Characterization of integrons, extended spectrum beta lactamases and genetic diversity among uropathogenic Escherichia coli isolates from Kerman, south east of Iran.

Background and Objectives: The study aimed to investigate the distribution of genes encoding integrons, extended spectrum beta-lactamase (ESBL) in E. coli isolated from UTIs, as well as the genetic diversity among the isolates. Materials and Methods: E. coli isolates were recovered from the patients with UTI in Kerman Iran. Antibiotic susceptibility was done according to CLSI guidelines. The presence of ESBL genes and integrons was evaluated using PCR. PCR and sequencing were applied for the evaluation of cassette content of integrons. Genotyping of the isolates was performed by multiple-locus variable-number tandem repeat analysis (MLVA). Results: Imipenem was the most effective antibiotic, while the highest resistance was observed to streptomycin. In total 40.2% of isolates were ESBL producers. Of 69 integron-positive isolates, 59 only had class I integrons, 4 only had class II integrons and 6 had both types. The most common gene cassette found within class I integrons was dfrA17-aadA5 (n=27). The E. coli isolates were divided into 16 MLVA clusters. Conclusion: The current study demonstrated the simultaneous presence of class I integrons and ESBLs involved in the resistance of UPEC isolates to antibacterial agents. Our finding also revealed that the E. coli isolates belonged to diverse clones.

Hydrogel nanocomposite based on alginate/zeolite for burn wound healing: In vitro and in vivo study.

Objectives: The main objective of the current assay was to evaluate the antibacterial and regenerative effects of hydrogel nanocomposite containing pure natural zeolite (clinoptilolite) integrated with alginate (Alg) as wound healing/dressing biomaterials. Materials and Methods: The zeolites were size excluded, characterized by SEM, DLS, XRD, FTIR, and XRF, and then integrated into Alg hydrogel followed by calcium chloride crosslinking. The Alg and alginate zeolite (Alg/Zeo) hydrogel was characterized by swelling and weight loss tests, also the antibacterial, hemocompatibility, and cell viability tests were performed. In animal studies, the burn wound was induced on the back of rats and treated with the following groups: control, Alg hydrogel, and Alg/Zeo hydrogel. Results: The results showed that the hydrodynamic diameter of zeolites was 367 ± 0.2 nm. Zeolites did not show any significant antibacterial effect, however, the hydrogel nanocomposite containing zeolite had proper swelling as well as hemocompatibility and no cytotoxicity was observed. Following the creation of a third-degree burn wound on the back of rats, the results indicated that the Alg hydrogel and Alg/Zeo nanocomposite accelerated the wound healing process compared with the control group. Re-epithelialization, granulation tissue thickness, collagenization, inflammatory cell recruitment, and angiogenesis level were not significantly different between Alg and Alg/Zeo nanocomposite. Conclusion: These findings revealed that although the incorporation of zeolites did not induce a significant beneficial effect in comparison with Alg hydrogel, using zeolite capacity in hydrogel for loading the antibiotics or other effective compounds can be considered a promising wound dressing.

Evaluation of aminoglycoside modifying enzymes, SCCmec, coagulase gene and PCR-RFLP coagulase gene typing of Staphylococcus aureus isolates from hospitals in Shiraz, southwest of Iran.

