Investigation of EpsA, OmpA, and Bap Genes among MDR and XDR Acinetobacter baumannii Isolates in Khorramabad, Iran.

BACKGROUND: Acinetobacter baumannii is an opportunistic hospital pathogen with high antibiotic resistance, and the ability to produce biofilm. This study aimed to investigate epsA, ompA, and bap genes involved in biofilm formation in MDR and XDR clinical isolates of Acinetobacter baumannii in Khorramabad, Iran. METHODS: In this study, 79 A. baumannii isolates were collected from various samples of the patients admitted to tertiary hospitals in Khorramabad city, Iran, between January and August 2019. After performing the semi-quantitative evaluation of biofilm production by microtiter plate assay, screening of isolates carrying epsA, ompA, and bap genes was done by PCR method. Finally, statistical analyses were conducted using SPSS 22. RESULTS: Among 79 A. baumannii isolates, 52% XDR, 40% MDR, and 16% non-XDRMDR isolates were found to be biofilm producers. All XDR and 94% MDR isolates had ompA and epsA genes, and bap genes were detected among > 80% of these isolates. Moreover, the presence of biofilm-related genes and biofilm production among non-XDRMDR isolates were less than among resistant isolates (p≤ 0.01). CONCLUSION: Based on the results, biofilm production and simultaneous presence of epsA, ompA, and bap genes among MDR, and XDR A. baumannii isolates have been found to be significantly more than non-XDR-MDR isolates.

Antibacterial effects and cellular mechanisms of iron oxide magnetic nanoparticles coated by piroctone olamine against some cariogenic bacteria.

Background: The present study aims to study antibacterial effects and cellular mechanisms of iron oxide magnetic nanoparticles loaded with piroctone olamine (Fe3O4@PO NPs) against some cariogenic bacteria (Streptococcus mutans and Actinomyces viscosus). Methods: Nanoparticles was synthesized by the coprecipitation method. Antibacterial effects of Fe3O4@PO NPs were performed by calculating the minimum inhibitory concentration (MIC). We also evaluated the level of reactive oxygen species (ROS) and protein leakage to assess whether antibacterial effects may be dependent on these mechanisms. Results: The results demonstrated that PO showed the lowest antibacterial effect compared to other drugs tested with MICs values of 53.33 and 64 µg/ml for S. mutans and A. viscosus, respectively. In contrast, the highest antibacterial effect was related to Fe3O4@PONPs with MICs values of 2.66 and 3.33 µg/ml for S. mutans and A. viscosus, respectively. Fe3O4@PONPs, Fe3O4MNP, and PO markedly increased (p < 0.001) ROS production and protein leakage of tested bacteria at ≥» MIC, ≥1/3 MIC, and ½ MIC, respectively. Conclusion: The findings of the present survey revealed the promising antibacterial effects of Fe3O4@PONP against some cariogenic bacteria; whereas it triggered the ROS production and protein leakage as the possible antibacterial mode of action of anti-infective agents. However, additional surveys are necessary to elucidate the accurate mechanisms of these nanoparticles.

Frequency of Helicobacter pylori Infection in Patients with Peptic Ulcer Referred to the Endoscopy Departments of Khorramabad City Hospitals, Iran, During 2013-2016.

BACKGROUND: The present study investigated the prevalence of Helicobacter pylori infection in peptic ulcer patients referred to the endoscopy departments in Khorramabad hospitals during 2013- 2016. METHODS: The early pool of the study included all patients who had been referred to the endoscopy department and whose endoscopic and pathology reports were available and complete. After recording endoscopic reports, 1224 peptic ulcer (gastric or duodenal ulcer) cases, in which biopsy assays were performed to examine the type of ulcer and the presence of Helicobacter pylori bacteria, were selected. Pathology reports were collected by referring to the pathology departments. The information in the pathology report, including demographic information, was included in a pre-designed questionnaire to match the endoscopic reports, the location of the pathology sample, and other details, including the presence or absence of Helicobacter pylori bacteria. Finally, the data were analyzed using SPSS, version 21. RESULTS: For all the 1224 patients studied, the mean age was 15.5 ± 17.5 years old. A total of 664 (54.2%) cases had gastric ulcers, 445 (36.4%) cases had duodenal ulcers, and 115 (9.4%) had both gastric and duodenal ulcers. Among gastric ulcer patients, 512 (65.7%) had a gastric ulcer in the antrum area, and 74.3% (579 patients) of the gastric ulcers were clean base type. CONCLUSION: The prevalence of infection was statistically significant in terms of the type, location, and number of peptic ulcers, including both gastric ulcer and duodenal ulcer.

