Novel mutation in efflux pump Rv1258c (Tap) gene in drug resistant clinical isolates of Mycobacterium tuberculosis in Iran.

INTRODUCTION: Tuberculosis (TB) remains a serious public health problem worldwide. Drug-resistant TB is considered a major and growing global threat. Despite the great variety of described mutations in Mycobacterium tuberculosis (MTB) resistance genes, the mechanisms of drug resistance are still controversial. Recently, a report on the role of efflux pump genes in drug resistance added to this complexity. Therefore, a thorough understanding of efflux pump genes in drug-resistant TB clinical isolates is needed. METHODOLOGY: We performed molecular analysis of the efflux pump gene (Rv1258c) in 33 drug-resistant and 20 drug-sensitive clinical MTB isolates by sequencing the amplicons' targets in both the forward and reverse directions. RESULTS: A novel mutation of the Rv1258c gene was identified at G442A (Ala148Thr) in rifampicin mono-resistant clinical strain, as compared to the H37Rv reference strain. In addition, a cytosine nucleotide insertion was found between the positions 580 and 581 (denominated Tap580) in two drug-sensitive strains at identical gene positions. CONCLUSIONS: These results indicated the possibility of mutation in the efflux pump genes and the important role of Tap efflux pump genes in drug-resistant MTB isolates. However, further research is required to determine the direct association of these mutations with resistant MTB.

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.

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.

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.

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.

Comparison of the Prevalence of Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) among Staphylococcus aureus Isolates in a Burn Unit with Non-Burning Units.

BACKGROUND: Staphylococcus aureus (S. aureus) is one of the most important pathogens in burn infections colonized in the nose and increase the risk of infections. METHODS: Overall, 85 S. aureus isolates were isolated from clinical and nasal hospitalized patients and health care workers (HCWs) in a burn unit and non-burn units in Isfahan from June 2016 and September 2016. Genes encoding penicillin-binding protein 2a (mecA) and adhesive surface proteins, including fibronectin-binding proteins (fnbA,fnbB), fibrinogen binding protein (fib), laminin-binding protein(eno), collagen binding protein (cna), elastin binding protein (ebps), intracellular adhesion operon (icaA and icaD) were detected using PCR method. RESULTS: The rate of methicillin-resistant S. aureus (MRSA) among burn and non-burn isolates were 62% (18/29) and 25% (14/56), respectively. The most prevalent MSCRAMMs genes in burn units were eno (86%) and fib (66%). The most common gene pattern in burn center was icaA+fib+eno. The frequency of icaD, fib and ebpS was higher in clinical samples than nasal samples. No relation was found between the MSCRAMMs genes in the burn unit and non-burn units. CONCLUSION: The high prevalence of MRSA in burn center can be a new challenge for clinicians. The higher frequency of icaD, fib and ebpS in clinical isolates than nasal isolates may reflect the important role of these genes in colonization and pathogenesis of S. aureus.

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.

Global Sequence Analysis and Expression of Azurin Gene in Different Clinical Specimens of Burn Patients with Pseudomonas aeruginosa Infection.

AIM: The purpose of this study was to analyze the sequence of azurin gene in relation to its expression in Pseudomanas aeruginosa strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of azurin gene using globally reported sequences was intended. MATERIALS AND METHODS: Fifty-nine multidrug-resistant P. aeruginosa isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the azurin gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The azurin gene sequences were compared with the sequence data from other countries. The expression level of azurin gene in P. aeruginosa isolates with different azurin sequences from different clinical specimens was evaluated by real-time PCR. RESULTS AND CONCLUSION: About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the azurin gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between azurin gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about azurin genetic variation and significance in P. aeruginosa pathogenesis.

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 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.

Inhibition of herpes simplex virus type 1 replication by novel hsa-miR-7704 in vitro.

Herpes simplex virus type 1 (HSV-1) infections are one of the most common diseases in human population. HSV-1 causes subclinical, mild to severe diseases, especially in immunocompromised patients. Acyclovir has been used to reduce manifestations of HSV-1 infections. The extensive use of this drug has led to the development of resistant strains. Thus, designing a novel anti-herpes drug with different mechanisms of action is urgently needed. Cellular microRNAs (miRNAs) have direct antiviral effects in addition to their regulatory functions. In this study we used a novel miRNA (hsa-miR-7704), expressed in macrophages, to inhibit HSV-1 lytic infection in HeLa cells. Synthesized hsa-miR-7704 mimics were transfected into HSV-1 infected HeLa cell. The inhibitory effects of the miRNA were evaluated by plaque assay, real time polymerase chain reaction and the viral titers were measured by the 50% tissue culture infective dose (TCID50). The viral titer and cell cytopathic effect were dramatically decreased in HeLa cells transfected with hsa-miR-7704 (50 and 100 nM), compared with HSV-1 infected cells alone or transfected with the mock miRNA control. These results suggest that hsa-miR-7704 inhibits HSV-1 replication efficiently in vitro. This may provide an alternative mechanism to prevent HSV-1 infections.