Staphylococcus aureus is an important human pathogen that causes various infections. Aminoglycosides are broad-spectrum antibiotics used to treat methicillinresistant S. aureus (MRSA) infections. Typing of S. aureus isolates by coagulase gene typing and PCR-RFLP coa gene is a fast and suitable method for epidemiological studies. Aim of the present study was to evaluate the resistance to aminoglycosides, staphylococcal chromosomal cassette mec (SCCmec) types, coagulation typing and PCR-RFLP coa gene in clinical isolates of S. aureus. 192 S. aureus isolates were collected from Namazi and Shahid Faghihi hospitals. Antibiotic resistance was measured by disk diffusion method and MIC was determined for gentamicin. The presence of genes encoding aminoglycoside modifying enzymes (AME) and mecA gene were assessed by PCR. Also the coagulase typing, PCR-RFLP coa gene, and SCCmec typing were performed. Out of 192 isolated S. aureus isolates, 83 (43.2%) MRSA isolates were identified. In this study, a high resistance to streptomycin and gentamicin (98.7%) were observed. Among the AME genes, the aac (6')-Ie-aph (2″) gene was the most common. Based on the SCCmec typing, it was determined that the prevalence of SCCmec type III (45.8%) was highest. From the amplification of the coa gene, 5 different types were obtained. Also, in digestion of coa gene products by HaeIII enzyme, 10 different RFLP patterns were observed. According to this study, aminoglycoside resistance is increasing among MRSA isolates. As a result, monitoring and control of aminoglycoside resistance can be effective in the treatment of MRSA isolates. Also, typing of S. aureus isolates based on coagulase gene polymorphism is a suitable method for epidemiological studies.

The Spread of Insertion Sequences Element and Transposons in Carbapenem Resistant Acinetobacter baumannii in a Hospital Setting in Southwestern Iran.

BACKGROUND: Acinetobacter baumannii is one of the most important hospital pathogenic bacteria that cause infectious diseases. The present study aimed to determine the frequency of carbapenem resistance genes in association with transposable elements and molecular typing of carbapenem-resistant A. baumannii bacteria collected from patients in Shiraz, Iran. MATERIALS AND METHODS: A total of 170 carbapenem-resistant A. baumannii isolates were obtained from different clinical specimens in two hospitals. The minimum inhibitory concentrations (MIC) of imipenem were determined and the prevalence of OXA Carbapenemases, Metallo-ß-lactamases genes, insertion sequences (IS) elements, and transposons were evaluated by the polymerase chain reaction (PCR) method. Finally, molecular typing of the isolates was performed by the Enterobacterial Repetitive Intergenic Consensus-PCR method. RESULTS: The MICs ranged from 16 to 1,024 µg/mL for imipenem-resistant A. baumannii isolates. Out of the 170 carbapenem resistant A. baumannii isolates, blaOXA-24-like (94, 55.3%) followed by blaOXA-23-like (71, 41.7%) were predominant. In addition, A. baumannii isolates carried blaVIM (71, 41.7%), blaGES (32, 18.8%), blaSPM (4, 2.3%), and blaKPC (1, 0.6%). Moreover, ISAba1 (94.2%) and Tn2009 (39.2%) were the most frequent transposable elements. Furthermore, (71, 44.0%) and (161, 94.7%) of the ISAba1 of the isolates were associated with blaOXA-23 and blaOXA-51 genes, respectively. Besides (3, 1.7%), (1, 0.6%) and (5, 2.9%) of blaOXA-23 were associated with IS18, ISAba4, and ISAba2, respectively. Considering an 80.0% cut off, clusters and four singletons were detected. CONCLUSION: According to the results, transposable elements played an important role in the development of resistance genes and resistance to carbapenems. The results also indicated carbapenem-resistant A. baumannii bacteria as a public health concern.

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate.

Background: There is limited information on the three-dimensional (3D) prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of phosphotransferase membrane receptor A/B (PmrA/B) in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods: The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and minimum inhibitory concentration assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results: Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 ß-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three ß-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 ß-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: The DNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii.

Frequency and molecular epidemiology of class A ESBLs producing Enteroinvasive Escherichia coli (EIEC) isolates among patients with diarrhea.

