Sub-minimum inhibitory concentrations (sub-MICs) of colistin on Acinetobacter baumannii biofilm formation potency, adherence, and invasion to epithelial host cells: an experimental study in an Iranian children's referral hospital.

Here, we described the efficacy of colistin sub-minimum inhibitory concentrations (sub-MICs) on biofilm-forming activity, host epithelial cell adherence, and invasion capacity of Acinetobacter baumannii strains collected from children admitted to the Children's Medical Center Hospital. Biofilm formation potency of A. baumannii clinical isolates was measured using a 96-well microtiter plate assay. Distribution of biofilm-related genes, including bap, abaI, ompA, csuE, and blaPER-1, was detected by PCR. The mRNA expression level of ompA and csuE was measured by qPCR in the presence of » and ½ MICs of colistin. A. baumannii adhesion and invasion to eukaryotic host cells were phenotypically assayed at sub-MICs of colistin. Eighty percent (56/70) and 35.7% (25/70) of A. baumannii isolates were multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes, respectively. The strong, moderate, and weak biofilm producers of A. baumannii were 37.1% (26/70), 32.8%, (23/70), and 22.8% (16/70), respectively. The frequencies of biofilm-associated genes were 100% for abaI, ompA, and csuE, followed by 22.8% (16/70) and 24.3% (17/70) for bap and blaPER-1, respectively. The downregulation of csuE and ompA expression levels was observed in the sub-MIC of colistin. In vitro cell culture study showed a decreased capability of A. baumannii to adhere to the human epithelial cells at sub-inhibitory doses of colistin; however, none of the isolates could invade HEp-2 cells. Our study showed that the genes encoding biofilm-associated proteins undergo downregulation in expression levels after exposure to sub-MICs of colistin in A. baumannii. Longitudinal in vivo studies are needed to fully understand the clinical aspects of pathogenicity mechanisms and evolutionary dynamics of drug resistance.IMPORTANCESince the toxicity of colistin is dose dependent, there is a focus on strategies that reduce the dose while maintaining the therapeutic effect of the drug. Our findings about sub-inhibitory doses of colistin provide a novel insight into the logical use of colistin to treat and control Acinetobacter baumannii-related infections in clinical practice.

Invasion of HeLa Cells by Shigella Species Clinical Isolates Recovered from Pediatric Diarrhea.

Shigella is considered a major public health concern, especially for children younger than 5 years of age in developing countries. The pathogenicity of Shigella is a complex process that involves the interplay of multiple genes located on a large, unstable virulence plasmid as well as chromosomal pathogenicity islands. Since various factors (including virulence and antibiotic resistance genes) are associated with the severity and duration of shigellosis, in this article, we aim to evaluate whether the invasion of HeLa cells is affected by Shigella spp. isolates with different characteristics (including serogroups, virulence gene profiles, and antibiotic resistance patterns) recovered from pediatric patients in Tehran, Iran. Cell invasion ability of 10 Shigella isolates with different serogroups (Shigella flexneri and Shigella sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin) were measured by plaque-forming assay in HeLa cell lines. The results show that all the selected Shigella spp. isolates recovered from pediatric patients were able to invade HeLa cells, but the total number and average size of plaques were different between the isolates. The higher invasion ability of S. flexneri isolates in HeLa cells compared to S. sonnei isolates was attributed to the presence of particular virulence genes; however, the role of each of these virulence factors remains to be determined.

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis.

BACKGROUND: The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. METHODS: MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. RESULTS: A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. CONCLUSIONS: The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells.

BACKGROUND: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1ß and TNF-α cytokines was measured by ELISA. RESULTS: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-ß as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). CONCLUSION: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Characterization of Sequence Types and Mechanisms of Resistance to Tigecycline Among Acinetobacter baumannii Isolated from Children.

