Alterations in the gut microbiota and their metabolites in human intestinal epithelial cells of patients with colorectal cancer.

BACKGROUND: The gut microbiota has become one of the main risk factors for the formation and development of colorectal cancer (CRC). CRC intensification may be due to the microbial pathogens' colonization and their released metabolites. Here, we analyzed Bacteroidetes and Clostridia bacteria in CRC patients and studied bacterial metabolome in cancerous tissues compared to their adjacent normal tissues. METHODS AND RESULTS: The population of selected bacteria in biopsy specimens of 30 patients with CRC was studied by RT-qPCR. The mutagenicity and cytotoxicity effects of microbiota metabolites were evaluated by Ames test and MTT Assay, respectively. Moreover, gene expression in carcinogenic pathways was studied by RT-qPCR, and genes with different expressions in tumor and non-tumor tissues were diagnosed. Based on microbiota analysis, the relative abundance of Clostridia and C. difficile was significantly higher in CRC tissue, whereas C. perfringens showed higher relative abundance in normal tissue. AIMES test confirmed the proliferation and mutagenicity effects of the bacterial metabolites in CRC patients. Significant upregulation of C-Myc, GRB2, IL-8, EGFR, PI3K, and AKT and downregulation of ATM were observed in CRC samples compared to the control. CONCLUSIONS: The influence of bacterial metabolites on inflammation and altered expression of genes in the cell signaling pathways was observed. The findings confirm the role gut microbiota composition and bacterial metabolites as key players in CRC onset and development.

EPIYA Motif Genetic Characterization from Helicobacter pylori Isolates in Distinct Geographical Regions of Iran.

Background: This study aimed to determine the current EPIYA motifs of the cagA gene in Helicobacter pylori isolates from patients with gastric disorders, and evaluate the association between these patterns and the clinical outcome of H. pylori infection in different geographical regions of Iran. Materials and Methods: We examined 150 patients with gastrointestinal disorders from the central and eastern regions of Iran. The detection of H. pylori and screening of cagA was performed by polymerase chain reaction (PCR). The pattern of the motifs was determined by PCR followed by sequencing. Results: The overall prevalence of H. pylori was 66.3% in eastern (Mashad) and 50.6% in the central (Isfahan) part of Iran. The frequency of cagA-positive strains in Mashad and Isfahan were 63.4% and 56.7%, respectively. The pattern of EPIYA motif was as follows: 43 (79.6%) ABC, 7 (12.9%) AB, 4 (7.4%) ABCC, and one (1.9%) ABCCC. We also identified a novel EPIYA C sequence motif which showed association with gastric cancer (GC). The relationship between the frequency of specific EPIYA motifs and GC was statistically significant (P < 0.05). Conclusions: This is the first report for the determination of the cagA EPIYA motif of H. pylori in the Northeast and center of Iran. The prevalence of cagA positive H. pylori between the two regions was significant (P ≤ 0.05). All isolates of the H. pylori cagA were western type (ABC). The increase in the number of EPIYA-C repeats was associated with GC (P ≤ 0.01).

Viral aetiology of acute central nervous system infections in children, Iran.

Introduction. Viral infections are increasingly an important cause of central nervous system (CNS) complications.Hypothesis/Gap Statement. There is no comprehensive insight about CNS infections due to viral agents among Iranian children.Aim. This study aimed to investigate the viral aetiology, clinical and epidemiological profile of children with acute infections of the CNS.Methodology. A prospective study was conducted on children at the referral hospital in Isfahan, Iran, from June 2019 to June 2020. A multiplex PCR assay was used to detect the viral causative agent in cerebrospinal fluid and throat/rectal swab samples.Results. Among 103 patients with eligible criteria, a confirmed or probable viral aetiology was detected in 41 (39.8 %) patients, including enteroviruses - 56.1 %, herpes simplex virus 1/2 (HSV-1/2) - 31.7 %, Epstein-Barr virus - 17.1 %, varicella-zoster virus (VZV) - 9.7 %, influenza A virus (H1N1) -4.9 % and mumps - 2.4 %. There was a higher proportion of PCR-positive samples in infants than in other age groups. Encephalitis and meningoencephalitis were diagnosed in 68.3 % (28/41) and 22 % (9/41) PCR-positive cases, respectively.Conclusion. The findings of this research provide insights into the clinical and viral aetiological patterns of acute CNS infections in Iran, and the importance of molecular methods to identify CNS viruses. HSV and VZV were identified as important causes of encephalitis in young children.

