Effect of phenylalanine arginyl ß-naphthylamide on the imipenem resistance, elastase production, and the expression of quorum sensing and virulence factor genes in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAßN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAßN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAßN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAßN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAßN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.

Antimicrobial photodynamic effect of the photosensitizer riboflavin, alone and in combination with colistin, against pandrug-resistant Pseudomonas aeruginosa clinical isolates.

INTRODUCTION: Development of multi-, extensively-, and pandrug-resistant (MDR, XDR, and PDR) strains of Pseudomonas aeruginosa remains a major problem in medical care. The present study evaluated the effect of antimicrobial photodynamic therapy (aPDT) as a monotherapy and in combination with colistin against P. aeruginosa isolates. METHODS: Two P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Minimum inhibitory concentration (MIC) of colistin was determined by the colistin broth disk elution (CBDE) and the reference broth microdilution (rBMD) methods. aPDT was performed using the photosensitizer (Ps) riboflavin at several concentrations and a light-emitting diode (LED) emitting blue light for different irradiation times with or without colistin at 1/2 × MIC concentration. RESULTS: Both PA1 and PA2 isolates were identified as colistin-resistant P. aeruginosa with a MIC ≥4 µg/mL by the CBDE and MICs of 512 µg/mL and 256 µg/mL, respectively, by the rBMD. In aPDT, neither riboflavin nor LED light alone had antibacterial effects. The values of colony forming units per milliliter (CFU/mL) in both isolates were significantly reduced by LED + Ps treatments in a time-dependent manner (LED irradiation time) and dose-dependent manner (Ps concentration). In comparison with control, treatment with Ps (50 µM) + LED (120 s) and Ps (100 µM) + LED (120 s) resulted in 0.27 log10 CFU/mL and 0.43 log10 CFU/mL reductions in PA1, and 0.28 log10 CFU/mL and 0.34 log10 CFU/mL reductions in PA2, respectively, (P < 0.01). The best results were obtained after the combination of aPDT followed by colistin, which increased bacterial reduction, resulting in a 0.41-0.7 log10 CFU/mL reduction for PA1 and 0.35-0.83 log10 CFU/mL reduction for PA2 (P = 0.001). CONCLUSIONS: This study suggests the potential implications of aPDT in combination with antibiotics, such as colistin for treatment of difficult-to-treat P. aeruginosa infections.

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.

Polymyxin combination therapy for multidrug-resistant, extensively-drug resistant, and difficult-to-treat drug-resistant gram-negative infections: is it superior to polymyxin monotherapy?

INTRODUCTION: The increasing prevalence of infections with multidrug-resistant (MDR), extensively-drug resistant (XDR) or difficult-to-treat drug resistant (DTR) Gram-negative bacilli (GNB), including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter species, and Escherichia coli poses a severe challenge. AREAS COVERED: The rapid growing of multi-resistant GNB as well as the considerable deceleration in development of new anti-infective agents have made polymyxins (e.g. polymyxin B and colistin) a mainstay in clinical practices as either monotherapy or combination therapy. However, whether the polymyxin-based combinations lead to better outcomes remains unknown. This review mainly focuses on the effect of polymyxin combination therapy versus monotherapy on treating GNB-related infections. We also provide several factors in designing studies and their impact on optimizing polymyxin combinations. EXPERT OPINION: An abundance of recent in vitro and preclinical in vivo data suggest clinical benefit for polymyxin-drug combination therapies, especially colistin plus meropenem and colistin plus rifampicin, with synergistic killing against MDR, XDR, and DTR P. aeruginosa, K. pneumoniae and A. baumannii. The beneficial effects of polymyxin-drug combinations (e.g. colistin or polymyxin B + carbapenem against carbapenem-resistant K. pneumoniae and carbapenem-resistant A. baumannii, polymyxin B + carbapenem + rifampin against carbapenem-resistant K. pneumoniae, and colistin + ceftolozan/tazobactam + rifampin against PDR-P. aeruginosa) have often been shown in clinical setting by retrospective studies. However, high-certainty evidence from large randomized controlled trials is necessary. These clinical trials should incorporate careful attention to patient's sample size, characteristics of patient's groups, PK/PD relationships and dosing, rapid detection of resistance, MIC determinations, and therapeutic drug monitoring.