The present study aimed to investigate the mechanisms of resistance to tigecycline and to determine sequence types of Acinetobacter baumannii isolates recovered from children, using the Multilocus Sequence Typing (MLST). A total of 74 A. baumannii isolates were recovered from patients at one of the children's hospital in Tehran, Iran. Antimicrobial susceptibility testing of the isolates was performed for different classes of antibiotics and minimum inhibitory concentrations of colistin and tigecycline were determined using broth microdilution method and E-test strips, respectively. The presence of ISAba1, AbaR, tet(39), and tetX and the expressions of adeB, adeG, and adeJ efflux pump genes were measured using Polymerase Chain Reaction (PCR) and quantitative real-time PCR (RT-PCR), respectively. The diversity of mutations across the regulatory genes of RND efflux pumps (adeRS, adeL, and adeN) and trm gene were determined using their PCR amplification and DNA sequencing in tigecycline-resistant isolates. In addition, STs of tigecycline-resistant isolates were determined using MLST method. Three A. baumannii isolates were resistant to tigecycline. Several amino acid substitutions were identified in AdeRS, AdeN, and Trm but no alteration was found in AdeL. Nevertheless, adeB, adeG, and adeJ overexpression were observed in 1, 2, and 1 isolates, respectively. The tigecycline-resistant isolates belonged to ST1720 and ST2285. This is the first study reporting on ST2285 in A. baumannii populations. Among 74 isolates, two tigecycline susceptible isolates carried tet(39) gene but no tetX gene was detected. We concluded that mutations in regulatory genes of RND efflux pumps and the trm gene may play some important role in A. baumannii resistance to tigecycline.

Prevalence of ß-lactamase-encoding genes and molecular typing of Acinetobacter baumannii isolates carrying carbapenemase OXA-24 in children.

BACKGROUND: ß-Lactam antibiotics have been broadly used for the treatment of Acinetobacter baumannii infections, resulting in development of ß-lactam inactivating ß-lactamases. Here, we described antibiotic resistance rate, prevalence of ß-lactamase-encoding genes, and clonal relationships of A. baumannii strains isolated from children referred to Children's Medical Center in Tehran, Iran, during 2019-2020. METHODS: A total of 60 non-replicate A. baumannii isolates were recovered from clinical specimens of pediatric patients. Antibiotic susceptibility testing was done by the disc diffusion method. Colistin susceptibility of isolates was performed by the broth microdilution method. ß-lactamase-encoding genes were characterized by PCR. The presence of ISAba1 element upstream of the several oxacillinase genes was also checked. Genetic relatedness of isolates was determined by using random amplification of polymorphic DNA (RAPD) typing. RESULTS: The antimicrobial susceptibility tests showed that 83.3% of A. baumannii isolates were MDR, and 40% XDR. Both MDR and XDR A. baumannii isolates were susceptible to colistin. The frequency of blaOXA-51-like, blaOXA-23-like, blaTEM, blaOXA-24-like, blaPER, blaSHV, blaCTX-M, blaOXA-58-like, and blaIMP was 100, 93.33, 60, 36.67, 28.33, 8.33, 5, 3.33, and 1.67%, respectively. Coexistence of ISAba1/blaOXA-23-like and ISAba1/blaOXA-51-like was observed in 65% and 85% of isolates, respectively. RAPD analysis revealed 4 common types and 2 single types of A. baumannii isolates. CONCLUSIONS: The multiple clones harboring blaOXA-23-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23-like were responsible for the spread of A. baumannii isolates in our clinical wards. Dissemination of the well-established clones is worrisome and would become therapeutic challenges due to the possible transferring genetic elements associated with resistance.

Detection of Enterovirus, Herpes Simplex, Varicella Zoster, Epstein-Barr and Cytomegalovirus in cerebrospinal fluid in meningitis patients in Iran.