Analysis of antibacterial and antibiofilm activity of purified recombinant Azurin from Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the antibacterial and antibiofilm activity of recombinant Azurin from Pseudomonas aeruginosa against different bacterial species. MATERIALS AND METHODS: The azurin gene was cloned in the pET21a vector. The pET21a-azurin construct was transformed into Escherichia coli BL21. The recombinant Azurin was expressed and purified using affinity chromatography and confirmed by Western blotting. The cytotoxicity of rAzurin was assessed on peripheral blood mononuclear cells. Antibacterial and antibiofilm activity of rAzurin with different concentrations were determined by micro-broth dilution and crystal violet methods, respectively. The effect of rAzurin on bacterial species was statistically analyzed by t-test and spearman correlation. RESULTS: The identity of purified protein was confirmed by blotting and distinguished as a 14 kDa band on 15% SDS-PAGE. The IC50 of rAzurin on Peripheral Blood Mononuclear Cell (PBMC) was determined as 377.91±0.5 µg/mL in 24 h. Vibrio cholerae and Campilobacter jejuni displayed the most sensitivity to rAzurin (27.5 and 55 µg/mL, respectively) and the highest resistance (220 µg/mL) was displayed by P. aeruginosa and E. coli. The MIC for other species was 110 µg/mL. The Minimum Biofilm Inhibition Concentration (MBIC) was determined as 220 µg/mL for Salmonella enterica and V. cholerae, 300 µg/mL for Shigella sonnei, Shigella flexneri and P. aeruginosa and 440 µg/mL for the other species. The antimicrobial effect of rAzurin on bacterial species were significant (p value<0.05) and correlation coefficient was negative. CONCLUSION: The rAzurin appears to be an appropriate choice and a new strategy for prevention of bacterial infection. It inhibits bacterial growth and biofilm formation and candidates as antimicrobial peptides.

Detection of antibiotic resistant Acinetobacter baumannii in various hospital environments: potential sources for transmission of Acinetobacter infections.

BACKGROUND: Antibiotic resistant Acinetobacter baumannii has emerged as one of the most problematic hospital acquired pathogens around the world. This study was designed to investigate the presence of antibiotic resistant A. baumannii in various hospital environments. METHODS: Air, water and inanimate surface samples were taken in different wards of four hospitals and analyzed for the presence of A. baumannii. Confirmed A. baumannii isolates were analyzed for antimicrobial susceptibility and also screened for the presence of three most common OXA- type carbapenemase-encoding genes. RESULTS: A. baumannii was detected in 11% (7/64) of air samples with the highest recovery in intensive care units (ICUs). A. baumannii was also detected in 17% (7/42) and 2% (1/42) of surface and water samples, respectively. A total of 40 A. baumannii isolates were recovered and analysis of antimicrobial susceptibility showed the highest resistance towards ceftazidime (92.5%, 37/40). 85% (34/40) and 80% (32/40) of the isolates were also resistant to imipenem and gentamicin, respectively. Resistance genes analysis showed that 77.5% (31/40) strains contained OXA-23 and 5% (2/40) strains contained OXA-24, but OXA-58 was not detected in any of the strains. CONCLUSION: Detection of antibiotic resistant A. baumannii in various samples revealed that hospital environments could act as a potential source for transmission of A. baumannii infections especially in ICUs. These results emphasize the importance of early detection and implementation of control measures to prevent the spread of A. baumannii in hospital environments.

Genetic Lineages of Mycobacterium tuberculosis Isolates in Isfahan, Iran.

In this study, we aimed to identify the genetic lineages of Mycobacterium tuberculosis isolates in Isfahan via the mycobacterial interspersed repetitive-unit-variable number tandem repeat typing method based on 15 loci. Forty-nine M. tuberculosis isolates were collected between 2013 and 2015 from Tuberculosis patients in Mollahadi Sabzevari Tuberculosis Center in Isfahan. All isolates were typed by 15-locus MIRU-VNTR typing. The highest percentage of isolates, 44.89 % (22/49), belonged to the Euro-American lineage, while the frequencies of the East-African-Indian, East-Asian, and Indo-Oceanic lineages were 28.57 % (14/49), 24.4 % (12/49), and 2.04 % (1/49), respectively. Among the 22 isolates of the Euro-American lineage, those belonging to the NEW-1 sub-lineage were most prevalent (24.4 %). Approximately, the same proportion of isolates belonging to the Delhi/CAS, Beijing, and NEW-1 sub-lineages were identified in Iranian and Afghan immigrant patients. The Delhi/CAS and Beijing sub-lineage isolates were prevalent among patients who had been previously treated for TB. Results showed that all of the 49 MIRU-VNTR patterns were unique and the clustering rate of the 15-locus MIRU-VNTR was 0.0 (minimum recent transmission). The results of this study show that the lineages of M. tuberculosis isolates in Isfahan are similar to those reported in the Eastern Mediterranean region (indicative of the epidemiological relationship between the countries in the region). The low clustering rate in our results reveals that transmission of tuberculosis in Isfahan is, in most cases, a reactivation of previous tuberculosis infection and the role of recently transmitted disease is minor.

