Inhibition of ERAP1 represses HLA-B27 free heavy chains expression on polarized macrophages and interrupts NK cells activation and function from ankylosing spondylitis.

BACKGROUND: We aimed to assess if Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphisms might impress Human leukocyte antigen (HLA)-B27-free heavy chains (FHCs) expression on macrophages and eventually NK cell activation in Ankylosing spondylitis (AS). METHODS: Blood samples were obtained from 10 HLAB27+ patients with protective and 10 HLAB27+ patients with non-protective genotype. Monocytes were isolated and polarized toward M1 and M2 macrophages. ERAP1 was inhibited in macrophages, which were then co-cultured with autologous NK cells. RESULTS: Expression of HLA-B27-FHCs on M1 and M2 macrophages was reduced in patients with protective ERAP1 genotype. Co-culturing ERAP1-inhibited M1 macrophages and NK cells from patients with protective genotype resulted in downmodulation of CD69 and CD107a markers on NK cells and reduced number of IFN-γ+ NK cells compared to that of patients with non-protective genotypes. CONCLUSION: Inhibition of ERAP1 activity, by diminishing NK activation, may have therapeutic value in treating AS patients.

Role of MexAB-OprM efflux pump in the emergence of multidrug-resistant clinical isolates of Pseudomonas aeruginosa in Mazandaran province of Iran.

BACKGROUND: Multidrug-resistant clinical isolates can cause many therapeutic problems. The MexAB-OprM efflux pump plays a significant role in expelling toxins and drugs from the bacterial cells resulting in multidrug-resistant Pseudomonas aeruginosa isolates. PURPOSE: This study aimed to investigate the effect of the MexAB-OprM efflux pump in the emergence of multidrug-resistant clinical isolates of P. aeruginosa. METHODS AND RESULTS: For the present study, 100 clinical isolates of P. aeruginosa were collected from different wards of teaching hospitals (2018-2019). After confirmation and detection of bacteria by standard methods, the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method. Also, the minimum inhibitory concentration (MIC) of ciprofloxacin was measured in the presence and absence of phenylalanine arginine beta-naphthylamide by the broth microdilution method. Then, the real-time PCR was used to investigate the expression level of the mexB gene compared to the standard PAO1 strain. Forty-one/100 isolates exhibited multidrug-resistant phenotype (MDR), while piperacillin-tazobactam and levofloxacin were the most and least effective antibiotics tested, respectively. Also, 54/100 isolates showed no increased expression of mexB gene compared to the standard PAO1 strain. However, among the 41 MDR isolates, 12 (29.26%) showed a more than three-fold increase in the expression level of the mexB gene. In this study, a significant relationship was observed between the resistance to tested antibiotics in MDR strains and the increased expression of the mexB gene. CONCLUSION: We found that increasing the expression of the mexB gene can cause the emergence of multidrug-resistant strains by increasing the minimum inhibitory concentration of the antibiotics. Then, we need to evaluate the resistance mechanisms separately in different area of a country to improve the antibiotic stewardship.

Molecular characterization of carbapenem-resistant Pseudomonas aeruginosa isolated from four medical centres in Iran.

BACKGROUND: Understanding the mechanisms of antibiotic resistance is important for designing new therapeutic options and controlling resistant strains. The goal of this study was to look at the molecular epidemiology and mechanisms of resistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from Tabriz, Iran. METHODS: One hundred and forty P. aeruginosa were isolated and antibiotic susceptibility patterns were determined. Overproduction of AmpC and efflux pumps were discovered using phenotypic techniques. Polymerase chain reaction (PCR) was used to determine the presence of carbapenemase-encoding genes. In addition, the expressions of OprD and efflux pumps were evaluated by the Real-Time PCR. Random amplified polymorphic DNA typing (RAPD) was performed for genotyping. RESULTS: Among 140 P. aeruginosa isolates, 74 (52.8%) were screened as CRPA. Overexpression of efflux systems was observed in 81% of isolates, followed by decreased expression of OprD (62.2%), presence of carbapenemase genes (14.8%), and overproduction of AmpC (13.5%). In most isolates, carbapenem resistance was multifactorial (60.8%). According to our results, the prevalence of CRPA is at alarming levels. Overexpression of efflux systems was the most common mechanism of carbapenem resistance. CONCLUSION: Most isolates may originate in patients themselves, but cross-infection is possible. Therefore, we suggest a pattern shift in the strategy of CRPA in our setting.