BACKGROUND: Despite medical advances, central nervous system (CNS) diseases put a pressure on the health care system. A number of risk factors, especially infectious agents can accelerate the progression of meningitis. As viruses probably account for most cases of meningitis, the diagnosis of them can reduce antibiotic prescriptions. Among various types of infectious diseases, the relationship between two important virus families, including Picornaviridae and Herpesviridae, and meningitis has attracted attraction. METHODS: In this study, one hundred and two samples were collected from patients who experienced symptoms, such as the loss of consciousness, seizures, muscle weakness, fever, headache, rash, and severe dementia, between November 2018 and September 2019. After RNA and DNA extraction, the prevalence of Enterovirus (EV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2), and Varicella zoster virus (VZV) was evaluated using PCR, multiplex PCR, and nested PCR. RESULTS: Results indicated that there were two VZV DNA-positive specimens, while six and five samples were infected with HSV-1 and EBV, respectively. CONCLUSION: We reported that the prevalence of EBV, HSV-1, and VZV in patients, suffering from meningitis cannot be ignored; however, further investigation is needed.

Genetic diversity and phylogenetic analysis of the surface layer protein A gene (slpA) among Clostridioides difficile clinical isolates from Tehran, Iran.

Clostridioides difficile is the most common causative agent of healthcare-associated diarrhea. C. difficile strains produce a crystalline surface layer protein (SlpA), encoded by the slpA gene. Previous studies have shown that SlpA varies among C. difficile strains. In this study, we used the SlpA sequence-based typing system (SlpAST) for the molecular genotyping of C. difficile clinical isolates identified in Iran; the PCR ribotypes (RTs) and toxin profiles of the isolates were also characterized. Forty-eight C. difficile isolates were obtained from diarrheal patients, and characterized by capillary electrophoresis (CE) PCR ribotyping and the detection of toxin genes. In addition, the genetic diversity of the slpA gene was investigated by Sanger sequencing. The most common RTs were RT126 (20.8%), followed by RT001 (12.5%) and RT084 (10.4%). The intact PaLoc arrangement representing cdu2+/tcdR+/tcdB+/tcdE+/tcdA+/tcdC+/cdd3+ profile was the predominant pattern and cdtA and cdtB genes were found in one-third of the isolates. Using the SlpA genotyping, 12 main genotypes and 16 subtypes were identified. The SlpA type 078-1 was the most prevalent genotype (20.8%), and identified within the isolates of RT126. The yok-1, gr-1, cr-1 and kr-3 genotypes were detected in 14.5%, 12.5%, 12.5% and 8.3% of isolates, respectively. Almost all the isolates with the same RT were clustered in similar SlpA sequence types. In comparison to PCR ribotyping, SlpAST, as a simple and highly reproducible sequenced-based technique, can discriminate well between C. difficile isolates. This typing method appears to be a valuable tool for the epidemiological study of C. difficile isolates worldwide.

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii.

BACKGROUND: This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. METHODS: Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAßN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR). RESULTS: Overall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected. CONCLUSIONS: Our findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

Involvement of the AcrAB Efflux Pump in Ciprofloxacin Resistance in Clinical Klebsiella Pneumoniae Isolates.

BACKGROUND: Increasing prevalence of multiple antibiotic resistance in Klebsiella pneumoniae strains confines the therapeutic options used to treat bacterial infections. OBJECTIVE: We aimed in this study to investigate the role of AcrAB and qepA efflux pumps and AAC(6')-Ib-cr enzyme in ciprofloxacin resistance and to detect the RAPD-PCR fingerprint of K. pneumoniae isolates. METHODS: A total of , 117 K. pneumoniae isolates were collected from hospitalized patients in three hospitals in Tehran, Iran, from August 2013 to March 2014. Antimicrobial susceptibility tests were performed by the disk diffusion method. Molecular identification and expression level of encoding quinolone resistance genes, acrA, acrB, qepA, and aac(6')-Ib-cr, were performed by PCR and real-- time PCR assays, respectively. All the K. pneumoniae isolates containing the mentioned genes were used simultaneously for RAPD-PCR typing. RESULTS: Colistin and carbapenems were the most efficient antibiotics against the clinical isolates of K. pneumoniae. PCR assay demonstrated that among the 117 isolates, 110 (94%) and 102 (87%) were positive for acrA and acrB gene and 5 (4%) and 100 (85%) isolates showed to have qepA and aac(6')-Ib-cr genes, respectively. Determination for AcrAB pump expression in 21% of strains demonstrated an increased expression, and the mean increase expression for acrB genes was 0.5-81. The results of RAPD-PCR reflected that in 95% CI, all isolates belonged to a clone. CONCLUSION: A high prevalence of genes encoding quinolone resistance in K. pneumoniae was detected in clinical samples. Therefore, the control of infection and prevention of drug-resistant bacteria spread need careful management of medication and identification of resistant isolates.

