Geni virulencije i antimikrobna osjetljivost izolata bakterije Escherichia coli dobijenih od krava s mastitisom u pokrajini Mashhad, Iran – kratko priopćenje

The purpose of this study was to determine the virulence genes and antimicrobial resistance patterns of Escherichia coli isolated from milk samples of cows with bovine mastitis. Forty-seven E. coli isolates from clinical mastitis milk samples, from five dairy farms in Northeast of Iran, were subjected to multiplex PCR to determine virulence genes stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC, and rfbE. In addition, antimicrobial susceptibility was assessed by applying disk diffusion methods. The eaeA and stx1 genes were most frequently detected in 42 (89.3%) and 34 (72.3%) isolates, respectively. However, the least frequent gene was F41 as it was found in only one isolate (2.1%). Furthermore, 9 out of 47 isolates were hlyA positive, and four isolates harbored the sta gene. The antimicrobial susceptibility demonstrated the highest resistance against lincomycin (100%) and neomycin (91.4%). Since these bacteria represent a high-risk pathogen on farms, the emergence of multiple antibiotic-resistant and pathogenic E. coli strains should be of great concern for public health.Cilj je istraživanja bio odrediti gene virulencije i antimikrobnu rezistencije bakterije Escherichia coli izolirane iz uzoraka mlijeka krava s mastitisom. Ukupno 47 izolata bakterije E. coli iz uzoraka mlijeka krava s kliničkim mastitisom, s pet mliječnih farmi u sjeveroistočnom Iranu, podvrgnuto je protokolu multipleks PCR-a kako bi se odredili geni virulencije stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC i rfbE. Antimikrobna je osjetljivost procijenjena primjenom disk-difuzijske metode. Najčešće određeni geni jesu gen eaeA, u 42 izolata (89,3 %) i gen stx1, u 34 izolata (72,3 %). Najrjeđi gen bio je F41, koji je pronađen u jednom izolatu (2,1 %). Nadalje, 9 od 47 izolata bilo je hlyA pozitivno, a četiri su izolata sadržavala gen sta. Procjena antimikrobne je osjetljivosti pokazala je najveću rezistenciju na linkomicin (100 %) i neomicin (91,4 %). Nalazi upućuju da se radi o visokorizičnim patogenima na farmama krava, stoga bi pojava višestruko rezistentnih i patogenih sojeva E. coli trebala izazvati veliku javnozdravstvenu zabrinutost

Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii

Background: Acinetobacter baumanniiis commonly resistant to nearly all antibiotics due to presence of antibiotic resistance genes and biofilm formation. In this study we determined the presence of certain antibiotic-resistance genes associated with biofilm production and the influence of low iron concentration on expression of the biofilmassociated protein gene (bap) in development of biofilm among multi-drug-resistant A. baumannii (MDRAB). Methods: Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR). Biofilm formation was assayed by the microtiter method. Results: The sequence of bap was determined and deposited in the GenBank database (accession no. KR080550.1). Expression of bap in the presence of low iron was analyzed by relative quantitative real time PCR (rqRT-PCR). Nearly half of the isolates belonged to RAPD-types A and B remaining were either small clusters or singleton. The results of biofilm formation revealed that 23 (35.4%), 18 (27.7%), 13 (20%), and 11 (16.9%) of the isolates had strong, moderate, weak, and no biofilm activities, respectively. ompA and csuE genes were detected in all, while bap and blaPER-1 were detected in 43 (66%) and 42 (64%) of the isolates that showed strong and moderate biofilm activities (p ≤ 0.05), respectively. Analysis ofbapexpression by rqRT-PCR revealed five isolates with fourfold bap overexpression in the presence of low iron concentration (20 µM). Conclusion: The results suggest thatbapoverexpression may influence biofilm formation in presence of low iron concentration

Prevalence of mecA Gene of Methicillin Resistant Staphylococcus spp. Isolated from Nosocomial Infections and Environmental Specimens in Sanandaj Hospitals, Kurdistan, Iran