A Phage Cocktail To Control Surface Colonization by Proteus mirabilis in Catheter-Associated Urinary Tract Infections.

Proteus mirabilis is a biofilm-forming bacterium and one of the most common causes of catheter-associated urinary tract infections (CAUTIs). The rapid spread of multidrug-resistant P. mirabilis represents a severe threat to management of nosocomial infections. This study aimed to isolate a potent phage cocktail and assess its potential to control urinary tract infections caused by biofilm-forming P. mirabilis. Two lytic phages, Isf-Pm1 and Isf-Pm2, were isolated and characterized by proteome analysis, transmission electron microscopy, and whole-genome sequencing. The host range and effect of the phage cocktail to reduce the biofilm formation were assessed by a cell adhesion assay in Vero cells and a phantom bladder model. The samples treated with the phage cocktail showed a significant reduction (65%) in the biofilm mass. Anti-quorum sensing and quantitative real-time PCR assays were also used to assess the amounts of transcription of genes involved in quorum sensing and biofilm formation. Furthermore, the phage-treated samples showed a downregulation of genes involved in the biofilm formation. In conclusion, these results highlight the efficacy of two isolated phages to control the biofilms produced by P. mirabilis CAUTIs. IMPORTANCE The rapid spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacterial strains and biofilm formation of bacteria have severely restricted the use of antibiotics and become a challenging issue in hospitals. Therefore, there is a necessity for alternative or complementary treatment measures, such as the use of virulent bacteriophages (phages), as effective therapeutic strategies.

Alhagi maurorum extract modulates quorum sensing genes and biofilm formation in Proteus mirabilis.

Proteus mirabilis (P. mirabilis) is a frequent cause of catheter-associated urinary tract infections. This study aims to investigate the anti-infective effect of Alhagi maurorum extract (AME), the traditional medicinal plant in the middle east, on the biofilm-forming P. mirabilis isolates. Hydroalcoholic extract and oil of A. maurorum were characterized by HPLC and GC-MS. The antiproliferative, anti-biofilm, and bactericidal activity of AME at various concentrations were assessed by turbidity, crystal violet binding, and agar well diffusion assays, respectively. The AME's effect on adhesion and quorum sensing (QS) were investigated by in vitro adhesion assay on cell culture and agar overlay assay using Janthinobacterium lividum (ATCC 12472) as a biosensor strain. In addition, the expression level of selected genes involved in QS and biofilm regulation were determined by quantitative Real-Time PCR. Furthermore, the bladder phantom model was created to evaluate the assays and investigate the catheter's calcium deposition. The most effective chemical compounds found in AME were tamarixetin, quercetin, and trans-anethole. Although AME did not inhibit swarming motility, it reduced biofilm production and exerted a concentration-dependent anti-adhesive and anti-QS activity against P. mirabilis. AME also downregulated the expression level of selected genes involved in biofilm formation and QS. This study showed that AME as a natural compound reduced biofilm formation of P. mirabilis by targeting virulence factor genes, quorum sensing, and other strategies that include preventing the adhesion of P. mirabilis to the cells. The results suggest that A. maurorum extract might have the potential to be considered for preventing UTIs caused by P. mirabilis.

Genetic Diversity of Helicobacter pylori Isolates from Patients with Gastric Diseases in Isfahan.