First Report on the Characteristics of Methicillin-Resistant Staphylococcus Capitis Isolates and an NRCS-A-clone Related Isolate Obtained from Iranian Children.

BACKGROUND: Methicillin-resistant staphylococcus capitis (MRSC) NRCS-A clone (Multi- resistant and vancomycin-non susceptible) has been recently described as an emerging cause of nosocomial bacteremia, especially in neonatal intensive-care units (NICUs). OBJECTIVE: The objective of this study was to evaluate the antibiotic and antiseptic resistance patterns, biofilm-producing ability and the prevalence of SCCmec and ACME types among MRSC isolates as well as to check the possible presence of NRCS-A clone at Tehran's Children's Medical Center, Iran. METHODS: A total of 256 coagulase-negative Staphylococcal isolates were collected, of which 10 S. capitis isolates were obtained and tested for susceptibility against 13 antimicrobial and 3 antiseptic agents, as well as biofilm production. The presence of 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs) were tracked. RESULTS: Seven out of 10 S. capitis isolates were MRSC (MIC90 van=8µg/mL) and resistant to trimethoprim/sulfamethoxazole, produced biofilm, (3 as strong biofilm producers) and carried ACME types I and II. Despite the identification of mec and ccr complexes in some isolates, all the SCCmec cassettes were untypeable (UT). CONCLUSION: According to the studied features, only one isolate belonged to the NRSC-A clone. The results indicate that MRSC with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients, particularly children.

A comparative characterization of nasal and clinical isolates of Staphylococcus aureus from west of Iran.

BACKGROUND AND OBJECTIVES: Recently, the rise of methicillin-resistant Staphylococcus aureus (MRSA) isolated from hospital healthcare workers (HCWs) and various infectious samples has become one of the main concerns in hospital settings. Therefore, epidemiological studies are necessary to monitor antibiotic resistance patterns in each region and to study the pathogenesis of this strain to control infections. MATERIALS AND METHODS: In this cross-sectional study, a total of 100 S. aureus isolates, including 50 isolates obtained from the anterior nares of healthcare workers, as well as 50 other isolates cultured from the various clinical specimens from the referral hospitals in Khorramabad (West of Iran) were tested. All isolates were examined to determine antibiotic resistance pattern, and the presence of staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb) and mecA genes. RESULTS: The mecA gene was found among 36% (18/50) of the clinical S. aureus isolates (CSIs) and 14% (7/50) of nasal S. aureus isolates (NSIs), with statistically significant difference (X2 = 6.53; p = 0.011). The difference between the frequency rate of sea gene among MRSA strains isolated from clinical specimens (46.6%, 7/15) was significant compared to strains isolated from nostrils (14.3%, 1/7) (X2 = 3.85; p = 0.049). CONCLUSION: The frequency of mecA, sea, and seb genes among the clinical samples was more than strains isolated from the nostrils of healthcare personnel.

Molecular Detection of Carbapenem Resistance in Acinetobacter Baumannii Isolated From Patients in Khorramabad City, Iran.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen, which causes a wide range of infections in hospitals, especially in intensive care units. Nowadays, due to the high resistance of Acinetobacter bumanni to antibiotics, this study, in addition to the phenotypic and genotypic investigations of drug resistance, focused on determining the molecular types of Acinetobacter baumannii isolated from patients in Khorramabad city by the pulsed-field gel electrophoresis (PFGE) method. MATERIALS AND METHODS: In this cross-sectional study, 50 samples of Acinetobacter baumannii were collected from educational hospitals in Khorramabad city, Iran, from January to August 2015. They were identified in the laboratory using biochemical tests and culture methods. After determining the drug resistance pattern by the disc diffusion method and percentage of resistance genes to carbapenems, Acinetobacter baumannii isolates were analyzed using the PFGE method using the Apa1 enzyme. RESULTS: The highest antibiotic resistance observed for Acinetobacter baumannii strains was against ampicillin-sulbactam (100%) and aztreonam (98%). The highest sensitivity was to polymixin B (100%) and colistin (94%), and also to the OXA-51-like gene present in all samples. The OXA-23-like gene was positive in 44 (88%) samples. PFGE results showed that Acinetobacterbaumannii strains had 33 different pulsotype patterns, of which 27 patterns had more than one strain and 23 had only one strain. CONCLUSION: Due to the high resistance of Acinetobacter baumannii and its ease of spread and its ability to transfer resistance genes, resistance control methods should be used in the disinfection of hospital areas. Hospital staff should observe hygiene standards and there should also be a reduction in antibiotic use.