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran / Resistencia antimicrobiana, factores de virulencia y genotipos de aislamientos clínicos de Pseudomonas aeruginosa de Gorgan, norte de Irán

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed β-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.(AU)

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran.

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed ß-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.

In vitro activities of cellulase and ceftazidime, alone and in combination against Pseudomonas aeruginosa biofilms.

BACKGROUND: Biofilms are a main pathogenicity feature of Pseudomonas aeruginosa and has a significant role in antibiotic resistance and persistent infections in humans. We investigated the in vitro activities of antibiotic ceftazidime and enzyme cellulase, either alone or in combination against biofilms of P. aeruginosa. RESULTS: Both ceftazidime and cellulase significantly decreased biofilm formation in all strains in a dose-dependent manner. Combination of enzyme at concentrations of 1.25, 2.5, 5, and 10 U/mL tested with 1/16× MIC of antibiotic led to a significant reduction in biofilm biomass. Cellulase showed a significant detachment effect on biofilms at three concentrations of 10 U/mL, 5 U/mL, and 2.5 U/mL. The MIC, MBC, and MBEC values of ceftazidime were 2 to 4 µg/mL, 4 to 8 µg/mL, and 2048 to 8192 µg/mL. When combined with cellulase, the MBECs of antibiotic showed a significant decrease from 32- to 128-fold. CONCLUSIONS: Combination of the ceftazidime and the cellulase had significant anti-biofilm effects, including inhibition of biofilm formation and biofilm eradication in P. aeruginosa. These data suggest that glycoside hydrolase therapy as a novel strategy has the potential to enhance the efficacy of antibiotics and helps to resolve biofilm-associated wound infections caused by this pathogen.

Antiviral Therapeutic Potential of Curcumin: An Update.

The treatment of viral disease has become a medical challenge because of the increasing incidence and prevalence of human viral pathogens, as well as the lack of viable treatment alternatives, including plant-derived strategies. This review attempts to investigate the trends of research on in vitro antiviral effects of curcumin against different classes of human viral pathogens worldwide. Various electronic databases, including PubMed, Scopus, Web of Science, and Google Scholar were searched for published English articles evaluating the anti-viral activity of curcumin. Data were then extracted and analyzed. The forty-three studies (published from 1993 to 2020) that were identified contain data for 24 different viruses. The 50% cytotoxic concentration (CC50), 50% effective/inhibitory concentration (EC50/IC50), and stimulation index (SI) parameters showed that curcumin had antiviral activity against viruses causing diseases in humans. Data presented in this review highlight the potential antiviral applications of curcumin and open new avenues for further experiments on the clinical applications of curcumin and its derivatives.

Detection of extensively drug-resistant and hypervirulent Klebsiella pneumoniae ST15, ST147, ST377 and ST442 in Iran.

In this study, we focused on the emergence of extensively drug-resistant (XDR), pandrug-resistant (PDR), and hypervirulent Klebsiella pneumoniae (hvKP) in Iran. During 2018 to 2020 a total of 52 K. pneumoniae isolates were collected from different clinical specimens. The hvKP isolates were identified by PCR amplification of virulence and capsular serotype-specific genes. Hypermucoviscous K. pneumoniae (hmKP) were identified by string test. Carbapenem-resistant hvKP (CR-hvKP), multidrug-resistant hvKP (MDR-hvKP), extensively drug-resistant hvKP (XDR-hvKP), and pandrug-resistant hvKP (PDR-hvKP) were determined by disc diffusion method, Carba-NP test and PCR method. XDR-hvKP isolates were typed by multilocus sequence typing (MLST). Among all K. pneumoniae isolates 14 (26.9%) were identified as hvKP and 78.6% (11/14) of them were hmKP however, none of the classic K. pneumoniae (cKP) isolates were hmKP. The predominant capsular serotype of hvKP was K2 (42.85%) followed by K1 (35.71%). The prevalence of MDR-hvKP, XDR-hvKP and PDR-hvKP isolates were 6 (42.9%), 5 (35.7%) and 1 (7.1%), respectively. ESBL production was found in 85.7% of hvKP isolates and most of them carried bla TEM gene (78.6%) and 6 isolates (42.9%) were CR-hvKP. Among hvKP isolates, 1 (7.1%), 2 (14.3%), 3 (21.4%), 8 (28.6%), and 11 (78.6%) carried bla NDM-6, bla OXA-48, bla CTX-M, bla SHV, and bla TEM genes, respectively. According to MLST analysis, 2, 1, 1, and 1 XDR-hvKP isolates belonged to ST15, ST377, ST442, and ST147, respectively. The occurrence of such isolates is deeply concerning due to the combination of hypervirulence and extensively drug-resistance or pandrug-resistance.