Phylogenetic Analysis of Prevalent Tuberculosis and Non-Tuberculosis Mycobacteria in Isfahan, Iran, Based on a 360 bp Sequence of the rpoB Gene.

BACKGROUND: Taxonomic and phylogenetic studies of Mycobacterium species have been based around the 16sRNA gene for many years. However, due to the high strain similarity between species in the Mycobacterium genus (94.3% - 100%), defining a valid phylogenetic tree is difficult; consequently, its use in estimating the boundaries between species is limited. The sequence of the rpoB gene makes it an appropriate gene for phylogenetic analysis, especially in bacteria with limited variation. OBJECTIVES: In the present study, a 360bp sequence of rpoB was used for precise classification of Mycobacterium strains isolated in Isfahan, Iran. MATERIALS AND METHODS: From February to October 2013, 57 clinical and environmental isolates were collected, subcultured, and identified by phenotypic methods. After DNA extraction, a 360bp fragment was PCR-amplified and sequenced. The phylogenetic tree was constructed based on consensus sequence data, using MEGA5 software. RESULTS: Slow and fast-growing groups of the Mycobacterium strains were clearly differentiated based on the constructed tree of 56 common Mycobacterium isolates. Each species with a unique title in the tree was identified; in total, 13 nods with a bootstrap value of over 50% were supported. Among the slow-growing group was Mycobacterium kansasii, with M. tuberculosis in a cluster with a bootstrap value of 98% and M. gordonae in another cluster with a bootstrap value of 90%. In the fast-growing group, one cluster with a bootstrap value of 89% was defined, including all fast-growing members present in this study. CONCLUSIONS: The results suggest that only the application of the rpoB gene sequence is sufficient for taxonomic categorization and definition of a new Mycobacterium species, due to its high resolution power and proper variation in its sequence (85% - 100%); the resulting tree has high validity.

Analysis of DNA gyrA Gene Mutation in Clinical and Environmental Ciprofloxacin-Resistant Isolates of Non-Tuberculous Mycobacteria Using Molecular Methods.

BACKGROUND: During the past several years, nontuberculous mycobacteria (NTM) have been reported as some of the most important agents of infection in immunocompromised patients. OBJECTIVES: The aim of this study was to evaluate the ciprofloxacin susceptibility of clinical and environmental NTM species isolated from Isfahan province, Iran, using the agar dilution method, and to perform an analysis of gyrA gene-related ciprofloxacin resistance. MATERIALS AND METHODS: A total of 41 clinical and environmental isolates of NTM were identified by conventional and multiplex PCR techniques. The isolates were separated out of water, blood, abscess, and bronchial samples. The susceptibility of the isolates to 1 µg/mL, 2 µg/mL and 4 µg/mL of ciprofloxacin concentrations was determined by the agar dilution method according to CLSI guidelines. A 120-bp area of the gyrA gene was amplified, and PCR-SSCP templates were defined using polyacrylamide gel electrophoresis. The 120-bp of gyrA amplicons with different PCR-SSCP patterns were sequenced. RESULTS: The frequency of the identified isolates was as follows: Mycobacterium fortuitum, 27 cases; M. gordonae, 10 cases; M. smegmatis, one case; M. conceptionense, one case; and M. abscessus, two cases. All isolates except for M. abscessus were sensitive to all three concentrations of ciprofloxacin. The PCR-SSCP pattern of the gyrA gene of resistant M. abscessus isolates showed four different bands. The gyrA sequencing of resistant M. abscessus isolates showed 12 alterations in nucleotides compared to the M. abscessus ATCC 19977 resistant strain; however, the amino acid sequences were similar. CONCLUSIONS: This study demonstrated the specificity and sensitivity of the PCR-SSCP method for finding mutations in the gyrA gene. Due to the sensitivity of most isolates to ciprofloxacin, this antibiotic should be considered an appropriate drug for the treatment of related diseases.