysis of Enterococcus faecalis and Enterococcus faecium strains isolated from clinical samples and traditional cheese types in the Northwest of Iran: antimicrobial susceptibility and virulence traits.

This research was conducted using 50 samples of popular traditional cheeses and 160 enterococcal clinical isolates. Phenotypic and genotypic methods used for identification of enterococci. Then, the incidences of antibacterial resistance and virulence traits were investigated. In total, 165 E. faecalis and 43 E. faecium obtained from traditional cheeses and different clinical isolates were analyzed in the study. Antibiotic susceptibility testing revealed 175(84.1%) isolates with multi-drug resistance (MDR) patterns, which was more common among clinical sources. The predominant virulence profile, including gelE, asa1 and cpd was detected within 47 (22.6%) of the MDR isolates. Our results showed that traditional cheeses and clinical E. faecalis isolates have distinct patterns of virulence traits. The identified enterococci with antibiotic resistance and associated virulence factors, could provide a potential risk to the public health.

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.

Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran.

Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes in animal-source foods (ASF). Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates. Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples. Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.

Effect of acidic and alkali shocks on expression of efaA gene in Enterococcus faecalis, isolated from root canal infection.

Enterococcus faecalis is an important opportunistic infectious agent involving the oral cavity and endodontics. The aim of this study was to evaluate the expression ratio of efaA gene in biofilm producer E. faecalis before and after receiving acidic and alkali shocks. One hundred E. faecalis isolates were gathered from 170 infectious root canals. After analysis of biofilm formation by the Microtiterplate method, the presence of efaA gene was examined by PCR and its expression was evaluated by Real-time PCR, one before applying any stressed to isolates and another by applying acidic and alkali shock. Chi-square method was used for statistical analysis. Eighty-two percent of samples had efaA gene. Evaluation of biofilm formation, 49% of the isolates were strong biofilm producer, 42% moderate and 10 % of them had no biofilm. 59% overexpression of efaA gene was observed in biofilm producer isolates, while there were no significant changes in samples with acidic stress and decreased expression after alkali shock. Findings of the present study, indicates importance of efaA gene in biofilm formation and pathogenesis of E. faecalis. Acid had no effect of expression of this gene but alkali reduced expression of this gene in a significant level. These results indicate the importance of efaA and acidic conditions in biofilm production by E. faecalis.