Background: Helicobacter pylori (H. pylori), a spiral-shaped bacterium colonizing the human stomach, is generally acquired in childhood. This pathogen is highly diverse and can be used as genetic markers for predict the history of human migrations. This study aimed to determine the genetic diversity of H. pylori isolates from patients with dyspepsia by the multi-locus sequence typing (MLST) and update data on the prevalence of H. pylori among Iranian dyspeptic patients. Materials and Methods: In this descriptive cross-sectional study, 165 gastric biopsy specimens were obtained from patients with dyspepsia referred to Dr. Shariati Hospital of Isfahan, Iran, from April to July 2018. The status of H. pylori infection was determined by FISH in paraffin-embedded biopsy specimens. MLST of seven housekeeping genes was performed for 20 H. pylori isolates. The phylogenetic tree was plotted using CLC v8 and iTol software. Results: The overall prevalence of H. pylori infection was 53.3%. In the results of the analysis of MLST, a total of 14 new STs were recorded. The results of the global analysis showed that all the isolates, with a wide diversity, have a genetic affinity with members of the European population, such as Italy and Russia, and are in the hpEurope haplotype. Conclusion: Given the high prevalence of H. pylori infection in this region, early and accurate identification of patients seems necessary. Sequence analysis and determination of the origin of the phylogeny of strains can be effective in clinical management and monitoring of risk factors for chronic and recurrence of infection.

The characteristic and potential therapeutic effect of isolated multidrug-resistant Acinetobacter baumannii lytic phage.

BACKGROUND: Widespread misuse of antibiotics caused bacterial resistance increasingly become a serious threat. Bacteriophage therapy promises alternative treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated and characterized a novel, potent lytic bacteriophage against multi-drug resistant (MDR) Acinetobacter baumannii and described the lytic capability and endolysin activity of the phage to evaluate the potential in phage therapy. METHODS: A novel phage, pIsf-AB02, was isolated from hospital sewage. The morphological analysis, its host range, growth characteristics, stability under various conditions, genomic restriction pattern were systematically investigated. The protein pattern of the phage was analyzed, and the endolysin activity of the phage was determined under the non-denaturing condition on SDS-PAGE. The optimal lytic titer of phage was assessed by co-culture of the phage with clinical MDR A. baumannii isolates. Finally, HeLa cells were used to examine the safety of the phage. RESULTS: The morphological analysis revealed that the pIsf-AB02 phage displays morphology resembling the Myoviridae family. It can quickly destroy 56.3% (27/48) of clinical MDR A. baumannii isolates. This virulent phage could decrease the bacterial host cells (from 108 CFU/ml to 103 CFU/ml) in 30 min. The optimum stability of the phage was observed at 37 °C. pH 7 is the most suitable condition to maintain phage stability. The 15 kDa protein encoded by pIsf-AB02 was detected to have endolysin activity. pIsf-AB02 did not show cytotoxicity to HeLa cells, and it can save HeLa cells from A. baumannii infection. CONCLUSION: In this study, we isolated a novel lytic MDR A. baumannii bacteriophage, pIsf-AB02. This phage showed suitable stability at different temperatures and pHs, and demonstrated potent in vitro endolysin activity. pIsf-AB02 may be a good candidate as a therapeutic agent to control nosocomial infections caused by MDR A. baumannii.

Significance of the coexistence of non-codon 315 katG, inhA, and oxyR-ahpC intergenic gene mutations among isoniazid-resistant and multidrug-resistant isolates of Mycobacterium tuberculosis: a report of novel mutations.