AIM: This study aimed to investigate the frequency and molecular epidemiology of class A ESBLs producing Enteroinvasive Escherichia coli (EIEC) isolates among patients with diarrhea. BACKGROUND: Antibiotic resistance is widespread among diarrheagenic Escherichia coli (DEC) in developing countries. Information regarding Extended-Spectrum ß-Lactamases (ESBLs) in diarrheagenic pathogens should be considered in clinical management when an optimal treatment is required. METHODS: A total of 581 stool samples were collected from patients with diarrhea in Ahvaz, Iran. PCR was used for the presence of the ipaH gene to confirm EIEC strains. The antibiotic resistance pattern of all EIEC isolates was determined by the disk diffusion method. EIEC isolates were screened for class A ß-lactamase genes. Genotyping of harboring ß-lactamase genes was performed by Multi-Locus VNTR Analysis (MLVA). RESULTS: Among 13 EIEC isolates, 9 isolates (69.2%) were found ESBL positive by double-disk synergy test (DDST) and PCR. Furthermore, bla CTX-M-15 and bla CTX-M-1 genes were detected in 77.8% (n=7) and 44.5% (n=4) of the bla CTX-M-1 group. On the other hand, the bla TEM-1 gene was detected in 66.6% (n=6). None of the isolates had bla SHV-1, bla KPC, or bla GES genes. Six MLVA genotypes were identified. CONCLUSION: The current study revealed that the presence of ESBLs genes mediates the resistance of EIEC isolates to the majority of antibiotics in this region. The presence of ESBLs genes in different MLVA types showed that one specific clone was not responsible for spreading the EIEC isolates.

Distribution of Genes Encoding Virulence Factors and the Genetic Diversity of Enteroinvasive Escherichia coli (EIEC) Isolates from Patients with Diarrhea in Ahvaz, Iran.

BACKGROUND: Entero-invasive E. coli (EIEC) is one of the causes of bacillary dysentery in adults and children. The ability of EIEC to invade and colonize the surface of epithelial cells is influenced by many virulence factors. This study aimed to investigate the distribution of virulence factor genes in EIEC strains isolated from patients with diarrhea in Ahvaz, Iran, as well as the genetic diversity between these isolates by Multilocus variable-number tandem repeat analysis (MLVA). MATERIALS AND METHODS: A total of 581 diarrheic stool samples were collected from patients with diarrhea attending two hospitals, in Ahvaz, Iran. The E. coli strains were identified by biochemical methods. Subsequently, all E. coli isolates were identified as EIEC by polymerase chain reaction (PCR) for the ipaH gene. The EIEC isolates evaluated by PCR for the presence of 8 virulence genes (ial, sen, virF, invE, sat, sigA, pic, and sepA). All EIEC strains were genotyped by the MLVA typing method. RESULTS: A total of 13 EIEC isolates were identified. The presence of ial, virF, invE, sen, sigA, pic, and sat genes was confirmed among 92.3%, 84.6%, 84.6%, 76.9%, 69.2%, and 15.3% of EIEC isolates, respectively. On the other hand, none of the isolates were positive for the sepA gene. The EIEC isolates were divided into 11 MLVA types. CONCLUSION: Our results showed a high distribution of virulence genes among EIEC isolates in our region. This study showed that MLVA is a promising typing technique for epidemiological studies. MLVA can supply data in the form of codes that can be saved in the database and easily shared among laboratories, research institutes, and even hospitals.

Virulence Factors, Capsular Serotypes and Antimicrobial Resistance of Hypervirulent Klebsiella pneumoniae and Classical Klebsiella pneumoniae in Southeast Iran.