Molecular characterization of pathogenicity locus (PaLoc) and tcdC genetic diversity among tcdA+B+Clostridioides difficile clinical isolates in Tehran, Iran.

Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs). Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene. Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene. The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/toxinotype 0 and the RT 126/toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

Characterization of biofilm formation and virulence factors of Staphylococcus aureus isolates from paediatric patients in Tehran, Iran.

OBJECTIVES: Staphylococcus aureus can cause several infections. Its capability to form biofilm has been reported to be a vital property involved in the bacteria's pathogenesis. Various genes contributing to biofilm formation have not yet been completely clarified. This study was designed to evaluate the factors influencing adherence and biofilm formation in S. aureus isolated from paediatric patients. MATERIALS AND METHODS: One hundred and ninety-seven S. aureus isolates were obtained from pediatric patients and confirmed with phenotypic and molecular examinations. Antimicrobial susceptibility testing and biofilm formation were evaluated using standard methods. The genes encoding adhesion and virulence factors were investigated by the PCR method. RESULTS: The most efficient antibiotics against S. aureus isolates were vancomycin and linezolid. Approximately, 54.2% of MSSA and 85.6% of MRSA isolates were biofilm producers according to the microtiter test. Our analysis indicated that MRSA isolates are better able to form biofilm compared with MSSA isolates. All isolates harbored clfA, fnbpA, icaA, icaB, icaC, and icaD, while clfB, fnbB, hlg, and pvl were detected in 99.5%, 42.1%, 97.5%, and 5.6% of isolates, respectively. In addition, a significant difference was found in fnhB gene and biofilm formation. CONCLUSION: Our findings showed a significant correlation between mecA and pvl genes and MRSA and biofilm formation in S. aureus isolates. Additionally, this study indicated the significant role of the fnhB gene as a major marker for S. aureus biofilm formation. Therefore, further experiments are warranted to exactly elucidate the function of the fnhB gene in the formation of biofilm.

Comparing open conventional carpal tunnel release with mini-incision technique in the treatment of carpal tunnel syndrome: A non-randomized clinical trial.

BACKGROUND: Carpal tunnel syndrome (CTS) is the most common nerve entrapment neuropathy which is the result of the compression of the median nerve in the wrist. Currently, there is no consensus about the best treatment option. The purpose of this clinical trial was to compare the clinical outcomes of patients undergoing open CT release with mini-incision CT release. PATIENTS AND METHODS: This clinical trial included 75 patients with CTS who were divided into two groups of 45 and 30 patients to undergo open-CT release or mini incision CT release respectively. Patients were evaluated pre-operatively, days after the surgery and then five months after the operation to record outcomes. At follow-up, the visual analogue scale (VAS) scores for pain, patients' satisfaction, return to work, length of scar, paresthesia, grip and opposition strength were measured. RESULTS: A total of 75 patients (mean age: 52.13 years, 73.3% female) underwent CTS surgery. Forty-five patients (60%) had open-CT release and 30 patients (40%) had mini-incision CT release. Postoperative pain and scar length were significantly lower in the mini incision group compared to open group (p < 0.001). The mini-incision CT group returned to work earlier than open group with higher satisfaction (p < 0.001). No significant differences were observed between two groups in respect to the improvement of the opposition, grip and paresthesia (p > 0.05). CONCLUSION: Our study demonstrated that mini-incision CT release improves pain more effectively and has better quality of life because of smaller length of scar, immediate return to work and higher overall satisfaction. Neurosensory and motor improvements were also seen in both techniques with the same clinical impact.