Detection of NDM-1 producing Klebsiella pneumoniae ST15 and ST147 in Iran during 2019-2020.

Carbapenems are employed to treat infections caused by Gram-negative bacteria including Klebsiella pneumoniae. This research is aimed to perform phenotypic detection of ß-lactamases and molecular characterization of NDM-1 positive K. pneumoniae isolates. Another objective is to investigate NDM-1 producing K. pneumoniae among children in Iran. From 2019 to 2020, altogether 60 K. pneumoniae isolates were acquired from various patients in certain Iranian hospitals. Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution methods. In addition, mCIM and eCIM were used to confirm the production of carbapenemases and metallo-beta-lactamases (MBLs), respectively. Detection of resistance genes namely, blaNDM-1, blaIMP, blaVIM, blaKPC, blaOXA-48-like, blaCTX-M, blaSHV, blaTEM, and mcr-1 was performed by PCR and confirmed by DNA sequencing. Multilocus sequence typing (MLST) was employed to determine the molecular typing of the strains. According to the findings, the highest rate of carbapenem resistance was detected against doripenem 83.3% (50). Moreover, 31.7% (19) were resistant to colistin. Further to the above, altogether 80% (48) were carbapenemase-producing isolates and among them 46.7% (28) of the isolates were MBL and 33.3% (20) isolates were serine ß-lactamase producer. According to the PCR results, 14 isolates produced blaNDM-1. Remarkably, four blaNDM-1 positive isolates were detected in children. In addition, these isolates were clonally related as determined by MLST (ST147, ST15). Altogether ten blaNDM-1 positive isolates were ST147 and four blaNDM-1 positive isolates were ST15. Based on the results, the emergence of NDM-producing K. pneumoniae among children is worrying and hence, it is necessary to develop a comprehensive program to control antibiotic resistance in the country.

Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates.

OBJECTIVE: The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. RESULTS: All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.

Molecular characteristics of antibiotic-resistant Escherichia coli and Klebsiella pneumoniae strains isolated from hospitalized patients in Tehran, Iran.

BACKGROUND: We evaluated the distribution of carbapenem and colistin resistance mechanisms of clinical E. coli and K. pneumoniae isolates from Iran. METHODS: 165 non-duplicate non-consecutive isolates of K. pneumoniae and E. coli were collected from hospitalized patients admitted to Iran's tertiary care hospitals from September 2016 to August 2018. The isolates were cultured from different clinical specimens, including wound, urine, blood, and tracheal aspirates. Antibiotic susceptibility testing was performed by disc diffusion and microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The presence of extended spectrum ß-lactamases (ESBLs) genes, carbapenemase genes, as well as fosfomycin resistance genes, and colistin resistance genes was also examined by PCR-sequencing. The ability of biofilm formation was assessed with crystal violet staining method. The expression of colistin resistance genes were measured by quantitative reverse transcription-PCR (RT-qPCR) analysis to evaluate the association between gene upregulation and colistin resistance. Genotyping was performed using the multi-locus sequencing typing (MLST). RESULTS: Colistin and tigecycline were the most effective antimicrobial agents with 90.3% and 82.4% susceptibility. Notably, 16 (9.7%) isolates showed resistance to colistin. Overall, 33 (20%), 31 (18.8%), and 95 (57.6%) isolates were categorized as strong, moderate, and weak biofilm-producer, respectively. Additionally, blaTEM, blaSHV, blaCTX-M, blaNDM-1, blaOXA-48-like and blaNDM-6 resistance genes were detected in 98 (59.4%), 54 (32.7%), 77 (46.7%), 3 (1.8%), 17 (10.30%) and 3 (1.8%) isolates, respectively. Inactivation of mgrB gene due to nonsense mutations and insertion of IS elements was observed in 6 colistin resistant isolates. Colistin resistance was found to be linked to upregulation of pmrA-C, pmrK, phoP, and phoQ genes. Three of blaNDM-1 and 3 of blaNDM-6 variants were found to be carried by IncL/M and IncF plasmid, respectively. MLST revealed that blaNDM positive isolates were clonally related and belonged to three distinct clonal complexes, including ST147, ST15 and ST3299. CONCLUSIONS: The large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of diverse carbapenem- and colistin-resistant isolates in the future.