Tuberculosis (TB) is a global threat due to the emergence and spread of drug-resistant Mycobacterium tuberculosis (MTB). Isoniazid (INH) is the main antibiotic used for prevention and treatment of TB. Evidence shows that accumulated mutations can produce INH resistant (INHR) strains, resulting in the progression of multidrug-resistant (MDR) TB. Since point mutations in katG gene, inhA gene, and oxyR-ahpC region correlated with the INH resistance, in this study, we aimed to identify mutations in these three genes in INHR and MDR clinical isolates of MTB by Sanger DNA sequencing analysis. Thirty-three out of 438 isolates were resistant, including 66.7% INHR and 30.3% MDR isolates. In the katG gene, 68.2% INHR isolates had non-synonymous point mutations, mainly R463L (63.6%), and non-synonymous point mutation KatG L587P was seen in one of the MDR isolate. A novel silent substitution L649L was identified in the inhA gene of the MDR isolates. The oxyR-ahpC intergenic region g-88a common mutations (63.6%) in INHR and two distinct novel mutations were found at positions -76 and -77 of the oxyR-ahpC intergenic region. The coexistence of katG non-codon 315 with oxyR-ahpC intergenic region mutations was highly frequent in INHR 59.1% and MDR isolates 70%. Since mutations of all three genes 95.5% lead to the detection of INHR, they might be useful for molecular detection. Our results indicated the continuous evolution and region-specific prevalence of INH resistance. Overall, identification of new mutations in INH resistance can improve the available strategies for diagnosis and control of TB.

Epstein-barr virus/Helicobacter pylori coinfection and gastric cancer: the possible role of viral gene expression and shp1 methylation.

Background and Objectives: Among the various factors involved in the development of gastric cancer (GC), infectious agents are one of the most important causative inducers. This study aimed to investigate the possible role of EBV gene expression on SHP1 methylation in co-infection with Helicobacter pylori in patients with GC. Materials and Methods: Formalin-fixed paraffin-embedded samples were obtained from 150 patients with gastrointestinal disorders. The presence of the H. pylori and EBV genome were examined by PCR. The expression level of viral gene transcripts and methylation status of the SHP1 cellular gene was assessed by quantitative real-time PCR and methyl-specific PCR. Results: EBV and H. pylori coinfection were reported in 5.6% of patients. The mean DNA viral load was significant in patients coinfected with cagA-positive H. pylori (P= 0.02). The expression of BZLF1 and EBER was associated with GC. Also, the expression level of BZLF1in GC tissues was significantly higher in coinfection (P = 0.01). SHP1 methylation frequency was higher in the GC group than in the control group (P = 0.04). The correlation between the methylation rate and the H. pylori infection was highly significant (P<0.0001). The strongest positive correlation was observed in GC specimens between SHP1 methylation and H. pylori cagA-positive strains (p= 0.003). Conclusion: Our results suggested that cagA might involve in the elevation of EBV lytic gene expression and SHP1 methylation, and the development of gastric cancer. Understanding the mechanism of EBV H. pylori - cagA + coinfection, as well as host epigenetic changes, can play an important role in diagnosing and preventing gastric cancer.

Determination of Capsular Serotypes, Antibiotic Susceptibility Pattern, and Molecular Mechanism of Erythromycin Resistance among Clinical Isolates of Group B Streptococcus in Isfahan, Iran.

BACKGROUND: Documented streptococcal resistance to erythromycin has recently been raised. The aim of this study is to identify the molecular mechanism of erythromycin resistance among group B Streptococcus (GBS) strains and to correlate with the clinical origin of strains. MATERIALS AND METHODS: A total number of 134 colonizing (n = 36), invasive (n = 36), noninvasive (n = 46), and asymptomatic (n = 16) GBS isolates were characterized by the detection of dltS gene, capsular serotyping, antibiotic susceptibility profiles using disc diffusion method, and screening of the ermB, ermTR, and mefA resistance genes. RESULTS: The distribution of capsular serotypes was as follow: serotype III (24.6%), Ia (21.6%), V (17.9%), Ib (14.9%), II (8.9%), IV (8.9%), VI (1.5%), and VII (1.5%). From 134 GBS isolates, 51 (38%) isolates were resistant to erythromycin. The constitutive macrolide lincosamide streptogrmin B (MLSB) was the most common resistance phenotype (62.7%), followed by inducible MLSB (27.4%) and M phenotype (9.8%). Erythromycin resistance rate was higher among asymptomatic GBS strains (13/16, 81.2%). Serotype III was the most prevalent type among resistant isolates (41.1%). The ermB gene highly distributed among resistant strains (64.7%), followed by ermTR (21.5%) and mefA (9.8%). The ermB gene was related to constitutive MLSB phenotype (84.3%, P < 0.05) and serotypes III (61.9%), Ib (87.5%), and V (83.3%). All M phenotype strains harbored mefA gene and were in association with serotype Ia (90%). CONCLUSION: The current study suggests that ribosomal modification with erm genes is the main mechanism of erythromycin resistance. Because of relatively high prevalence of erythromycin resistance, double disc test highly recommended for GBS disease treatment and intrapartum prophylaxis among penicillin intolerant patients in our region.