Chronic Suppurative Otitis Media: A Case Report.

BACKGROUND CONTEXT: Chronic Suppurative Otitis Media (CSOM) is a common cause of hearing impairment and disability. CSOM caused by Pseudomonas aeruginosa is usually treated with topical ciprofloxacin and resistance to ciprofloxacin in CSOM isolates has rarely been reported. CASE PRESENTATION: A 24-year-old male patient with CSOM due to p. aeruginosa was reported. CSOM was prolonged for ten years and physician prescribed topical ciprofloxacin drops, pus suctioning and ear pH alteration. The treatment wasn't effective and the patient came back to the clinic with relapse of suppurative otitis media. P. aeruginosa was isolated as the cause of CSOM and the isolate was resistant to ciprofloxacin, aztreonam, imipenem, gentamicin, doripenem, cefepime, levofloxacin, amikacin and susceptible to colistin and ceftazidime. There were two mutations in gyrA and eight mutations were observed in nfxB genes. Finally, tympanomastoidectomy was done. CONCLUSION: Usually topical antibiotics, especially ciprofloxacin, is effective against ear infections but our case was different and the P. aeruginosa isolated from CSOM was resistant to most of the antibiotics. One reason for CSOM recurrence might be surgery failure. The routine and primary treatment for CSOM did not seem sufficient and tympanomastoidectomy is suggested to be the best treatment approach for these patients.

High Rates of Antimicrobial Resistance and Virulence Gene Distribution Among Shigella spp. Isolated from Pediatric Patients in Tehran, Iran.

BACKGROUND: Shigella continues to be important causes of acute pediatric diarrhea worldwide. Shigella produces numerous virulence factors involved in colonization and invasion into epithelial cells which eventually result in the disease. The present study was conducted to evaluate the prevalence of virulence genes and to investigate antibiotic resistance profiles among Shigella isolates obtained from pediatric patients in Iran. METHODS: A total of 141 Shigella isolates were collected between March 2017 and September 2018 from stool of children under 14 who were suspected to have shigellosis. Shigella isolates were identified using standard microbiological and serological tests and antimicrobial susceptibility testing was carried out via Kirby-Bauer disk diffusion method. In addition, the presence of seven virulence determinants including ipaH, ipaB, ipaC, ipaD, ipgD, sen, and virA were evaluated using PCR. RESULTS: S. sonnei (78.7%) was the most prevalent shigella spp. among children with shigellosis followed by S. flexneri (19.9%) and S. boydii (1.4%). Antimicrobial susceptibility testing revealed that most of the isolates were considered as multidrug-resistant (MDR) strains. Our findings also showed a high resistance rate against trimethoprim-sulfamethoxazole in Shigella isolates. The prevalence of ipaH, ipaC, sen, ipaD, virA, ipaB, and ipgD were 100%, 95.7%, 95.7%, 94.3%, 93.6%, 92.9%, and 80.8%, respectively. CONCLUSION: The current study revealed that S. sonnei was the predominant species isolated from children with shigellosis in Iran. Our results also indicated a high distribution of type III secretion system effector protein-encoding genes and high multidrug-resistance among shigella spp. in Iran. Therefore, it is suggested that antimicrobial susceptibility testing be performed prior to antibiotic prescription.