Background: Methicillin resistant Staphylococcus aureus (MRSA) is one of the major agents for increasing number of serious hospital and community acquired infections. The aim of this study was to investigate the occurrence of the MRSA and mecA gene among nosocomial and environmental specimens in Kurdistan hospitals and determining the antibiotic resistance of the isolates. Materials and Methods: A total of 264 clinical and environmental Staphylococcus was isolated from Kurdistan medical University Hospitals, in February 2011 to June 2012 Iran, and their susceptibility patterns to different antibiotics were determined. Furthermore, agar screen method was used to determine oxacillin resistant isolates. Finally, using PCR, the oxacillin resistant isolates were tested for the presence of mecA gene. Results: In this study, from 88 (93.18%) Staphylococcus aureus isolates, 82 were found resistant to oxacillin using agar screen method and mecA gene was detected in 66 strains (75%). Our results showed that the agar screen method is more reliable in determination of MRSA strains compared to PCR. Conclusion: In this research the studied MRSA were found with high prevalence and mecA was widespread in S. aureus isolates in Sanandaj

Design of peptides interfering with iron-dependent regulator (IdeR) and evaluation of Mycobacterium tuberculosis growth inhibition

Objective(s): Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), stayed a global health thread with high mortality rate. Since TB has a long-term treatment, it leads high risk of drug resistant development, and there is an urgent to find new drugs. The aim of this study was designing new inhibitors for a new drug target, iron dependent regulator, IdeR. Materials and Methods: Based on the interaction most populated amino acids of IdeR to the related gene operators, 50 short peptides were modeled. Bonding affinity of short peptides toward DNA were studied by docking. Top 10 best predicted bonding affinity were selected. DNA binding assay, microplate alamar blue assay, colony counting, quantitative real time- PCR (qRT-PCR) of IdeR corresponding genes, cell wall-associated mycobactin and whole-cell iron estimation were done to prove the predicted mechanism of in silico potent constructs. Results: Amongst the 10 synthesized short peptide candidates, glycine-valine-proline-glycine (GVPG) and arginine-proline-arginine (RPR) inhibited Mtb in vitro at 200 µM concentration. qRT-PCR showed mbtB 58-fold over expression that resulted in Mtb growth inhibition. Cell wall-associated mycobactin and whole-cell iron estimation confirmed the results of qRT-PCR. Conclusion: We introduced two new lead compounds to inhibit Mtb that are promising for the development of more potent anti-tubercular therapies

Investigation of NDM-1 and OXA-48 producing carbapenem resistant Klebsiella pneumoniae ST15 in Iran

The aim of this study was to determine the frequency of carbapenem resistant Klebsiella pneumoniae (CRKP) sequence types (STs) in Iran. Samples were collected from three university hospitals in Sanandaj, Iran, from December 2016 to March 2018. Antibiotic susceptibility testing, phenotypic and genotypic detection of carbapenemases were performed. Common K. pneumoniae capsular types were sought for all isolates. The genetic relatedness of isolates was investigated by multilocus sequence typing (MLST). Plasmids were detected by PCR-based Replicon Typing (PBRT). During the study, 67 K. pneumoniae isolates were identified. Of which, 18 (26.9%) isolates were detected as carbapenemresistant. The most effective antibacterial agent was tigecycline (97%, 65 isolates) followed by imipenem and ertapenem (73.13%, 49 isolates). PCR showed that 13 isolates (19.4%) had bla(NDM-1) gene and 5 (7.5%) harbored bla(OXA-48). Examination of common capsular types showed that 2 isolates had K2 and 2 others had K54. REP-PCR revealed 10 clones and 11 singleton strains. MLST analysis of CRKP found ST15 as the most common type (13 isolates, 72.2%), but other STs were also detected namely, ST19, ST117, ST1390, and ST1594. ColE1 and IncL/M plasmids were the carriers of blaNDM- 1 and blaOXA-48, respectively. The results showed that CRKP spread in our health centers. Our results, therefore, indicate a worrying trend of resistance to carbapenems in K. pneumoniae

Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran. During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods. The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps