Isolation of lytic bacteriophages and their relationships with the adherence genes of Staphylococcus saprophyticus.

OBJECTIVE: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. RESULTS: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens.

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64 M. tuberculosis and 26 M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms.

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Cross-reactivity between Mycobacterium avium subspecies paratuberculosis 4027 peptide and Human IRF5 may contribute to Multiple Sclerosis in Iranian patients.

Background: The etiology of Multiple sclerosis (MS) is complicated and can be affected by several environmental factors, such as Mycobacterium avium subspecies paratuberculosis (MAP) infection in genetically predisposed individuals. The link between MAP and MS depends on host genetic and epigenetic aspects and population-based features that require further investigation. We aimed to study the possible role of MAP in triggering MS using molecular and serological methods. Materials and methods: This case-control study examined 200 blood samples (100 MS patients and 100 HCs) to search for the MAP-specific IS900 gene. In addition, ELISA was conducted to determine the humoral response against MAP_402718-32 and its human IRF5424-434 peptide homolog. Results: The frequency of MAP detection based on the molecular method in MS patients and HCs was 48 % and 13 %, respectively (p < 0.0001). The presence of antibodies against MAP_402718-32 and IRF5424-434 was 55 % and 65 % in MS patients versus 9 % and 7 % in HCs, respectively (p < 0.0001). A good correlation was observed between MAP_4027 and IRF5 antibodies (r = 0.5782, p < 0.0001), indicating that the same antibodies recognized common peptide epitopes. Conclusion: Our research revealed a significant association between MAP and MS, highlighting the possible role of MAP as an important infection trigger factor of MS. It is hypothesized that cross-reactivity between MAP4027 and IRF5 may dysregulate immune homeostasis.

Detection of Mycobacterium avium Subspecies paratuberculosis in the Blood of Patients with Rheumatoid Arthritis by Using Serological and Molecular Techniques.

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in triggering rheumatoid arthritis (RA) could be a population-specific phenomenon. This study explored the relationship between MAP and RA using serological and molecular techniques; In this case-control study, 239 Iranian participants, including 120 RA patients and 119 controls, were enrolled. The indirect ELISA was designed to diagnose antibodies against MAP3865c125-133 and Zinc transporter 8 (ZnT8)178-186. The Nested-Polymerase Chain Reaction (PCR) detected MAP in blood; The frequency of MAP in RA patients and controls was 31.9% and 12.5%, respectively (P = 0.002). The antibodies against MAP3865c125-133 and ZnT8178-186 were 42.9% and 37% in RA patients and 14.2% and 11.7% in the controls, respectively (P < 0.0001). Interestingly, positive ELISA results in previously diagnosed (PD) RA were more common than newly diagnosed (ND) RA patients (P < 0.05).; The findings showed a higher frequency of MAP and its antibodies in the RA patients than in the controls. This data indicated MAP as one of RA's predisposing factors. Also, this first report implies the high positivity of MAP in Iranian RA patients.

Detection of virulence genes among Staphylococcus saprophyticus isolated from women with urinary tract infections: first report from Iran.

OBJECTIVE: The purpose of the present study was to investigate the biofilm production, and the presence of virulence genes and biochemical characteristics among the S. saprophyticus clinical isolates. A total of 35 clinical isolates of S. saprophyticus were collected from patients referred to several hospitals. By the crystal violet staining method, the capability of biofilm formation was performed. The genes associated with surface of S. saprophyticus were investigated by the PCR-sequencing techniques. Hemagglutination and lipase activity assays were also performed. RESULTS: The results of crystal violet staining assay showed that 32 isolates (91%) form biofilm. Moreover, seven (20%), 13 (37%), and 12(34%) isolates were categorized as weak, moderate, and strong biofilm producers, respectively. virulence genes including UafA, Aas and Ssp had an overall prevalence of 88%, 91% and 80%, respectively. None of the isolates exhibited lipolytic activities. Regarding hemagglutination properties, only 11 (31%) isolates demonstrated hemagglutination of sheep erythrocytes. The results of this study indicate a high prevalence of UafA and Aas genes that can enhance the pathogenicity of S. saprophyticus, and Identification and better understanding of the functions of these genes can be used for therapeutic purposes. Maybe in the future we will be switch to anti-adhesion therapy because of drug resistance.

Evaluation of Mutations Related to Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates.