BACKGROUND: The present study was conducted to investigate the distribution of virulence factors, capsular serotypes and antibiotic resistance properties of classical Klebsiella pneumoniae (cKP) and hypermucoviscous/hypervirulent Klebsiella pneumoniae (hvKP) isolated from different clinical specimens in Kerman, south-east of Iran. MATERIALS AND METHODS: A total of 146 K. pneumoniae isolates were obtained from different clinical specimens. HvKP isolates were identified using the string test. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence-associated genes, rmpA, kfu, fimH, mrkD, allS, iutA, magA, entB and ybtS were evaluated by PCR. Antimicrobial susceptibility was also determined using the disc diffusion method. RESULTS: Out of 146 K. pneumoniae isolates, 22 (15.1 %) were hvKP. More than half of the hvKP isolates, 13 (59.1%), belonged to non-K1, K2, K5, K20, K54, K57 serotypes. Out of 22 hvKP isolates, 3 and 3 had K1 and K2 serotypes respectively. Among all isolates, entB 140 (95.9%) and mrkD 138 (94.5%) were the most common virulence genes. RmpA, iutA and kfu were associated with hvKP isolates (P-value <0.05). However, no significant difference was found in fimH, allS, mrkD, entB and ybtS genes between hvKP and cKP strains. HvKP exhibited significantly lower resistance rates to all antimicrobial agents than cKP, except to trimethoprim-sulphamethoxazole and ampicillin (P-value <0.05). CONCLUSION: The frequency of hvKP was low, but overall, the prevalence of virulence-related genes was higher in hvKP than cKP. HvKP was not related to specific serotypes. Furthermore, hvKP isolates were more susceptible to antimicrobial agents compared to cKP isolates.

Antibiotic resistance, ESBL genes, integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran.

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum ß-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1-15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for blaTEM and blaCTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.

Endemic dissemination of different sequence types of carbapenem-resistant Klebsiella pneumoniae strains harboring bla NDM and 16S rRNA methylase genes in Kerman hospitals, Iran, from 2015 to 2017.

INTRODUCTION: The emergence and spread of Klebsiella pneumoniae strains resistant to multiple antimicrobial agents are considered as a serious challenge for nosocomial infections. MATERIALS AND METHODS: In this study, 175 nonrepetitive clinical isolates of K. pneumoniae were collected from hospitalized patients in Kerman, Iran. Extended-spectrum ß-lactamases (ESBLs), AmpC, and carbapenemase-producing isolates were recognized by phenotypic methods. The resistance genes including efflux pumps oqxA/oqxB, 16S rRNA methylase, ESBL, AmpC, and carbapenemase were detected by PCR-sequencing method. Molecular typing was performed by enterobacterial repetitive intergenic consensus-PCR and multilocus sequence typing methods among bla NDM-positive isolates. RESULTS: Thirty-seven (21.14%) isolates along with sequence types (STs): ST43, ST268, ST340, ST392, ST147, and ST16 were harbored bla NDM. ST43 in 2015 and ST268 during 2016-2017 were the most frequent STs among New Delhi metallo-beta-lactamase (NDM)-positive isolates. We found the distribution of some isolates with bla NDM, bla CTX-M, bla SHV, bla OXA, bla TEM, bla CMY, rmtC, and oqxA/oqxB. Enterobacterial repetitive intergenic consensus-PCR represented seven clusters (A-G) plus four singletons among NDM-positive isolates. This study provides the first report of bla NDM-1-positve K. pneumoniae along with ST268 as well as the spread of nosocomial infections with six different STs harboring bla NDM-1 and other resistance genes in hospital settings especially neonatal intensive care unit. CONCLUSION: The dissemination of various clones of NDM-producing K. pneumoniae can contribute to increase the rate of their spread in health care settings. Therefore, molecular typing and detection of resistance genes have an important role in preventing and controlling infection by limiting the dissemination of multidrug-resistant isolates.

Distribution of Aminoglycoside-Modifying Enzymes and Molecular Analysis of the Coagulase Gene in Clinical Isolates of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus.