Risk factors for mortality in hospitalized patients infected with carbapenem-resistant Pseudomonas aeruginosa in Iran.

Introduction: Mortality due to carbapenem-resistant Pseudomonas aeruginosa (CRPA) infection has increased worldwide in recent years. The risk factors associated with hospital settings in Iran and the role of strain resistance mechanisms in many studies are unclear. Methods: A retrospective study was conducted on consecutive non-repetitive patients with CRPA infections isolated from seven major hospitals from northwest of Iran. We evaluated different risk factors and characteristics of bacteria for the death or survival of patients. Results: In this study, 116 CRPA isolates were obtained from patients admitted to seven hospitals. Forty-one (35.3%) patients were enrolled in the study of mortality risk factors. Significant risk factors associated with mortality included the site of infection, hospitalization in different wards, the use of invasive devices, and the type of carbapenem resistance mechanisms. Conclusions: ICU admission, the use of mechanical ventilation and chest tube and infection with pandrug-resistant strains were the most important factors in increasing mortality due to CRPA infection. These results suggested that the clinicians should emphasize the proper use of antibiotic and invasive procedures.

In-vitro Effect of Imipenem, Fosfomycin, Colistin, and Gentamicin Combination against Carbapenem-resistant and Biofilm-forming Pseudomonas aeruginosa Isolated from Burn Patients.

The aim of this study was to investigate in-vitro antibacterial and antibiofilm effect of colistin, imipenem, gentamicin, and fosfomycin alone and the various combinations against carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa). Eight carbapenem-resistant and biofilm-forming P. aeruginosa isolates from burn patients were collected. The mechanisms of resistance to carbapenem were determined by the phenotypic, PCR, and Real-Time PCR assays. The minimum inhibitory concentration (MIC) of antimicrobial agents was determined by the broth micro dilution. To detect any inhibitory effect of antibiotics against the biofilm, the biofilm inhibitory concentration was determined. To detect synergetic effects of the combinations of antibiotics, the checkerboard assay and the fractional inhibitory concentration (FIC) were used. The highest synergic effect was observed in colistin/fosfomycin and gentamicin/fosfomycin (5 of 8 isolates), and the lowest synergic effect was found in gentamicin/imipenem and colistin/gentamicin (1 of 8 isolates). Colistin/fosfomycin, imipenem/fosfomycin, colistin/imipenem, gentamicin/fosfomycin, and gentamicin/imipenem were shown synergic effect for 3, 2, 2, 2 and 1 isolates, respectively. The combination of antibiotics had different effects on biofilm and planktonic forms of P. aeruginosa. Therefore, a separate determination of inhibitory effects of the antibiotic in the combination is necessary. Fosfomycin/colistin and fosfomycin/gentamicin were more effective against planktonic form and fosfomycin/colistin against biofilm forms.

Molecular epidemiology and carbapenem resistance of Pseudomonas aeruginosa isolated from patients with burns.