Systematic Review and Meta-analysis of Carbapenem Resistance of Acinetobacter baumannii in Iran.

BACKGROUND: The prevalence of carbapenem resistance in Acinetobacter baumannii has been increasing worldwide, and therapeutic options are extremely limited. We performed a systematic review to evaluate the phenotypic and genotypic carbapenem resistance in A.baumannii reported in Iran. METHODS: We systematically searched Pub Med, Web of Science Direct, and Google scholar databases to identify studies addressing the carbapenem resistance of A. baumannii. The selected papers were published between 2005 and 2016, but the sample collection period was between 2002 and 2016. To estimate the prevalence, the Der Simonian and Laird randomized models, a 95% confidence interval, was used. For the heterogeneity check, I2 test was used. The Egger test was used to check the propagation bias. RESULTS: Analysis of data indicates that there was an increase in resistance to carbapenems from 4.5% in 2005 to a 100% prevalence rate in 2016 (65.4 (95% CI: 58.8 - 71.6). CONCLUSION: According to the results of this study, the rate of resistance to carbapenem in A.baumannii has been increasing in Iran. The presence of carbapenem-resistant isolates is a major concern, because carbapenem is the main drug used against Multi Drug Resistant (MDR) isolates.

Mentha piperita essential oils loaded in a chitosan nanogel with inhibitory effect on biofilm formation against S. mutans on the dental surface.

Mentha piperita essential oils (MPEO) were loaded into chitosan nanogel to use as antibiofilm agent against Streptococcus mutans and to protect its dental plaque. Chitosan nanoparticles (CsNPs) were prepared by sol-gel method using linking bridge of tripolyphosphate (TPP). Physiological properties of MPEO-CNs were assessed by FTIR, SEM/EDX, DLS and zeta potential. Release kinetics, MIC and MBC were determined for MPEO-CNs. Expression of biofilm-associated genes including 8 genes: grfB, C and D, brpA, spaP, gbpB, relA and vicR was investigated at the presence of sub-MIC of MPEO-CNs. Most abundant bioactive compounds of MPEO were l-menthol (45.05%) and l-menthal (17.53%). SEM/EDX exhibited successful entrapment of MPEO into CsNPs followed by the changes in abundance of elemental peaks. A signal at 1737 cm-1 on chitosan spectrum was attributed to the carboxylic (CO) groups overlapped by MPEO incorporation. A new signal at 2361 cm-1 was assigned to electrostatic interactions of amine groups in chitosan with phosphoric units of TPP within the MPEO-chitosan. MPEO incorporation into porous nanogel decreased monodispersity of the nanoparticles and then raises z-average. Maximum release of MPEO was about 50% during 360 h in a hydroalcoholic solvent at ambient temperature. The adherence of bacterial cells showed high sensitivity to the nanoformulation of MPEO compared with unloaded chitosan-nanogel. Antibiofilm inhibition of S. mutans occurred in 50 and 400 µg/mL for MPEO-CNs and unloaded-nanogel, respectively. Among biofilm synthesis genes, gtfB, gtfC, gtfD were slightly affected by MPEO-CNs treatment, while gbpB, spaP, brpA, relA, and vicR genes underwent significant down-regulation in the presence of both unloaded-nanogel and MPEO-loaded-nanogel. This study demonstrated that the MPEO-CNs promised an efficient nanoformulation with the greatest inhibitory action against some glycosyltransferase genes (gtfB, C and D) as important enzymes involved in extracellular polymers. Finally, the results concluded that MPEO-CNs have a potential use as antibiofilm agent in toothpaste or mouth washing formulations.

Distribution of Class D Carbapenemase and Extended-Spectrum ß-Lactamase Genes among Acinetobacter Baumannii Isolated from Burn Wound and Ventilator Associated Pneumonia Infections.