Tetracycline resistance mediated by tet efflux pumps in clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii is one of the most frequent nosocomial pathogen capable of acquiring resistance to different antimicrobials. The aim of this study was to investigate the activity of tetracycline, doxycycline and minocycline, the prevalence of tet(A) and tet(B) determinants, and the role of efflux pump in tetracycline resistance among the A. baumannii clinical isolates. Susceptibility of 98 A. baumannii isolates to tetracyclines was evaluated by disk diffusion method. The presence of active efflux pump was investigated by determination of the minimum inhibitory concentration (MIC) of tetracycline using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Polymerase chain reaction (PCR) was performed to investigate the presence of tet(A) and tet(B) determinants in tetracycline-resistant isolates. The rate of resistance to tetracycline, doxycycline and minocycline was 47.95%, 0%, and 30.61%, respectively. Among the 47 tetracycline-resistant isolates, 29.79% were originated from burned patients and showed MIC ranging from 128-256 µg/mL with both MIC 50 and MIC90 values of 256 µg/mL, while 70.21% were from ventilator-associated pneumonia (VAP) patients and had MIC values ranging from 32-1024 µg/mL, with MIC50 and MIC90 of 512 µg/mL and 1024 µg/mL, respectively. The tet(B) gene was found in 61.7% of tetracycline-resistant isolates, while none of the isolates carried the tet(A) gene. CCCP led to 2-128-fold reduction in tetracycline MIC of the tested isolates. The results showed that doxycycline and minocycline are promising agents for the treatment of A. baumannii infections. This study has also revealed the role of efflux activity in the resistance to tetracycline of A. baumannii isolates. The emergence of resistance to these agents is likely due to the spread of clones presenting with a higher prevalence of resistance determinants.

Decreased carO gene expression and OXA-type carbapenemases among extensively drug-resistant Acinetobacter baumannii strains isolated from burn patients in Tehran, Iran.

A major challenge in the treatment of infections has been the rise of extensively drug resistance (XDR) and multidrug resistance (MDR) in Acinetobacter baumannii. The goals of this study were to determine the pattern of antimicrobial susceptibility, blaOXA and carO genes among burn-isolated A. baumannii strains. In this study, 100 A. baumannii strains were isolated from burn patients and their susceptibilities to different antibiotics were determined using disc diffusion testing and broth microdilution. Presence of carO gene and OXA-type carbapenemase genes was tested by PCR and sequencing. SDS-PAGE was done to survey CarO porin and the expression level of carO gene was evaluated by Real-Time PCR. A high rate of resistance to meropenem (98%), imipenem (98%) and doripenem (98%) was detected. All tested A. baumannii strains were susceptible to colistin. The results indicated that 84.9% were XDR and 97.9% of strains were MDR. In addition, all strains bore blaOXA-51 like and blaOXA-23 like and carO genes. Nonetheless, blaOXA-58 like and blaOXA-24 like genes were harbored by 0 percent and 76 percent of strains, respectively. The relative expression levels of the carO gene ranged from 0.06 to 35.01 fold lower than that of carbapenem-susceptible A. baumannii ATCC19606 and SDS - PAGE analysis of the outer membrane protein showed that all 100 isolates produced CarO. The results of current study revealed prevalence of blaOXA genes and changes in carO gene expression in carbapenem resistant A.baumannii.

Evaluation of antimicrobial resistance, biofilm forming potential, and the presence of biofilm-related genes among clinical isolates of Pseudomonas aeruginosa.