From the Urinary Catheter to the Prevalence of Three Classes of Integrons, ß-Lactamase Genes, and Differences in Antimicrobial Susceptibility of Proteus mirabilis and Clonal Relatedness with Rep-PCR.

INTRODUCTION: Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum ß-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. METHOD: Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (intI1, intI2, intI3, bla TEM, bla CTX-M, bla SHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR. RESULT: From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was blaTEM . Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1-G39) of 40 isolates that 38 isolates had unique patterns. CONCLUSION: In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The intI1 and bla TEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future.

Prevalence, population structure, distribution of serotypes, pilus islands and resistance genes among erythromycin-resistant colonizing and invasive Streptococcus agalactiae isolates recovered from pregnant and non-pregnant women in Isfahan, Iran.

BACKGROUND: The information on antibiotic resistance and molecular features of Group B Streptococcus (GBS) are essential for epidemiological purposes as well as vaccine development. Therefore, we aimed to assess the antimicrobial resistance profiles and molecular characteristics of GBS isolates in Isfahan, Iran. A total number of 72 colonizing and invasive GBS were collected from pregnant and non-pregnant women. The GBS isolates were analyzed for resistance profiles, capsular genotyping, and detection of PI-1, PI-2a, PI-2b, hvgA, ermB, ermTR, lnuB and, mefA genes. Besides, erythromycin-resistant strains were subjected to multilocus sequence typing (MLST). RESULTS: The prevalence of colonizing and invasive GBS were 11 and 0.05%, respectively. The frequency of capsular serotypes was as follows: III (26.3%), Ia (20.83%), Ib and V (each 15.2%), IV (9.7%), II (8.3%), VII (2.7%), and VI (1.3%). Overall frequencies of PIs were as follows: PI-1, 37.5%, PI-1 + PI-2a, 30.5%, PI-1 + PI-2b, 29.1% and PI-2b, 2.7%. Two maternal colonizing GBS (2.6%) were hvgA positive and were belonged to ST-17/CPS-III/PI-1 + PI-2b lineage. Among 30(41.6%) erythromycin resistant GBS, 21 isolates (70%) harbored ermB gene, followed by ermTR (23.3%) and mefA (10%). One clindamycin-resistant isolate harbored the lnuB gene. MLST analysis revealed the following five clonal complexes (CCs) and nine STs: (CC-19/ST-335, ST-19, and ST-197), (CC-12/ST-43, ST-12), (CC-23/ST-163, ST-23), (CC-17/ST-17) and (CC-4/ST-16). CONCLUSION: The study shows an alarmingly high prevalence of erythromycin-resistant GBS in Iran. In addition, we report dissemination of ST-335/CPS-III clone associated with tetracycline and erythromycin resistance in our region. The distribution of capsular and pilus genotypes varies between invasive and colonizing GBS that could be helpful for vaccine development.

Cellular miR-101-1 Reduces Efficiently the Replication of HSV-1 in HeLa Cells.