Enzymatic alteration of aminoglycosides by aminoglycoside-modifying enzymes (AMEs) is the major mechanism of resistance to aminoglycosides. The purpose of this study was to determine the frequency of AME genes, staphylococcal chromosomal cassette mec (SCCmec) types, and molecular analysis of the coagulase (coa) gene in Staphylococcus aureus strains isolated from clinical specimens. Totally, 102 S. aureus were tested by disk diffusion and microbroth dilution methods for susceptibility to aminoglycosides. AMEs genes and SCCmec types were determined by multiplex polymerase chain reaction (PCR). For polymorphism analysis, the 3' end region of the coa gene was amplified by PCR and the products were then subjected to restriction digestion with HaeIII enzyme. Of the 102 S. aureus, 42 (41.2%) were methicillin-resistant S. aureus (MRSA). Thirty-five (83%) of MRSA strains were resistant to kanamycin, 32 (76.2%) to tobramycin, 30 (71.4%) to gentamicin, 25 (59.5%) to amikacin, and 10 (23.8%) to netilmicin. The aac(6')-Ie-aph(2″) was the most frequent gene among MRSA isolates 19 (45.2%), followed by aph(3')-IIIa 8 (19%), ant(4')-Ia 6 (14.3%), and aph(2″)-Id 2 (4.8%). SCCmec types included type I 10 (23.8%), II 1 (2.4%), III 21 (50%), and IV 7 (16.7%). Three (7.2%) isolates were nontypeable. Digestion of the PCR products of the coa gene yielded 19 distinct restriction fragment length polymorphism patterns. In conclusion, given the alarming rate of resistance to aminoglycosides among MRSA, the monitoring of aminoglycoside resistance and AME genes should be performed to limit the spread of aminoglycoside resistance among MRSA isolates. Several variants of the coa gene were found in the studied isolates, although the majority of the MRSA isolates belonged to a limited number of coagulase types.

A Systematic In Silico Analysis of the Legionellaceae Family for Identification of Novel Drug Target Candidates.

The family Legionellaceae consists of Gram-negative bacteria that are widely distributed in aquatic environments around the world. This family consists of a single genus, Legionella, that is recognized as an important cause of community-acquired pneumonia and hospital-acquired pneumonia. Legionella consists of intracellular pathogens, thus cellular pharmacokinetic and pharmacodynamic properties of an antibiotic against these bacteria as well as uptake and subcellular distribution into macrophages should be considered for a successful outcome of disease. Treatment strategies for Legionella infection require a combination of multiple antibiotics. Hence, because of the possible development of resistance to the drugs during therapy, a new alternative targeted therapy is yielding promising results. In this study, a comprehensive in silico target identification pipeline was performed on members of the family Legionellaceae to identify the best targets. Using a homology-based computational pipeline method, new drug targets were identified. Of 4,358 analyzed proteins, 18 proteins, including proteins involved in metabolism (amino acid, energy, and lipid metabolisms), cellular transport, cell division, and cell motility, were selected as the final putative drug targets. These proteins play an important role in the survival and propagation of Legionella infection. In conclusion, homology-based methods could improve the identification of novel drug targets and the drug discovery process, which can potentially be effective for the prevention and treatment of Legionella infections.

Emergence of co-existence of blaNDM with rmtC and qnrB genes in clinical carbapenem-resistant Klebsiella pneumoniae isolates in burning center from southeast of Iran.

Dissemination of carbapenemase-producing Klebsiella pneumoniae along with 16S rRNA methyltransferase (16S-RMTase) has been caused as a great concern for healthcare settings. The aim of this study was to investigate the prevalence of resistance genes among K. pneumoniae isolates. During October 2015 to February 2016, 30 non-duplicative K. pneumoniae strains were isolated from clinical specimens in a burn center in Kerman, Iran. Antibiotic susceptibility tests of isolates, carbapenemase, extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamase-producing isolates were determined by phenotypic methods. The beta-lactamase, oqxA/B efflux pumps, qnr A, B, S, 16S-RMTase (rmt A, B, and C), and mcr-1 resistance genes were determined by PCR. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for molecular typing. According to our findings, tigecycline has been shown the most active agent against K. pneumoniae isolates. Antibiotic resistance genes, blaTEM-1, blaSHV-12, blaCTX-M-15, blaCTX-M-2, blaNDM-1, blaFOX, blaMOX, blaEBC, blaACC, blaCIT, rmtC, qnrB, qnrS, oqxA, and oqxB, were detected in 11 (36.7%), 13 (43.3%), 11 (36.6%), 5 (16.6%), 9 (30%), 1 (3.3%), 1 (3.3%), 1 (3.3%), 1 (3.3%), 2 (6.7%), 1 (3.3%), 9 (30%), 2 (6.7%), 18 (60%), and 13 (43.3%) of isolates, respectively. The blaNDM-1 with rmtC was simultaneously observed in one isolate. ERIC-PCR results revealed 25 distinct patterns in eight clusters (A-H) and five singletons. This study highlights the high prevalence of blaNDM and emergence of rmtC among carbapenem-resistant K. pneumoniae. The resistance genes are often co-located on the conjugative plasmids, so it might be the reason of the rapid spread of them. The prevalence of multidrug-resistant K. pneumoniae isolates limits the available treatment options and presents tremendous challenges to public health.