INTRODUCTION: Resistance to Acinetobacter baumannii is dramatically on the rise in Iran. Therefore, it is important to study resistance pattern among Acinetobacter isolates which is a common cause of nosocomial infections. AIM: To investigate antibiotic resistance patterns and the role of resistant genes and biofilm formation in the induction of resistance among Acinetobacter baumannii isolated from burn wound and ventilator associated pneumonia infections. MATERIALS AND METHODS: Total 103 isolates such as 33 burn samples from Rasool Akram Hospital and 70 isolates from ventilated patients in Shahid Motahhari Hospital were identified with A. baumannii using biochemical method, and then identified to species level with PCR of gyrB and blaOXA-51 gene. Antibiotic sensitivity pattern for ß-lactam and carbapenem antibiotics was assessed using Agar disc diffusion test and E-test. The presence of different carbapenemase and metalo-ß-lactamase (blaOXA-51-like, gyrB, blaOXA-23-like, blaOXA-24-like, blaOXA-58, blaVEB, blaPER, blaGIM, blaSIM, blaIMP, blaVIM), extended-spectrum ß-lactamases (blaTEM, blaSHV) and two insertion sequences genes (ISaba1, IS1113) was assessed. Biofilm formation of all isolates was then assessed. Chi-square analysis or Fisher's-exact tests were used for statistical analysis. A p-value <0.05 was considered statistically significant. RESULTS: Colistin was the most effective antimicrobial agents, although 10.7% (11/103) of the isolates were resistant. The high rate of resistance to meropenem (93.2%) and imipenem (90.3%) was determined. Also, with exception of ampicillin-sulbactam, surprisingly the resistant rate was 28.2%, the resistance to ß-lactam antibiotic was dramatically increased. Co-existence of two and three blaOXA genes was also determined. The blaOXA-58 was detected in only one isolate. The blaTEM and blaOXA-23 was the most prevalent Extended-Spectrum ß-Lactamases (ESBL) gene. All isolates were biofilm producers. CONCLUSION: Antibiotic resistance is increasing among A. baumannii isolates which is due to excessive use of antibiotics and also acquired resistant genes and biofilm production. Resistance to nearly all antimicrobial agents especially colistin as end choice for treatment of multiple drug resistance A. baumannii is a big concern.

Phytotherapy in Streptococcusagalactiae: An Overview of the Medicinal Plants Effective against Streptococcus agalactiae.

Streptococcus agalactiae is a spherical and Gram-positive bacterium that causes postpartum sepsis, endometritis, chorioamnionitis and premature delivery in pregnant women. The use of herbs and natural ingredients for the treatment of various disorders has been common. The present review is a report on the medicinal plants with anti-Streptococcus agalactiae effects. In this review, the search was carried out in Web of Science, PubMed, Scopus, Google Scholar and Science direct by keywords such as bacteria, Streptococcus agalactiae and medicinal plants. According to the search results, 10 medicinal plants are used as anti-bacterial against Streptococcus agalactiae. Results of this study suggest that the active ingredients listed in this review paper used for pharmacological studies on Streptococcus agalactiae so it can produce effective natural antibiotic for the future.

Synergistic Effect of Colistin and Rifampin Against Multidrug Resistant Acinetobacter baumannii: A Systematic Review and Meta-Analysis.

The existence of infections caused by multidrug resistant (MDR) Acinetobacter baumannii is a growing problem because of the difficulty to treat them. We examined the published literature and focused our analysis on the investigation of the synergism of colistin and rifampin against MDR A. baumannii isolates via systematic review and meta-analysis. A systematic literature search was performed using the following 4 databases (PubMed, Scopus, EMBASE and ISI Web of Sciences). The related articles were evaluated during the period from December 2014 to January 2015. Information based on resistance and sensitivity to antibiotics, the minimum inhibitory concentration and the effects of two antibiotics on each other including synergism, antagonism, relative synergism and additive antagonism were extracted. A meta-analysis of 17 studies including 448 samples was brought into process and 2% (95% CI 0-4%) and 72% (95% CI 56-89%) resistance to colistin and rifampin were observed, respectively. 42% of all isolates showed MIC = 4 µg/ml (95% CI 14-69%) to rifampin and 30% MIC= 2 µg/ml to colistin (95% CI 3.8-78%). MIC50 and MIC90 for both rifampin and colistin were 2 µg/ml and 4 µg/ml, respectively. 63% of the strains demonstrated synergy (95% CI 37-90%), 7% were highlighted as relative synergism (95% CI 0.0- 13%), 3% showed an additive effect (95% CI -0.0-7%) and 14% were indifferent (95% CI 6-23%). The antagonistic effect was not observed in this combination. Synergy rates of time-kill assay in rifampin and colistin combinations were generally higher than those of check bored microdilution and E-test method. The results demonstrated that the combination therapy could be more useful when compared to monotherapy and that this strategy might reduce the resistance rate to rifampin in MDR A. baumannii isolates.