Antimicrobial Resistance and Molecular Characteristics of Methicillin-resistant Staphylococcus aureus Isolates from Children Patients in Iran.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) causes high rates of mortality and a substantial burden to health systems worldwide. Here, we investigated the antimicrobial susceptibility and molecular characteristics of MRSA isolated from children referred to Children's Medical Center in Tehran. MATERIALS AND METHODS: A total of 98 MRSA isolates were collected from children. Antimicrobial resistance patterns were determined using the disk diffusion and E-test methods. The presence of biofilm encoding genes and the pvl gene were determined by PCR. We used the microtiter plate method to assess the ability of biofilm formation. The MRSA isolates were further analyzed using PFGE and SCCmec typing. RESULTS: Antibiotic susceptibility testing showed that the highest and the lowest antibiotic resistance percentage were related to erythromycin (62%) and minocycline (10%), respectively. Overall, 63% of MRSA isolates were biofilm producers. Resistance to two antibiotics such as erythromycin (72% vs 28%, P=0.01) and clindamycin (71% vs 29%, P=0.04) was higher among biofilm producers than non-biofilm producers. All strains had biofilm-forming genes and the prevalence of pvl gene was 41%. Most MRSA isolates belonged to SCCmec IVa (75%) and SCCmec III (18%). In PFGE technique, 5 common types and 2 single types were identified; Common type 1 with 37 isolates was dominant clone. CONCLUSION: We thus report preliminary data on the prevalence and distribution of MRSA genotypes in Tehran Children's Hospital. These findings characterize the MRSA colonization dynamics in child patients in Iran and may aid the design of strategies to prevent MRSA infection and dissemination.

Transcriptional Regulation of Epithelial to Mesenchymal Transition Related Genes by Lipopolysaccharide in Human Cervical Cancer Cell Line HeLa.

Objective: Cancer is one of the common diseases in the world, and cervical cancer is the fourth one. In this type of cancer, many risk factors, especially infectious diseases, such as human papilloma virus (HPV) and gram-negative bacteria can have important effects on the expression of epithelial to mesenchymal transition related genes like Snail, E-cadherin, and ZEB-1, responsible for connecting cell tissues. In this study, we have investigated the effect of Escherichia coli O111:B4 Lipopolysaccharide (LPS) on HPV positive cell line (HeLa), the expression level of the (Snail, E-cadherin, and ZEB-1), HPV oncogenes (E6, E7) and also microRNA-9, 192. Materials and Methods: HeLa cell line was treated with LPS to analyze Snail, E-cadherin, ZEB-1, E6, E7 and also microRNA-9, 192 expression by quantitative real-time PCR in 24, 48 and 72 hours. Results: Quantitative real-time PCR revealed a significant reduction in E-cadherin mRNA level at 10ug/L of LPS in three time-points and after 24 hours at 5ug/L of LPS; however, ZEB-1 at 10ug/L of LPS and Snail at 5, 10ug/L of LPS are up-regulated. E7 also illustrated a slight increase, but we did not find any relationship between E7 and LPS treatment. Additionally, there are upward trends in microRNA-9, 192 levels. Conclusion: The result of this study, LPS is able to reduce E-cadherin expression, caused by increase in repressor E-cadherin protein expression and some microRNAs, probably. Since bacterial infection can be in cervical site, it is likely to be effective in reducing the E-cadherin expression in the EMT and enhance cancer process, therefore; removing these infections by using the appropriate antibiotics may result in slowing down this process, which requires more research.

Clonal dissemination of Staphylococcus aureus isolates causing nosocomial infections, Tehran, Iran.

OBJECTIVES: In the current research, the prevalence of Staphylococcus aureus clones and genes encoding antimicrobial resistance and toxins were examined among 120 S. aureus strains from nosocomial infections in tehran, Iran. MATERIALS AND METHODS: Antimicrobial susceptibility was examined, based on disk diffusion and PCR method to identify resistance and toxin-encoding genes. Based on the polymorphisms in SCCmec, agr, spa, and MLST, the isolates were typed. RESULTS: Among 120 S. aureus isolates, 85 (70.8%) were methicilin resistant S. aureus (MRSA), and 35 (29.2%) were methicilin sensetive S. aureus (MSSA). The tested isolates contained resistance genes, including ant(4΄)-Ia (90%), aac(6΄)-Ie/aph(2˝) (80%), aph(3΄)-IIIa (30%), erm(A) (26.7%), erm(B) (10.8%), erm(C) (11.7%), msr(A) (40.8%), msr(B) (14.2%), tet(M) (45.8%), and mupA (8.3%). The MRSA strains were clustered into six different clones. The most common genotypes included ST239-SCCmec III/t037 (23.3%), ST239-SCCmec III/t388 (22.5%), ST22-SCCmec IV/t790 (8.3%), ST15-SCCmec IV/t084 (7.5%), ST585-SCCmec III/t713 (5%), and ST239-SCCmec III/t924 (4.2%), respectively. ST182/t196 (8.3%) and ST123/t171 (5%) belonged exclusively to MSSA strains. Overall, 10 (66.7%) and 5 (33.3%) out of 15 isolates with pvl genes were attributed to clones ST22-SCCmec IV/t790 and ST15-SCCmec IV/t084, respectively. ST22-SCCmec IV/t790, ST239-SCCmec III/t037, and ST15-SCCmec IV/t084, were related to high-level mupirocin-resistant phenotypes. CONCLUSION: The genetic diversity of S. aureus was confirmed in our hospitals, and ST239-SCCmec III/t037 showed a relatively high prevalence in our study. It seems that assessment of resistance and virulence genes in different S. aureus molecular types is necessary for proper antibiotic consumption.