Distribution of genes encoding virulence factors and multilocus variable-number tandem-repeat analysis (MLVA) of entero-aggregative Escherichia coli (EAEC) isolated in Iran from patients with diarrhoea.

PURPOSE: Entero-aggregative Escherichia coli (EAEC) is one of the main causes of diarrhoea worldwide. Several virulence factors have been identified in EAEC. This study was conducted to investigate the distribution of virulence factor genes in EAEC strains isolated in Iran from children with diarrhoea, as well as the genetic similarity of these isolates. METHODOLOGY: A total of 37 EAEC isolates were tested for the presence of 11 virulence genes by PCR, and the genetic relatedness of these strains was further determined by multilocus variable-number tandem-repeat analysis (MLVA). RESULTS: All EAEC isolates were typical EAEC. pic, set1A and set1B were the most prevalent genes, detected in 54.1 % of the isolates, followed by sat (43.2 %), astA (32.4 %), pet (24.3 %), agg4A (24.3 %), sepA (18.9 %), agg3A (13.5 %), sigA (8.1 %), aggA (8.1 %) and aafA (5.4 %). Using MLVA, the 37 isolates were divided into 32 types and classified into five clonal complexes. CONCLUSION: This study showed that EAEC is a heterogeneous group of E. coli possessing a broad range of virulence factors. There was no notable association between MLVA patterns and virulence profiles.

Development, physicochemical characterization, and antimicrobial evaluation of niosomal myrtle essential oil.

Myrtus communis (myrtle) is well known for its therapeutic effects pertaining to the major secondary metabolites including essential oils (EOs). EOs are composed of volatile compounds and simply evaporate or decompose leading to their instability. Preparation of EOs niosomal formulation may be a promising approach to deal with these obstacles. Niosomal formulations of myrtle essential oil (nMEO) were provided using non-ionic surfactants and cholesterol (Chol). In the next steps, vesicle size, zeta potential, percentage of entrapment efficiency (EE%) and physical stability of nMEO were investigated. Finally, the effect of myrtle essential oil (MEO) and nMEO on microbial growth inhibition were assessed. Values for nMEO size and zeta potential ranged from 6.17 ± 0.32 to 7.24 ± 0.61 (µm) and -20.41 ± 0.17 to -31.75 ± 0.45 (mV), respectively. Higher degrees of EE% were obtained by F6 formulation (Span/Tween 60:Chol (50:50 molar ratio)). Moreover, niosomes have been reported to be stable at 4 °C during a three-month time period. It was revealed that nMEO F6 formulation inhibited growth of Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, and Bacillus subtilis at concentrations lower than that of MEO. Overall, it was found that stable multilamellar vesicles were formed in the presence of 0.5% MEO and F6 formulation. This formulation also exhibited better antibacterial activity than MEO.

Serogroups, subtypes and virulence factors of shiga toxin-producing Escherichia coli isolated from human, calves and goats in Kerman, Iran.