Drug resistance to streptomycin in the clinical isolates of Mycobacterium tuberculosis (MTB) needs special consideration. It can mostly be caused by mutations in four genes with the names rpsL, rrs, gidB, and whiB7. The main objective of this study was the evaluation of the type and frequency of mutations in these mentioned genes using the PCR-sequencing method. This study was performed on 15 streptomycin-resistant and five streptomycin-sensitive isolates. Among resistant isolates, 11 samples contained mutations in codon 43 of the rpsL gene, which caused the lysine to be converted to arginine. Additionally, all of the isolates had mutations in the gidB. Missense mutations in codons 92 and 20 of this gene result in the amino acids Glutamic acid or Arginine being changed to Aspartic acid or Proline, respectively. No mutations in the rrs or whiB7 were found in any of the samples. Simultaneous mutations of rpsL and gidB were found in 10 isolates, the majority of which were Beijing strain. The results showed that the mutations of rpsL and gidB genes are mostly responsible for the streptomycin resistance in the evaluated MTB isolates. Furthermore, the discovery of dual mutations in Beijing strains highlights the strain's considerable potential for developing Tuberculosis drug resistance.

Mycobacterium avium subsp. paratuberculosis and Hashimoto's thyroiditis: Is MAP the trigger?

Hashimoto's thyroiditis (HT) is an autoimmune disorder of the thyroid gland that can cause hypothyroidism. As HT is a multifactorial disorder, activation of immune responses in genetically predisposed individuals exposed to some environmental factors can contribute to it. Microorganisms, as environmental factors, including Mycobacterium avium ssp. paratuberculosis (MAP) by molecular mimicry, can be important in this autoimmune disorder. This study aimed to investigate the association between MAP and HT. This case-control study included 110 participants consisting of 60 HT patients and 50 healthy controls (HCs). Blood samples were collected. Nested PCR of the IS900 gene determined the presence of MAP DNA. The enzyme-linked immunosorbent assay (ELISA) was designed to identify antibodies (Abs) against the MAP3865c epitope, which has a homologous sequence with ZnT8 in the sera. The demographic information of all participants was recorded. Anti-TG, anti-TPO, TSH, anemia, and ruminant exposure were higer in HT patients than in the HCs (p < 0.05). MAP IS900 was detected significantly more in the patients (46.6% consisting of 30, 8.3, and 8.3% in clinical, subclinical, and unknown) than in the HCs (14%). The sera showed a remarkable frequency of reactivity against MAP3865c in the patients (38.3%) in comparison to the HCs (10%) (p = 0.0001). Furthermore, a significantly higher rate of livestock contact and traditional dairy consumption was found in individuals with MAP or anti-MAP3865c Abs positive result (p < 0.05). This study suggests a possible link between MAP and HT. These findings indicated that MAP frequency was not statistically different in the severity of HT and its shift into the clinical and subclinical forms; therefore, it could be assumed that MAPs are the initiators of the process. The results imply on a possible zoonosis transmission route of MAP from livestock products to humans. Further research is needed to confirm these results in larger groups of HT patients.

The Role of Genital Mycoplasmas in Preterm Labor.

Background: Growth of Mycoplasma in genital tract can cause problems such as infertility, pelvic inflammatory disease (PID), and preterm labor. This study was designed to evaluate the role of these bacteria in preterm labor among individuals in Gorgan city which is located in north of Iran. Methods: The study included 100 women with complaints of pain in preterm labor before 37 weeks of pregnancy (case group) and 100 women with term labor (control group) who were referred to Shahid Sayyad Shirazi Teaching Hospital in Gorgan city, north of Iran. Vaginal swabs, collected from all of these women, were evaluated for genital Mycoplasma sp. by molecular method using specific primers with polymerization chain reaction (PCR). The comparison of results was done by conducting X 2 and p<0.05 was considered significant. Results: Genital Mycoplasma was detected in 78 cases (39%) of 200 vaginal samples. Genital Mycoplasma colonization rates in the preterm and term samples were 60% and 18%, respectively, with relative risk of 2.05 (1.78-2.37) (p=0.001). The proportion of Ureaplasma parvum (44% and 15%), Ureaplasma urealyticum (11%, 3%), and Mycoplasma homins (5%, 0%) was significantly higher in women with preterm birth (PTB) than term labor. No cases of Mycoplasma genitalum were detected in this study. Conclusion: There is a significant relationship between presence of genital Mycoplasma in vaginal secretion and the risk of preterm labor.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates.