Protection of mice against Staphylococcus aureus infection by a recombinant protein ClfA-IsdB-Hlg as a vaccine candidate.

Staphylococcus aureus is one of the most important causes of nosocomial infections. An effective vaccine to prevent S. aureus infections is urgently required due to the dramatic increase in the number of antibiotic-resistant strains. In this report, we evaluated a newly recombinant protein composed of selected antigenic regions of clumping factor A (ClfA), iron surface determinant B (IsdB) and gamma hemolysin B (HlgB) of S. aureus and sequence coding for hydrophobic linkers between three domains. The recombinant gene was constructed in pET-28a (+) and expressed in Escherichia coli BL21. In addition, sequence coding for a His(6)-tag was added followed by a hybrid procedure of nickel chelate protein purification. Immunization of BALB/c mice with the recombinant protein ClfA-IsdB-Hlg evoked antigen-specific antibodies that could opsonize S. aureus cells, enhancing in vitro phagocytosis by macrophages. Vaccination with the recombinant protein also reduced the bacterial load recovered from mice spleen samples and increased survival following the intraperitoneal challenge with pathogenic S. aureus compared to the control mice. Our results showed that the recombinant protein ClfA-IsdB-Hlg is a promising vaccine candidate for the prevention of S. aureus bacteremia infections.

In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus.

PURPOSE: Staphylococcus aureus is one of the most important causes of nosocomial and community-acquired infections. The increasing incidence of multiple antibiotic-resistant S. aureus strains and the emergence of vancomycin resistant S. aureus strains have placed renewed interest on alternative means of prevention and control of infection. S. aureus produces a variety of virulence factors, so a multi-subunit vaccine will be more successful for preventing S. aureus infections than a mono-subunit vaccine. MATERIALS AND METHODS: We selected three important virulence factors of S. aureus, clumping factor A (ClfA), iron-regulated surface determinant (IsdB), and gamma hemolysin (Hlg) that are potential candidates for vaccine development. We designed synthetic genes encoding the clfA, isdB, and hlg and used bioinformatics tools to predict structure of the synthetic construct and its stabilities. VaxiJen analysis of the protein showed a high antigenicity. Linear and conformational B-cell epitopes were identified. RESULTS: The proteins encoded by these genes were useful as vaccine candidates against S. aureus infections. CONCLUSION: In silico tools are highly suited to study, design, and evaluate vaccine strategies.

Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR.

Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.

Distribution of bla(TEM), bla(SHV), bla(CTX-M) genes among clinical isolates of Klebsiella pneumoniae at Labbafinejad Hospital, Tehran, Iran.

Extended-spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella pneumoniae have been increasingly recognized in the hospital settings in Iran as well as throughout the world. The aim of this study was to detect and determine the genes encoding the ESBLs including bla(TEM), bla(SHV), and bla(CTX-M) groups among the K. pneumoniae isolates at Labbafinejad Hospital by polymerase chain reaction (PCR) and characterize them by direct sequencing of PCR products. Eighty-nine isolates were isolated from patients at different wards during March 2008-March 2009. They were identified as K. pneumoniae using biochemical tests. Susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion method. The phenotypic confirmatory test was used to screen the isolates for production of ESBLs. To amplify the bla(SHV) the template DNA was extracted by boiling method. Plasmid DNA was extracted using minipreparation kit and used as template in PCR for detection of bla(TEM) and bla(CTX-M). The selected PCR products were sequenced and analyzed. All 89 strains were susceptible to imipenem. The rates of resistance to different antibiotics were in the following order: aztronam (79.7%), cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%). The phenotypic confirmatory test detected 62 isolates (69.7%) as ESBL-producing K. pneumoniae. The prevalence of genes encoding ESBLs were as follows: bla(TEM) 54% (n = 48), bla(SHV) 67.4% (n = 60), bla(CTX-M-I) 46.51% (n = 40), and bla(CTX-M-III) 29% (n = 25). The bla(CTX-M-II) and bla(CTX-M-IV) were not detected. All bla(TEM) types were characterized as bla(TEM-1) and all bla(CTX-M-I) were identified as bla(CTX-M-15). The SHV types were characterized as SHV-5, SHV-11, and SHV-12. The rate of ESBL at Labbafinejad Hospital was 25% increase in a 4-year study that ended in March 2009. It appears that bla(TEM-1), bla(SHV-5), bla(SHV-11), bla(SHV-12), and bla(CTX-M-15) are the dominant ESBLs among the resistant strains of K. pneumoniae in Iran.