AIM: The present study was conducted to detect the occurrence, serogroups, virulence genes and phylogenetic relationship of shiga toxin-producing Escherichia coli (STEC) in human, clave and goat in Kerman (southeast of Iran). BACKGROUND: STEC have emerged as the important foodborne zoonotic pathogens causing human gastrointestinal disease and confirming the risk to public health. METHODS: A total of 671 fecal samples were collected from diarrheic patients (n=395) and healthy calves (n=156) and goats (n=120) and screened for the presence of stx gene. Furthermore, the prevalence of stx1 and stx2 variants, serotypes (O157, O145, O103, O26, O111, O91, O128, and O45), phylogenetic groups and the presence of ehxA, eae, hylA, iha and saa virulence genes were studied. RESULTS: Prevalence of STEC in human diarrheic isolates was 1.3% (5 isolates), in claves was 26.3% (41 isolates) and in goats was 27.5% (33 isolates). stx1 gene was the most prevalent variant and detected in 75 isolates. Furthermore, stx1c was the most predominant stx subtype, found in 56 isolates. The ehxA identified in 36 (45.6%) isolates, followed by iha 5 (6.3%), eaeA 4 (5.1%), hlyA 2 (2.5%) and saa 2 (2.5%). Most of the isolates belonged to phylogroup B1. Only two O26 and one O91 isolates were detected in our study. CONCLUSION: Our results show that STEC strains were widespread among healthy domestic animals in the southeast of Iran.

Genome-Wide Identification of Potential Drug Target in Enterobacteriaceae Family: A Homology-Based Method.

The Enterobacteriaceae is a large family of Gram-negative, facultative anaerobic, non-spore forming rod-shaped bacteria that includes harmless and pathogenic organisms. The emergence and development of drug resistance in Enterobacteriaceae is complicating the treatment of serious infections. The aim of this study is to predict and characterize putative drug targets in Enterobacteriaceae family employing a homology-based computational method. The final putative drug targets were qualitatively characterized via cellular function prediction, subcellular localization prediction, broad-spectrum, and druggability analyses. Of 6,327 analyzed proteins, 35 proteins were selected as final putative drug targets in Enterobacteriaceae family. These putative drug targets were involved in different vital pathways like metabolism, biosynthesis of macromolecule, and cell division. Predicted drug targets were also localized in the cytoplasm and cytoplasmic membrane of the pathogen that acts as antimicrobial or vaccine targets. Of 35 drug targets, 5 targets were druggable and 30 targets were not druggable and were predicted as novel drug targets, which should be further evaluated to develop new antimicrobial. Thirteen drug targets were considered as broad-spectrum targets. It is expected that results of our study could facilitate the production of novel antibacterial for efficient treatment of infections caused by Enterobacteriaceae pathogens.

Role of antigen-43 on biofilm formation and horizontal antibiotic resistance gene transfer in non-O157 Shiga toxin producing Escherichia coli strains.

BACKGROUND AND OBJECTIVES: The objectives of this study were to evaluate the antibiotic resistance profiles, biofilm formation, presence of antigen 43 (Ag43) gene, and transfer of antibiotic resistance phenotype among non-O157 Shiga toxin producing Escherichia coli (STEC). MATERIALS AND METHODS: From October 2014 to November 2015 a total of 276 stool samples were collected from healthy calves, goats and 395 patients with the sign of nonbloody diarrhea and screened for presence of stx and serotype O157 genes by polymerase chain reaction (PCR) technique. Susceptibility to 14 antibiotics was determined as per CLSI guideline. Presence of Ag43 and intimin (eaeA) genes were detected by PCR. Biofilm formation was measured by microtiter plate method. Conjugation was carried out by membrane filter technique. RESULTS: We isolated 74 (93.6%) non-O157 STEC strains from 41 calves, 33 goats and 5 (6.3%) patients' stools, however, no O157 serotype was detected in our study. Resistance was observed most commonly to tobramycin (66.2%), kanamycin (48.6%), and amikacin (29.7%) and less frequently to ciprofloxacin (4.1%), amoxicillin-clavulanic acid (5.4%), and ceftriaxone (9.5%) in isolates recovered from calves and goats fecal samples, whereas, all human isolates were sensitive to ceftazidime, ciprofloxacin, tobramycin and imipenem, respectively. Furthermore, Ag43 was detected in 60 STEC isolated from animals and 5 human origins (no eaeA gene was found in this study). Biofilm formation from Ag43+ and Ag43- colonies showed 20 isolates with strong biofilm activities. Cefotaxime resistance phenotype was transferred to E. coli ATCC 25922.1 (Nalr) by conjugation at a frequency of 1.6×10-4. CONCLUSION: From the above results we concluded that, human infections with non-O157 STEC were significantly low in Kerman. Ag43 was insignificant with biofilm quantity in most cases.