BACKGROUND: Mycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. MATERIALS & METHODS: Among 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. RESULTS: Data analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. CONCLUSION: esat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

Occurrence and risk factors of nontuberculous mycobacteria in tuberculosis-suspected patients in the north of Iran.

BACKGROUND AND OBJECTIVES: Some Nontuberculous Mycobacteria (NTM) can occasionally infect the human population and cause infections having symptoms similar to tuberculosis (TB). This study tried to provide updated data about the frequency and diversity of NTM species. MATERIALS AND METHODS: Suspicious samples of Mycobacterium tuberculosis (MTB) with both positive results in Ziehl-Neelsen (ZN) staining and Löwenstein-Jensen medium culturing were evaluated during January 2016 and December 2018 in Gorgan, Iran. After determination of MTB isolates by the growth rate, pigmentation status, the niacin test, and the insertion sequence 6110 (IS6110) PCR assay, other unknown isolates (presumably NTM) were detected by the 16S rDNA sequencing method and drawing the phylogenetic tree. Based on the patients' demographic information, their risk factors were also assessed. RESULTS: Among 226 culture-positive samples, obtained from 2994 individuals with suspected symptoms of TB, the analyses found 12 (5.3%) NTM and three Mycobacterium caprae isolates. Mycobacterium simiae (6/12) was the most prevalent NTM species. The average nucleotide similarity value was 98.2% ± 3.7. In comparison to patients with MTB (211 confirmed cases), other mycobacterium infections were more common in patients over 65 years old (Odd ratio (95% convenience interval): 2.96 (0.69 - 12.59), P = 0.14). CONCLUSION: Although the NTM species has a small portion in TB suspected patients, their prevalence has increased, mainly in elderly patients. Moreover, M. simiae was the most prevalent NTM species in our region. Therefore, identification of common species in each region is recommended and clinicians should pay more attention to them in each region.

Mycobacterium tuberculosis typing using Allele-specific oligonucleotide multiplex PCR (ASO-PCR) method.

Mycobacterium tuberculosis (M. tuberculosis) genotyping provides valuable information related to the origin and the evolution of the isolates. This study aimed to evaluate the applicability of single-nucleotide polymorphisms (SNPs) technique for lineages identification of M. tuberculosis and compare it with mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The lineages of 162 clinically isolates were evaluated using six pair primers by Multiplex-PCR based on SNPs. Among 162 isolates, 70 (43.2%) isolates were lineage 4, following that 62 (38.3%) and 22 (13.6%) isolates were lineage 3 and 2, respectively. The method could not type 8 (4.9%) isolates. Moreover, we could identify 71 out of 79 unknown isolates resulted from the MIRU-VNTR method. The results showed that the SNP typing method has the potential to determine the lineages of M. tuberculosis as a rapid laboratory screening test.

CRISPR-Cas systems in Proteus mirabilis.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a bacterial defense mechanism against bacteriophages composed of two different parts: the CRISPR array and the Cas genes. The spacer acquisition is done by the adaptation module consisting of the hallmark Cas1 Cas2 proteins, which inserts new spacers into the CRISPR array. Here we aimed to describe the CRISPR-Cas system in Proteus mirabilis (P. mirabilis) isolates. CRISPR loci was observed in 30 genomic contents of 109 P. mirabilis isolates that each locus was consisted of two CRISPR arrays and each array had a different preserved leader sequences. Only the type I-E CRISPR-Cas system was common in these isolates. The source of the spacers was identified, including phages and prophages. CRISPR spacer origin analysis also identified a conserved PAM sequence of 5'-AAG-3' nucleotide stretch. Through collecting spacers, CRISPR arrays of P. mirabilis isolates were expanded mostly by integration of bacteriophageal source of spacers. This study shows novel findings in the area of the P-mirabilis CRISPR-Cas system. In this regard, among analyzed genome of P. mirabilis isolates, Class I CRISR-Cas systems were dominant, and all belonged to type I-E. In the flanks of the CRISPR, some other elements with regulatory role were also found. A motif of 11 nt size was found to be preserved among the analyzed genome. We believe that it might has a CRISPR-Cas system transcription facilitator by targeting the Rho element.

Comparison of hspX gene sequence in the Beijing and non-Beijing Mycobacterium tuberculosis.