INTRODUCTION: Herpes simplex viruses (HSVs) are widely distributed in the human population. HSV type 1 (HSV-1) is responsible for a spectrum of diseases, ranging from gingivostomatitis to keratoconjunctivitis, and encephalitis. The HSVs establish latent infections in nerve cells, and recurrences are common. Their frequent reactivation in elderly and immunosuppressed patients causes serious health complications. OBJECTIVES: Due to the growing resistance to its main drug, acyclovir, alternative treatments with different mechanisms of action are required. MicroRNAs regulate host and viral gene expression posttranscriptionally. Previous studies reported that mir-101-2 expression has widely participated in the regulation of HSV-1 replication. In this study, we investigate the effect of hsa-miR-101-1 in the replication of HSV-1. METHODS: We found that transfection of miR-101-1 into HeLa cells could reduce effectively HSV-1 replication using plaque assay and real-time PCR methods. RESULTS: We showed that overexpression of miR-10-1 produced less viral progeny and manifested a weaker cytopathic effect, without affecting cell viability. DISCUSSION/CONCLUSION: This result can give us new insights into the control of HSV-1 infections.

Efficient process development for high-level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli.

Overproduction of recombinant mecasermin was achieved by investigation of effect of three factors, temperature, inducer amount, and culture media, at three levels according to the Taguchi statistical design in Escherichia coli in a bench-scale bioreactor. In optimal conditions (induction temperature 28 °C, terrific broth with glucose (TB+G) medium, with 0.1 mM IPTG as inducer) 0.84 g/L mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values of recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn-HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF-1 (mecasermin) was formulated with arginine. Mecasermin protein remained t stable at 4 °C for up to 2 years. The quantitative and qualitative control indicated that mecasermin is expressed correctly (without the initial methionine by mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF-HPLC), monomer form (SEC-HPLC), and active (bioactivity test). Also, the purification results revealed that expression at low temperature results in the efficient purification of the overproduced mecasermin with high quantity and quality.

Characterization of Hypervirulent Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae Among Urinary Tract Infections: The First Report from Iran.

INTRODUCTION: This study was conducted to identify the hypermucoviscosity, iron acquisition, and capsule serotypes of K. pneumoniae strains isolated from urinary tract infections among community-acquired patients (CA) and assess the frequency of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum beta-lactamases (ESBL) genes between classic and hypervirulent strains. MATERIALS AND METHODS: A total of 105 K. pneumoniae were isolated from CA-UTI. Demographic data related to the underlying diseases and clinical manifestations were further collected. Antibiotic resistance pattern and molecular characterization were compared among ESBL-positive, ESBL-negative, hypervirulent, and classic isolates. RESULTS: The results revealed that 52.4% of the isolates were confirmed as ESBL producers and 11 (10.5%) were considered as hypervirulent K. pneumoniae (hvKp). Ciprofloxacin and nalidixic acid were the most inactive antibiotics with resistance rates of 68.6% and 64.8%, respectively. Molecular characterization revealed that 7.6% of all the isolates carried k1 and 66.6% carried K2 genes. The most frequent ESBL gene was blaSHV 63.8%, followed by blaTEM 59.0%, and blaCTX-M 58.1%. ESBL genes were significantly more in hvKp than in cKp. Moreover, 61 (84.7%), 47 (65.2%), and 16 (22.2%) of isolates harbored qnrB, qnrS, and qnrA. ESBL genes were detected in all hvKps, and blaSHV was observed in 90.9% of hvKp (P value= 0.048, 95%). DISCUSSION: This study reported the high frequency of antimicrobial and multidrug resistance among hvKp isolates. Coexistence of PMQR and ESBL genes in hvkp indicates the necessity to enhance the clinical knowledge and management of hvKp infections.

Sequence-based detection of first-line and second-line drugs resistance-associated mutations in Mycobacterium tuberculosis isolates in Isfahan, Iran.