Virulence Gene Profile and Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) of Enteroinvasive Escherichia coli (EIEC) Isolates From Patients With Diarrhea in Kerman, Iran.

BACKGROUND: Enteroinvasive Escherichia coli (EIEC) isolates cause dysentery in humans. Several virulence factors associated with EIEC pathogenesis have been characterized. Multilocus variable-number tandem-repeat analysis (MLVA) is a PCR-based method that has been used for genotyping bacterial pathogens. OBJECTIVES: The aim of this study was to investigate the distribution of virulence factor genes in EIEC isolates from patients with diarrhea in Kerman, Iran, as well as the genetic relationships between these isolates. PATIENTS AND METHODS: A total of 620 diarrheic stool samples were collected from patients attending two hospitals in Kerman from June 2013 to August 2014. All isolates were confirmed as EIEC by PCR for the ipaH gene. The EIEC isolates were evaluated by PCR for the presence of nine virulence genes (ial, set1A, sen, virF, invE, sat, sigA, pic, and sepA). MLVA was performed for all EIEC isolates. RESULTS: A total of 11 EIEC isolates were identified, and all were positive for the ial gene. The invE and virF genes were observed in 81.8% of the isolates, while sen, sigA, and pic were detected in 72.7%, 63.6%, and 27.3% of the isolates, respectively. None of the isolates were positive for the sat, set, and sepA genes. Using MLVA, the 11 total isolates were divided into five types. CONCLUSIONS: By studying the profiles of virulence genes and MLVA, it can be concluded that EIEC isolates do not have high heterogeneity and are derived from a limited number of clones.

Molecular diagnosis and anti-microbial resistance patterns among Shigella spp. isolated from patients with diarrhea.

AIM: This study aims to determine the serogroup distribution and molecular diagnosis, as well as antimicrobial resistance profiles among Shigella spp. isolated from patients with diarrhea in Kerman, southeast of Iran. BACKGROUND: Shigella species are frequent cause of bacterial dysentery worldwide. Previous studies have been reported that S. sonnei and S. flexneri are the most prevalent serogroups in various parts of Iran. PATIENTS AND METHODS: A total of 624 stool samples were randomly collected from patients with diarrhea from June 2013 to August 2014. Biochemical and serological characterizations were performed for identifying Shigella spp. In addition, the multiplex PCR assay was carried out for the detection and differentiation of three pathogenic Shigella spp. Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. RESULTS: Fifty six (9%) Shigella strains were isolated from stool samples. The most common species were S. flexneri 31(55.4%), followed by S . sonnei 18(32.1%) and S. boydii 7(12.5%). S. dysentery was not detected in the present study. All the isolates that identified by serological test as Shigella spp. were confirmed by the multiplex PCR method. The highest rate of resistance was observed for ampicillin and trimethoprim-sulphamethoxazole antibiotics with 52(92.9%) resistant, followed by tetracycline 44(78.6%) and cefotaxime 33(58.9%). All Shigella isolates were susceptible to ciprofloxacin. A significant relationship was found between the Shigella species and cefotaxime resistance (p<0.05). CONCLUSION: S. flexneri was found as the most prevalent serogroup causing shigellosis. The high rate of resistance to third-generation cephalosporins limits the treatment options available for the management of shigellosis in Kerman, Iran.