PURPOSE: The pathogenicity of various lineages of Mycobacterium tuberculosis (MTB) is different. This could be due to the difference in survival ability within the host macrophage. The alpha crystalline secretion protein, a product of the hspx gene, is one of the bacterial protection factors in these stressful situations. The Beijing family, part of the East Asian lineage, was reported to be more virulent. Regarding the importance of this protein in pathogenicity, this study was conducted to investigate the polymorphism of the hspx gene in Beijing family compared to non- Beijing strains. METHOD: DNA of 50 MTB isolates were extracted by boiling method. The existence of hspx gene was determined using PCR-specific primer and finally PCR product was sequenced to examine the polymorphism in both direct and reverse directions. Sequencing results were aligned by chromas software. RESULTS: The hspx gene was detected in all of the Beijing and non-Beijing isolates. The polymorphism in the sequences of this gene were not observed in all of the MTB isolates. DISCUSSION: This study indicated that hspx gene is protected. Also it has showed that lineage type was not related to the sequence of hspX gene, but the expression of this protein may be different, which requires further studies.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVES: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. MATERIALS AND METHODS: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. RESULTS: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. CONCLUSION: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

Isolation, Characterization and Genomic Analysis of a Novel Bacteriophage VB_EcoS-Golestan Infecting Multidrug-Resistant Escherichia coli Isolated from Urinary Tract Infection.

Escherichia coli (E. coli) is one of the most common uropathogenic bacteria. The emergence of multi-drug resistance among these bacteria resulted in a worldwide public health problem which requires alternative treatment approaches such as phage therapy. In this study, phage VB_EcoS-Golestan, a member of Siphoviridae family, with high lytic ability against E. coli isolates, was isolated from wastewater. Its burst size was large and about 100 plaque-forming units/infected cell, rapid adsorption time, and high resistance to a broad range of pH and temperatures. Bioinformatics analysis of the genomic sequence suggests that VB_EcoS-Golestan is a new phage closely related to Escherichia phages in the Kagunavirus genus, Guernseyvirinae subfamily of Siphoviridae. The genome size was 44829 bp bp that encodes 78 putative ORFs, no tRNAs, 7 potential promoter sequences and 13 Rho-factor-independent terminators. No lysogenic mediated genes were detected in VB_EcoS-Golestan genome. Overall VB_EcoS-Golestan might be used as a potential treatment approach for controlling E. coli mediated urinary tract infection, however, further studies are essential to ensure its safety.

In Vitro Synergistic Effect of Vancomycin and Some Antibacterial Agents Against Clinical Methicillin-Resistant and Sensitive Staphylococcus aureus Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) can be responsible for serious long-term infections. Sometimes monotherapy can be ineffective for the treatment of these infections; hence, it is hypothesized that combined drug treatment can be more potent in these cases. The aim of this study was to investigate the synergistic effect of vancomycin and eight other antibacterial agents to identify the best combination pattern in the management of MRSA. Ameri-Ziaee double synergism test (AZDAST), double-disc, checkerboard, and time-kill methods were used to assess the synergistic effect in 24 isolates of S. aureus, including 22 MRSA and two methicillin-sensitive S. aureus (MSSA). Furthermore, based on the results, handmade combined antibiotic discs were prepared to evaluate the results of the checkerboard and time-kill methods at the plate level. All the isolates were sensitive to vancomycin, linezolid, and daptomycin. Furthermore, penicillin had the highest resistance (100%) in all isolates. The synergistic activities were observed, when the vancomycin was combined with the imipenem, using three double-disc, checkerboard, and time-kill methods. The sub-minimum inhibitory concentration (MIC) amount of the combined discs could increase the diameter of the inhibition zone, confirming the results. The data obtained from this study suggested that vancomycin and imipenem together, even at sub-MIC, could be effective against MRSA and MSSA infections.

MIRU-VNTR 15 loci capability in diagnosis of Beijing M. Tuberculosis strains in comparison with Real Time PCR.