Genotype Distribution of Panton-Valentine Leukocidin (PVL)-Positive Staphylococcus aureus Strains Isolated from Wound-Related Infections: a Three-Year Multi-Center Study in Tehran, Iran.

The spread of Panton-Valentine leucocidin (PVL)-carrying S. aureus strains in patients with wound infections in both the community and hospitals is increasing in some areas of Iran. In the present study, we determined the molecular characteristics and distribution of PVL-producing S. aureus strains isolated from wound infections. Genes encoding resistance, toxins, and staphylococcal enterotoxins were analyzed by polymerase chain reaction assays. Genotyping was performed using multi-locus sequence typing. Aminoglycoside resistance genes including ant (4')-Ia (57.4%) and aac (6')-Ie/aph (2″) (45.7%) were the most prevalent genes in isolates. Staphylococcal enterotoxin type A, as the most frequent type, was present in 20.2% of isolates. Strains belonged to seven clonal complexes. The most frequent clonal complex was CC30 (ST30) (29.8%), followed by CC22 (ST22) (21.3%), CC8 (ST8 and ST931) (17%), CC88 (ST88) (10.6%), CC59 (ST59 and ST338) (8.5%), CC1 (ST772 and ST1) (7.5%), and CC15 (ST15) (5.3%). Our findings indicated an increasing trend of CC30, carrying a wide range of resistance and toxin genes, which could present an obstacle in the treatment of patients with wound infections. Further studies are required to investigate the carriage of resistance, the antibiotic susceptibility pattern, and toxins encoding genes in different molecular types.

Molecular analysis of accessory gene regulator functionality and virulence genes in Staphylococcus aureus derived from pediatric wound infections.

Staphylococcus aureus is a major human pathogen causing infections with high morbidity and mortality in both healthcare and community settings. The accessory gene regulator (Agr) is a key genetic element controlling the expression of numerous virulence factors in S. aureus. The significance of a functional Agr system in clinical S. aureus isolates derived from pediatric wound infections is still unclear. Therefore, the present study was conducted to identify virulence genes and determine Agr functionality from this cohort of patients. A total of 48 S. aureus wound isolates were collected from patients referred to Tehran Children's Medical Center Hospital from April 2017 to April 2018. In addition, in vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion and E-test methods. Conventional PCR was performed for the detection of toxins (tsst-1, hla, hlb, hld, eta, etb, etd, edin-A, edin-B, edin-C) and Agr typing (agrI, agrII, agrIII, agrIV). Agr functionality was assessed by quantitative reverse transcriptase real-time PCR (qRT-PCR). All S. aureus isolates were found to be susceptible to linezolid and vancomycin. The most frequently detected toxin gene was eta (100%), and the most prevalent Agr type was agrIII (56.3%). Importantly, qRT-PCR revealed that Agr was functional in 28 (58%) of wound isolates. Consequently, our data suggests that a functional Agr system may not be required for the development of S. aureus wound infections.