OBJECTIVES: Pseudomonas aeruginosa is known as a leading cause of nosocomial infections worldwide. Antimicrobial resistance and biofilm production, as two main virulence factors of P. aeruginosa, are responsible for the persistence of prolonged infections. In this study, antimicrobial susceptibility pattern and phenotypic and genotypic characteristics of biofilm of P. aeruginosa were investigated. RESULTS: A total of 80 clinical P. aeruginosa isolates were obtained. Isolates showed resistance to all antibiotics with a rate from 12.5% (n = 10) against amikacin and piperacillin/tazobactam to 23.75% (n = 19) to levofloxacin. Multidrug-resistant P. aeruginosa accounted for 20% (n = 16). 83.75% (n = 67) of isolates showed biofilm phenotype. All three biofilm-related genes were found simultaneously in 87.5% (n = 70) of P. aeruginosa and 13.5% (n = 10) of the isolates had none of the genes tested. From the results of the present study, combination therapy including an anti-pseudomonal beta-lactam (piperacillin/tazobactam or ceftazidime) and an aminoglycoside or carbapenems (imipenem, meropenem) with fluoroquinolones in conjunction with an aminoglycoside can be used against Pseudomonas infections. However, reasonable antimicrobial use and high standards of infection prevention and control are essential to prevent further development of antimicrobial resistance. Combination strategies based on the proper anti-pseudomonal antibiotics along with anti-biofilm agents can also be selected to eradicate biofilm-associated infections.

Heteroresistance to colistin in oxacillinase-producing carbapenem-resistant Acinetobacter baumannii clinical isolates from Gorgan, Northern Iran.

OBJECTIVES: Colistin resistance rates are rising globally among multidrug-resistant Gram-negative bacilli, including Acinetobacter baumannii (A. baumannii). A new type of resistance - heteroresistance - has also been reported to colistin in clinical A. baumannii isolates. This study investigated the presence of colistin heteroresistance in carbapenem-resistant A. baumannii clinical isolates. METHODS: Different clinical specimens from hospitalised patients were investigated for A. baumannii. The MICs to imipenem, meropenem and colistin were determined by broth microdilution. PCR was performed to detect OXA-type carbapenemase genes (blaOXA-23-like, blaOXA-24/40-like, blaOXA-51-like, blaOXA-58-like, and blaOXA-143-like). Heteroresistance to colistin was examined using the population analysis profiles method. Genotypic relatedness of the isolates was analysed by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). RESULTS: Overall, 71 A. baumannii isolates were recovered from clinical specimens. Of these, 27 (38.03%) and 44 (61.97%) isolates were carbapenem-susceptible and carbapenem-resistant, respectively. In addition, 67 (94.36%) isolates were susceptible to colistin, with MICs between 0.25-2 µg/mL. Among the 44 selected carbapenem-resistant colistin-susceptible isolates, the frequency of blaOXA-51-like, blaOXA-23-like and blaOXA-24/40-like genes was 100%, 77.27% and 43.18%, respectively. Nine of 44 (20.45%) isolates were characterised as colistin-heteroresistant with subpopulations growing at 6-8 µg/mL, whereas two of 44 (4.54%) presented heterogeneous subpopulations growing at up to 1 µg/mL of colistin. ERIC­PCR typing clustered A. baumannii isolates to 10 common types (CT1-CT10) containing isolates from different hospitals and 12 single types (ST1-ST12). CONCLUSIONS: A. baumannii with a colistin heteroresistance phenotype was common. This could be of great concern since colistin is often used as a last-resort drug for treating A. baumannii infections, highlighting that care is necessary with colistin monotherapy. In addition, more effective strategies and surveillance are required to confine and prevent the inter-hospital and/or intra-hospital dissemination of A. baumannii between therapeutic centres.

Plastic binding feature of polymyxins: the effect on MIC susceptibility measurements.

The loss of polycationic antimicrobial peptides, polymyxins, due to adhesion to plastics is an important subject matter that influences in vitro susceptibility testing, including minimum inhibitory concentration (MIC) assays. This review reminds us that this issue can serve as a significant source of variation in the MIC values for polymyxins against Gram-negative bacteria.

Molecular and serologic characterization of rotavirus from children with acute gastroenteritis in northern Iran, Gorgan.