Tuberculosis is an infectious disease, which requires special medical attention due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. The present study aimed to assess drug resistance to first-line anti-mycobacterial drugs, including rifampin (RIF), isoniazid (INH), and ethambutol (EMB), as well as second-line drugs, including ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP). The following eight loci were investigated to evaluate drug resistance: rpoB, katG, inhA, and embB, associated with resistance to RIF, INH, and EMB and gyrA, rrs, eis, and tlyA, associated with resistance to OFX, AMK, KAN, and CAP. A total of 482 patients with tuberculosis, who were referred to Molla Haadi Sabzevari Healthcare Center (Isfahan, Iran) during 2014-2017, were studied. Of 482 patients with tuberculosis, 32 (6.63%) Mycobacterium tuberculosis isolates were resistant to the first-line anti-mycobacterial drugs. Overall, 23 (71.8%), 13 (40.6%), and 3 (9.3%) isolates were resistant to INH, RIF, and EMB, respectively. Also, 13 (100%), 6 (46.1%), and 1 (7.6%) out of 13 MDR/RIF-resistant isolates were resistant to CAP and KAN, AMK, and OFX, respectively. Among the eight loci, non-synonymous substitutions were observed in rpoB (n = 7), katG (n = 10), inhA (n = 7), gyrA (n = 13), and rrs (n = 3), whereas synonymous substitutions were seen in tlyA and gyrA. On the other hand, no mutation was detected in embB or eis. Based on the present results, mutations in the eis promoter region and embB locus may not be involved in resistance to KAN and EMB in our study population. Also, the gyrA Asp94Asn mutation may be an indicator of resistance to OFX. We did not detect any XDR isolates, whereas MDR and pre-XDR isolates were found, which can be alarming.

Molecular Assessment of Resistance to Clarithromycin in Helicobacter pylori Strains Isolated from Patients with Dyspepsia by Fluorescent In Situ Hybridization in the Center of Iran.

BACKGROUND AND AIMS: Helicobacter pylori is a common infectious bacterium mostly found in gastroduodenal diseases. The increased prevalence of clarithromycin-resistant H. pylori strains is a major challenge in the successful treatment of infections caused by this organism. The present study is aimed at detecting the clarithromycin resistance pattern of H. pylori strains isolated from gastric biopsies and evaluating point mutations of the 23S rRNA gene. Patients and methods. In the present descriptive cross-sectional study, 165 patients with gastrointestinal disorders, who were referred to the Endoscopy Center of Dr. Shariati Hospital of Isfahan, Iran, were enrolled from April to July 2018. H. pylori infection was diagnosed by culture, and susceptibility of the isolates to clarithromycin was assessed by the E-test. Minimum inhibitory concentration (MIC) values were obtained based on EUCAST recommendations. Also, fluorescence in situ hybridization (FISH) was used to determine point mutations associated with clarithromycin resistance. RESULTS: By using culturing, H. pylori was isolated from 50.3% (83/165) gastric biopsy specimens. The overall frequency of resistance to clarithromycin was 25.3% (21/83) by the E-test. In the resistance genotypic analysis, 19 isolates had mutations. The prevalence of A2143G and A2144G mutations was 68.4% (13/19) and 31.5% (6/19), respectively. A2143C mutation was not tracked in any isolate. Two isolates with MIC > 0.5 µg/mL had no mutations that could be related to other mechanisms of resistance. CONCLUSION: As presented in the study, the high prevalence of clarithromycin-resistant H. pylori due to point mutations of the 23S rRNA gene indicates the necessity of revising the standard treatment regimen based on antibiotic susceptibility pattern of each region.

Molecular Characterization of Hospital- and Community-Acquired Streptococcus agalactiae Isolates among Nonpregnant Adults in Isfahan, Iran.