Beijing strain has great importance among Mycobacterium tuberculosis (MTB) genotypes due to their drug resistance and pathogenicity. Determination of MTB genotypes and comparison of MIRU-VNTR 15 loci and real-time PCR methods for Beijing family identification was the main objective in this study. This study was conducted on 92 confirmed MTB isolates, 31 and 61 of which previously were determined by real- time PCR as Beijing and non-Beijing, respectively. Allelic diversity, clustering rate, phylogenetic tree, clonal complexes and molecular genotypes of isolates were determined by MIRU-VNTR 15 loci on its online software. In addition MIRU-VNTR 15 loci were performed for 16 non-tuberculosis isolates. Concordance between MIRU-VNTR 15 loci and real Time PCR in determination of Beijing genotype was 95.6(89.2-98.8). 74 different pattern of MIRU-VNTR were detected in 92 MTB isolates. 69 patterns were unique and 5 clusters were determined. Largest clusters contain 11 and 6 members. The clustering rate was 19.56%. Among 15 loci, Mtub04 and MIRU10 have the highest and MIRU04 had the lowest discriminatory power. In non-Beijing isolates, New1 and Delhi/CAS with 25% and 16.3% were the most prevalent MTB genotypes. None of the non-Tuberculosis isolates had the complete MIRU-VNTR 15 loci pattern. The results of this study showed that MIRU-VNTR 15 loci, in addition to being able to differentiate MTB genotypes, can distinguish non-tuberculosis species from MTB strains, but for the exact differentiation of Beijing MTB genotypes, it may be necessary to increase the patterns known in MIRU-VNTR data base.

Carbapenem-resistant Acinetobacter baumannii isolates carrying blaOXA genes with upstream ISAba1: First report of a novel OXA subclass from Iran.

OBJECTIVES: Carbapenem-resistant Acinetobacter baumannii (CRAB) have emerged as a serious threat to public-health worldwide. This study aimed to determine the antimicrobial susceptibility of A. baumannii isolates in Iran and to investigate oxacillinase-encoding determinants and their association with insertion sequence ISAba1 in CRAB isolates. METHODS: This study was performed on A. baumannii isolates recovered from patients with burn wound infections during 2013. All isolates were evaluated for antimicrobial susceptibility by the disk diffusion method. Minimum inhibitory concentrations (MICs) of five antibiotics (imipenem, meropenem, polymyxin B, colistin and tigecycline) were determined for all CRAB isolates. PCR was performed to determine the distribution of blaOXA determinants and ISAba1 insertion upstream of each corresponding gene in the CRAB isolates. RESULTS: A total of 65 A. baumannii isolates were recovered during the 1-year period, with CRAB accounting for 63 (96.9%) of isolates. Polymyxin B, colistin and tigecycline were the most effective agents against CRAB isolates, with susceptibility rates of 100%, 87.3% and 65.1%, respectively. The proportion of CRAB isolates carrying oxacillinase determinants was as follow: blaOXA-51-like, 100%; blaOXA-23-like, 74.6%; blaOXA-24/40-like, 47.6%; and blaOXA-235-like, 12.7%. ISAba1, ISAba1-blaOXA-23-like and ISAba1-blaOXA-51-like were detected in 100%, 41.3% and 1.6% of CRAB isolates, respectively. Co-occurrence of blaOXA determinants or inserted ISAba1 upstream of the corresponding genes was associated with increased carbapenem MICs (≥128µg/mL). CONCLUSION: The emergence of high-level CRAB with blaOXA and ISAba1-blaOXA family in burn patients is a matter of increasing clinical concern, emphasising the need for infection control efforts to limit such problematic bacteria.

Genomic analyses of a novel bacteriophage (VB_PmiS-Isfahan) within Siphoviridae family infecting Proteus mirabilis.

Proteus mirabilis is one of the most common causes of complicated urinary tract infections (UTI), especially in catheter-associated UTIs. The increased resistance to antibiotics, among P. mirabilis isolates has led us to search for alternative antibacterial agents. In this study, genome of a lytic Proteus phage VB_PmiS-Isfahan, isolated from wastewater, and active against planktonic and biofilms of P. mirabilis, isolated from UTI, was analyzed. Accordingly, the genome was sequenced and its similarity to other phages was assessed by the Mauve and EasyFig softwares. "One Click" was used for phylogenetic tree construction. The complete genome of VB_PmiS-Isfahan was 54,836 bp, dsDNA with a G+C content of 36.09%. Nighty-one open reading frames (ORFs) was deduced, among them, 23 were considered as functional genes, based on the homology to the previously characterized proteins. The BLASTn of VB_PmiS-Isfahan showed low similarity to complete genome of Salmonella phages VB_SenS_Sasha, 9NA, and VB_SenS-Sergei. A comparison of Nucleic acid and amino acid sequence, and phylogenetic analyses indicated that the phage is novel, significantly differs, and is distant from other genera, within Siphoviridae family. No virulence-associated and antibiotic resistance genes were detected. Thus, VB_PmiS-Isfahan phage is suggested as a potential novel candidate for the treatment of diseases, caused by P. mirabilis.