OBJECTIVE: The aim of this study was to investigate the molecular epidemiology and carbapenem resistance mechanisms of Pseudomonas aeruginosa isolated from patients with burns in Azerbaijan, Iran. METHOD: Pseudomonas aeruginosa was isolated from 38 patients with burns. Disk diffusion and agar dilution methods were used to determine antibiotic susceptibility patterns. The overproduction of AmpC ß-lactamase and efflux pumps were detected by phenotypic methods. The presence of carbapenemase-encoding genes was detected by multiplex polymerase chain reaction (PCR). Expression of the OprD gene and MexAB efflux pumps were also evaluated with real-time PCR. Random amplified polymorphic DNA typing (RAPD-PCR) was used for genotyping of carbapenem-resistant Pseudomonas aeruginosa (CRPA). RESULTS: Minimum inhibitory concentration (MIC) assays demonstrated high levels of resistance to all classes of antibiotics except colistin and polymyxin B. The initial screening by carbapenem disks indicated 24 isolates (63.15%) as CRPA. Different mechanisms of carbapenem resistance were observed, including carbapenemase production (8.4%), overexpression of AmpC (25%) and decreased expression of OprD (75%). The overexpression of MexAB efflux pumps was detected in 19 (79.1%) isolates by phenotypic assay or real-time PCR. The resistance to carbapenem was multifactorial in most cases (58.3%). The RAPD genotyping revealed different patterns with nine clusters. CONCLUSION: According to our results, the prevalence of CRPA is at an alarming level. Our results did not demonstrate an epidemic clone. The most common mechanism of carbapenem resistance was decreased expression of OprD. Therefore, we suggest a reconsideration in the management of CRPA infections of patients in our burn care hospital in Azerbaijan, Iran.

Association of specific haplotype of tumor necrosis factor-α and interleukin-1ß polymorphisms with Helicobacter pylori infection and gastric carcinogenesis.

INTRODUCTION: The Helicobacter pylori infection and cytokine-mediated inflammatory responses play significant roles in the pathogenesis of gastric cancer (GC). This study was performed to determine the association between the risk of GC and genetic polymorphisms in interleukin (IL)-1ß and tumor necrosis factor-alpha (TNF-α). METHODS: The polymorphisms of IL-1ß and TNF-α genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 290 patients who underwent endoscopy. Infection with H. pylori was diagnosed by histological analysis, rapid urease test, and PCR of gastric biopsy samples. Quantitative real-time PCR was performed to determine the relative mRNA expression levels. RESULTS: No significant difference was detected in allele frequency and genotype of all studied polymorphisms between chronic gastritis (CG), GC and healthy individuals. IL-1ß mRNA was down-regulated in both gastritis (relative quantification (RQ)=0.447) and the GC groups (RQ=0.151). In contrast, the expression of TNF-α was up-regulated in the GC group (RQ=2.817) compared to the gastritis group (RQ=0.861). CONCLUSIONS: The studied single-nucleotide polymorphisms are not risk factors for development of CG and GC. However, H. pylori infection causes a huge increase in the TNF-α expression in GC patients.

Characterization of carbapenem-resistant but cephalosporin-susceptible Pseudomonas aeruginosa.

In this study, mechanisms of carbapenem resistance in carbapenem-resistant but cephalosporin-susceptible (Car-R/Ceph-S) Pseudomonas aeruginosa were investigated. A total of 243 P. aeruginosa isolates were studied. The disk diffusion and agar dilution methods were used for determination of antibiotic susceptibility patterns. AmpC and efflux pump overproductions were detected by phenotypic methods. The presence of carbapenemase-encoding genes was detected by polymerase chain reaction (PCR). The expression of OprD, MexAB-OprM, and MexXY-OprM efflux pumps was assessed by real-time PCR. According to disk diffusion method, altogether 116 P. aeruginosa isolates (47.7%) were carbapenem-resistant and among them, 23 isolates (19.8%) were cephalosporin-susceptible. Carbapenemase producer was not detected. Overexpression of AmpC was detected in one (4.3%) isolate that was ceftazidime-susceptible but cefepime-resistant. Overexpression of MexAB-OprM and MexXY-OprM efflux pumps was detected in 12 (60.9%) and 16 (68.8%) of isolates, respectively. A total of 16 (68.8%) isolates showed decreased expression of OprD. The Car-R/Ceph-S P. aeruginosa did not develop by carbapenemase production. The resistance to carbapenem was mediated in our clinical isolates by decreased expression of OprD and overexpression of MexAB-OprM and MexXY-OprM efflux systems or the combination of these mechanisms.

Genotypic Diversity of Multidrug Resistant Shigella species from Iran.