BACKGROUND: The pattern and distribution of human rotavirus genotypes in young children in developing countries play an important role in epidemiological studies, as well as providing a strategy for the development of future rotavirus vaccine. METHODS: We evaluated stool samples from 349 children with acute gastroenteritis from Northern Iran (Gorgan city, Golestan province). Polyacrylamide Gel Electrophoresis (PAGE) and Latex Agglutination Test (LAT) were utilized to determine the prevalence of human rotavirus in fecal samples. Moreover semi-multiplex RT-PCR technique was carried out in order to determine the P and G genotypes of human rotavirus in rotavirus-positive samples. RESULTS: A total of 46 rotavirus-positive samples were G and P genotyped. Whereas 28 (60.8%) fecal specimens contained only one rotavirus strain, 14 (30.4%) were mixed rotavirus infections and 4 (8.8%) was non-typeable. Overall, during the study, 57.82% of strains identified as genotype G1, G2 (18.70%), G3 (4.69%), G4 (3.13%), G8 (3.13%), G9 (6.26%) and non-typeable G (6.26%). From all these mentioned rotavirus strains, 46 were characterized as P [8] (97.80%) and P [4] (2.20%).Our analysis of the G and P genotyping of strains from all 46 rotavirus-infected children has revealed that 4/46(6.26%) of G type strains were non-typeable. The predominant single G/P combination was G1P [8] (57.82%), followed by, G2P [8] (16.98%), G2P [4] (1.72%), G3P [8] (4.69%), G4P [8] (3.13%) G8P [8] (3.13%), G9P [8] (6.26%) and four cases of non-typeable G (6.26%). Rotavirus was detected in 39 specimens (11.17%) by PAGE and in 38 specimens (10.88%) by LAT. Both tests were 100% specific; however, the LAT was 82.61% sensitive compared to the PAGE, which was 84.78% sensitive. CONCLUSIONS: The results suggest that to characterize rotavirus strains as well as design new effective vaccines for children with acute gastroenteritis, a large-scale study is needed in future.

Characterization of Phenotypic and Genotypic Diversity of Stenotrophomonas maltophilia Strains Isolated From Selected Hospitals in Iran.

Stenotrophomonas maltophilia is an environmental Gram-negative bacterium that has rapidly emerged as an important nosocomial pathogen in hospitalized patients. Treatment of S. maltophilia infections is difficult due to increasing resistance to multiple antibacterial agents. The purpose of this study was to determine the phenotypic and genotypic characterization of S. maltophilia isolates recovered from patients referred to several hospitals. A total of 164 clinical isolates of S. maltophilia were collected from hospitals in various regions in Iran between 2016 and 2017. Antibiotic susceptibility testing was performed by disc diffusion method and E-test assay according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The ability of biofilm formation was assessed with crystal violet staining and then, biofilm-associated genes were investigated by PCR-sequencing method. The presence of L1 (a metallo-ß-lactamase), L2 (a clavulanic acid-sensitive cephalosporinase), sul1 and sul2 (resistance to Trimethoprim/Sulfamethoxazole), Smqnr (intrinsic resistance to quinolones), and dfrA genes (dihydrofolate reductase enzyme that contributes to trimethoprim resistance) was also examined by PCR-sequencing. Relative gene expression of smeDEF efflux pump was assessed by real-time PCR. Genotyping was performed using the multi-locus sequencing typing (MLST) and repetitive extragenic palindromic-PCR (Rep-PCR). Isolates were resistant to imipenem (100%), meropenem (96%), doripenem (96%), and ceftazidime (36.58%). Notably, 5 (3.04%) isolates showed resistant to trimethoprim-sulfamethoxazole (TMP-SMX), an alarming trend of decreased susceptibility to TMP-SMX in Iran. Minocycline and levofloxacin exhibited the highest susceptibility of 91.46 and 99.39%, respectively. Using the crystal violet staining, 157 (95.73%) isolates had biofilm phenotype: 49 (29.87%), 63 (38.41%), and 45 (27.43%) isolates were categorized as strong-, moderate- and weak-biofilm producer while 7 isolates (4.26%) were identified a non-biofilm producer. Biofilm genes had an overall prevalence of 145 (88.41%), 137 (83.53%), and 164 (100%) of rmlA, rpfF, and spgM, respectively. L1, L2, Smqnr, sul1, and sul2 resistance genes were detected in 145 (88.41%), 156 (96.12%), 103 (62.80%), 89 (54.26%), and 92 (56.09%) isolates, respectively. None of the S. maltophilia isolates were positive for dfrA12, dfrA17, and dfrA27 genes. Gene expression analysis showed that smeD efflux system was overexpressed in two out of the five clinical isolates (40%) that showed resistance to TMP-SMX. Most of the isolates were genetically unrelated. Two new sequence types (ST139 and ST259) were determined. Our results showed that TMP-SMX was still an effective antibiotic against S. maltophilia. The findings of the current study revealed an increasing prevalence of antibiotic resistance and biofilm genes in clinical S. maltophilia isolates in Iran.