BACKGROUND: The increasing incidence of Group B Streptococcus (GBS) infection among nonpregnant adults has become of growing clinical and public health concern. The current study investigated the distribution of important virulence determinants and antibiotic susceptibility of GBS isolates causing community acquired (CA) and hospital acquired (HA) infections among nonpregnant adults. MATERIALS AND METHODS: A total of 62 GBS, including 31 CA GBS and 31 HA GBS, were collected from a teaching hospital in Isfahan, Iran. Capsular polysaccharide genotypes (CPS), PI 1, PI 2a, PI 2b, and hypervirulent GBS adhesin (hvgA) virulence genes and antibiotic resistance profiling were determined. RESULTS: There were 19 (30.6%) cases of underlying disease that diabetes mellitus (20.9%) was most common. The rate of multidrug resistant GBS strains was accounted for 29%. Distribution of macrolide resistant phenotypes was as follows: constitutive macrolides, lincosamides, and streptogramin B (MLSB) (15 isolates); inducible resistance to MLSB; and L phenotype (each 5 isolates) and M phenotype (1 isolate). V and Ia serotypes were the most predominant capsular type in HA GBS and CA GBS isolates, respectively. The most frequent pilus types were PI 1, PI 1+PI 2a, PI 1+PI 2b, and PI 2a. PI 1 and PI 1+PI 2a had significantly different distributions between CA and HA GBS isolates. Three CA GBS isolates (9.6%) were positive for hvgA gene that belonged to clonal complex 17/sequence type 17/CPS III/PI 1+PI 2b lineage. CONCLUSION: There was a significant difference in the distribution of PIs among CA GBS and HA GBS isolates in our region.

Influence of Heterologously Expressed azurin from Pseudomonas aeruginosa on the Adhesion and Invasion of Pathogenic Bacteria to the Caco-2 Cell Line.

This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp. and Escherichia coli) and epithelial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) on the human colorectal adenocarcinoma (Caco-2) cell line. Azurin protein was produced by cloning the azurin gene into pET21a and heterologous expression in E. coli BL21. The protein was purified using affinity chromatography and confirmed by Western blotting. The purified protein was evaluated by three experiments of adhesion and invasion assays, including exclusion, competition, and replacement. Azurin was observed to significantly inhibit the attachment and invasion of S. aureus, Salmonella spp., and E. coli, while no such inhibitory effects were observed on P. aeruginosa. In fact, the protein increased the adhesion of P. aeruginosa to the cell. In conclusion, our study proposes that azurin is a potential prophylactic or preventive helper candidate to inhibit the attachment and invasion of pathogenic bacteria to host cells and reduce the progression of the infection process. Our study also reveals the involvement of azurin in bacteria-host cell interactions, providing novel and important insights toward the elucidation of its biological function in this field. Thus, this study provides new opportunities to use azurin as an adjunct therapy against critical stages of infection by a wide range of pathogenic bacteria.

The rate and importance of Clostridium difficile in colorectal cancer patients.

AIM: The aim of this study was to analyze the Clostridium difficile and their toxins in cancerous tissues in comparison to their adjacent healthy tissues in patients with colorectal cancer (CRC) in Iran. BACKGROUND: Intestinal infection or colonization by microbial pathogens and their released metabolites may have a role in the exacerbation of CRC. METHODS: A total of 60 biopsy samples from 30 cancerous and 30 adjacent healthy tissues were collected from patients with CRC. Biopsies were homogenized and cultured in cycloserine cefoxitin fructose agar-agar medium to investigate the presence of C. difficile. DNA was extracted, PCR was performed on pure colonies for bacteria detection, and toxin genes were evaluated in each bacterium positive cases. Real-time PCR was performed on extracted DNA for quantitative comparison of Clostridium difficile in healthy and tumor tissues in CRC patients. RESULTS: Clostridium difficile was isolated from 18 of the cancerous tissue (60%) and 6 of their healthy adjacent tissue (20%) in the culture medium, but toxin genes were positive just in one sample in both groups. Real-time PCR showed the colonization in all samples. CONCLUSION: This study showed a higher prevalence of Clostridium difficile in cancerous lesions in comparison to healthy tissues. We suggest that the investigation of the rate of CD of colorectal cancer patients before surgery is critical for patients. Further studies with more samples size to study the importance of this bacterium and its toxins in the investigation of colorectal cancer patients survey is recommended.