BACKGROUND: In many developing countries, shigellosis is endemic and also occurs in epidemics and treatment of multidrug-resistant (MDR) isolates are important. The aims of this study were to determine the antimicrobial susceptibility, prevalence of class 1 and 2 integrons and the clonal relatedness of isolates. MATERIALS AND METHODS: Antimicrobial susceptibility tests were performed by disc diffusion method. Polymerase chain reaction (PCR)-sequencing technique was employed for detection and characterization of integrons. The genetic relatedness was evaluated by using enterobacterial repetitive intergenic consensus (ERIC) PCR. RESULTS: There was a high percentage of resistance to trimethoprim-sulfamethoxazole (TMP/SMX) (93.7%), ampicillin (AMP) (87.3%), streptomycin (STR) (84.5%) and tetracycline (TET) (78.9%). Multidrug resistant phenotype was seen in 95.1% of total isolates. Most common MDR profile was TMP/SMX/STR/AMP resistant pattern. Among the 142 Shigella spp. analyzed in this study, 28 isolates were positive for class 1 integron with two types of gene cassette arrays (dfrA17/aadA5 = 31.7% and dfrA7 = 3.8%). The class 2 integron was more frequently detected among the isolates (94.7%) with dfrA1/sat1/aadA1 (69.4%) and dfrA1/sat1 (30.6%) gene cassettes. ERIC-PCR results showed 6, 5, 4 and 3 main genotypes among S. flexneri, S. sonnei, S. boydii and S. dysenteriae isolates, respectively. CONCLUSIONS: Our findings revealed that multidrug resistant Shigella species with high prevalence of class 2 integron were very common in Iran. In addition, ERIC-PCR patterns showed limited variety of clones are responsible for shigellosis in the region of the study.

Characterization of integrons, extended-spectrum ß-lactamases, AmpC cephalosporinase, quinolone resistance, and molecular typing of Shigella spp. from Iran.

INTRODUCTION: The wide distribution of extended-spectrum ß-lactamase (ESBL) producing Shigella spp., along with the emergence of fluoroquinolone resistant isolates, is a serious threat to public health, posing a new challenge for the effective treatment of shigellosis. The purpose of this study was to determine the level of antimicrobial resistance, the presence of genes encoding resistance to cephalosporins, and plasmid-mediated quinolone resistance (PMQR) among the clinical isolates of Shigella spp. in Iran. MATERIALS AND METHODS: A total of 142 Shigella isolates were collected from different parts of Iran. All of the cephalosporin resistant Shigella strains were selected based on ESBL and AmpC production. The presence of PMQR regions was assessed in ciprofloxacin-resistant isolates, and genetic relatedness in the isolates was determined. RESULTS: Seventy-eight Shigella isolates were found to be resistant to extended-spectrum cephalosporin (ESC). The blaCTX-M15 was the most prevalent cephalosporinase. Four ESBL-producing isolates were also resistant to ciprofloxacin. Among the PMQR regions, aac(6')-lb-cr gene was the most prevalent, as it was seen in 83.3% of the ciprofloxacin resistant isolates, while qnrA was positive in 16.7%. Clonal relatedness showed a limited variety of clones was responsible for Shigella infection in the region studied. CONCLUSION: Overall, our findings indicated that a large number of ESBL producing Shigella spp. were mediated mainly by blaCTX-M15. This study is the first report on ciprofloxacin-resistant ESBL-producing Shigella isolates from patients in Iran.

Evaluation of Carbapenem Resistance Mechanisms and Its Association with Pseudomonas aeruginosa Infections in the Northwest of Iran.

The aims of this study were to determine carbapenem resistance mechanisms, molecular epidemiological relationship, clinical impact, and patient outcome of carbapenem-resistant Pseudomonas aeruginosa (CRPA) infections. A total of 42 nonduplicated CRPA were recovered from Urmia, Iran. Antimicrobial susceptibility tests were carried out using phenotypic methods. The carbapenem resistance mechanisms such as carbapenemase genes, efflux pump hyperexpression, AmpC overproduction, and OprD gene downregulation were determined by phenotypic and molecular methods. Eighteen metallo-ß-lactamase (MBL) producer isolates were found to be sensitive to amikacin. Among the CRPA, 52.3%, 26.1%, 26.1%, and 59.5% were identified as carbapenemase, efflux pump hyperexpression, AmpC overproduction, and reduced expression OprD gene, respectively. Random Amplified Polymorphic DNA analysis yielded 25 distinct profiles. Most MBL-positive isolates were recovered from patients hospitalized in urology and internal wards with urinary tract infections. Most of the strains showed downregulation of porin. The clonal distribution of the strains was related to carbapenem resistance mechanisms (most of MBL producers belong to the same clones) and the same hospital wards where the isolates were collected. The study demonstrates that the main risk factor of MBL-related infections was hospitalization in non-intensive wards. Amikacin was considered a very efficient antibiotic to treatment of MBL-producing CRPA isolates. Our results showed that OprD downregulation and IMP-type MBL are the main carbapenem resistance mechanisms in CRPA isolates from northwest of Iran.

Role of MexAB-OprM and MexXY-OprM efflux pumps and class 1 integrons in resistance to antibiotics in burn and Intensive Care Unit isolates of Pseudomonas aeruginosa.

BACKGROUND: The overexpression of efflux pumps and existence of class 1 integrons are the most important mechanisms that contribute to antimicrobial resistance in Pseudomonas aeruginosa especially in burn and Intensive Care Units (ICUs). The present study evaluated the role of MexAB-OprM and MexXY-OprM efflux pumps and class 1 integrons in resistance to antibiotics in burn and ICU isolates of P. aeruginosa. METHODS: Fifteen burn and forty-two ICU isolates were obtained from four hospitals in Northwest Iran. The isolates were identified and evaluated by the disk diffusion and agar dilution methods for determining antibiotic resistances. The presence of class 1 integrons and associated resistance gene cassettes were detected by PCR and sequencing of the products. The expression levels of efflux pumps were evaluated by phenotypic and genotypic (Quantitative Real-time PCR) methods. The isolates were genotyped by Random Amplified Polymorphic DNA Typing (RAPD-PCR). RESULTS: All burn isolates were integron positive and Multi-drug resistant (MDR), while 78.5% and 69% of ICU isolates were found as MDR and integron positive, respectively. The aadB gene was the most prevalent gene cassette (63.6%) followed by aacA4 (47.7%). Thirty-nine (68.4%) and 43 (75.4%) isolates exhibited an overexpression of MexAB-OprM and MexXY-OprM. Among burn isolates, 80% and 86.6% of them were mexB and mexY overexpressed, while 64.2% and 71.4% of ICU isolates exhibited mexB and mexY overexpression, correspondingly. The isolates were genotyped as 24 different RAPD profiles and were grouped into 15 clusters. CONCLUSIONS: The data suggested that class 1 integron had a more significant role than efflux pumps in resistance to beta-lactams and aminoglycosides in burn and ICUs except for gentamicin in burn isolates. Based on our data, it is possible that efflux pumps were not the main cause of high-level resistance to antibiotics.

Carbapenem inactivation: a very affordable and highly specific method for phenotypic detection of carbapenemase-producing Pseudomonas aeruginosa isolates compared with other methods.

This investigation was undertaken to compare phenotypic and molecular methods for detection of carbapenemase-producing Pseudomonas aeruginosa. A total of 245 non-duplicated isolates of P. aeruginosa were collected from hospitalized patients. Disc diffusion method was used to identify carbapenem-resistant bacteria. Three phenotypic methods, including Modified Hodge Test (MHT), Modified Carba NP (MCNP) test and Carbapenem Inactivation Method (CIM) were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was used to detect carbapenemase encoding genes. Of 245 P. aeruginosa isolates investigated, 121 isolates were carbapenem-resistant. Among carbapenem-resistant isolates, 40, 39 and 35 isolates exhibited positive results using MHT, MCNP test and CIM, respectively. PCR indicated the presence of carbapenemase genes in 35 of carbapenem-resistant isolates. MHT showed low sensitivity and specificity for carbapenemase detection among P. aeruginosa isolates in comparison to PCR. CIM was most affordable and highly specific than MCNP test compared with the molecular method.