Prevalence and molecular characterization of colistin resistance in Pseudomonas aeruginosa isolates: insights from a study in Ardabil hospitals.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates. RESULTS: The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains. CONCLUSION: This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.

Carbapenem-resistant Enterobacterales in wastewater resources and healthy carriers: A survey in Iran.

The carbapenem-resistant Enterobacterales (CRE) pose a pressing public health concern. Here, we investigated the frequency of CRE bacteria, carbapenemase-encoding genes, and the molecular epidemiology of carbapenemase-resistant Escherichia coli in wastewater resources and healthy carriers in Iran. Out of 617 Enterobacterales bacteria, 24% were carbapenem-resistant. The prevalence of CRE bacteria in livestock and poultry wastewater at 34% and hospital wastewater at 33% was significantly higher (P ≤ 0.05) than those in healthy carriers and municipal wastewater at 22 and 17%, respectively. The overall colonization rate of CRE in healthy individuals was 22%. Regarding individual Enterobacterales species, the following percentages of isolates were found to be CRE: E. coli (18%), Citrobacter spp. (24%), Klebsiella pneumoniae (28%), Proteus spp. (40%), Enterobacter spp. (25%), Yersinia spp. (17%), Hafnia spp. (31%), Providencia spp. (21%), and Serratia spp. (36%). The blaOXA-48 gene was detected in 97% of CRE isolates, while the blaNDM and blaVIM genes were detected in 24 and 3% of isolates, respectively. The B2 phylogroup was the most prominent group identified in carbapenem-resistant E. coli isolates, accounting for 80% of isolates. High prevalence of CRE with transmissible carbapenemase genes among healthy people and wastewater in Iran underscores the need for assertive measures to prevent further dissemination.

Molecular characterization and prevalence of ß-lactamase-producing Enterobacterales in livestock and poultry slaughterhouses wastewater in Iran.

Beta-lactamase-producing Enterobacterales bacteria cause severe hard-to-treat infections. Currently, they are spreading beyond hospitals and becoming a serious global health concern. This study investigated the prevalence and molecular characterization of extended-spectrum ß-lactamase and AmpC-type ß-lactamase-producing Enterobacterales (ESBL-PE, AmpC-PE) in wastewater from livestock and poultry slaughterhouses in Ardabil, Iran. A total of 80 Enterobacterales bacteria belonging to 9 species were identified. Among the isolates, Escherichia coli (n = 21/80; 26.2%) and Citrobacter spp. (n = 18/80; 22.5%) exhibited the highest frequency. Overall, 18.7% (n = 15/80) and 2.5% (n = 2/80) of Enterobacterales were found to be ESBL and AmpC producers, respectively. The most common ESBL producer isolates were E. coli (n = 9/21; 42.8%) and Klebsiella pneumoniae (n = 6/7; 85.7%). All AmpC-PE isolates belonged to E. coli strains (n = 2/21; 9.5%). In this study, 80% of ESBL-PE and 100% of AmpC-PE isolates were recovered from poultry slaughterhouse wastewater. All ESBL-PE and AmpC-PE isolates were multidrug-resistant. In total, 93.3% of ESBL-PE isolates harbored the blaCTX-M gene, with the blaCTX-M-15 being the most common subgroup. The emergence of ESBL-PE and AmpC-PE in wastewater of food-producing animals allows for zoonotic transmission to humans through contaminated food products and contaminations of the environment.

Occurrence and Characteristics of Toxigenic Staphylococcus aureus in Retail Foods in Iran.

Staphylococcus aureus causes various toxigenic and invasive diseases in humans worldwide. This study examined the prevalence, virulence genes, and antibiotic resistance of S. aureus isolates collected from 894 retail food samples in Ardabil, Iran. Staphylococcal cassette chromosome mec (SCCmec), spa, and multilocus sequence typing methods were employed to further investigate the molecular characteristics of methicillin-resistant S. aureus (MRSA) isolates. The results revealed that 11.18% (n = 100) of food samples exhibited contamination with S. aureus (10.50% methicillin-sensitive S. aureus [MSSA] and 0.67% MRSA). Notably, raw minced meat (29.41%), Faloodeh (25%), and Olivier salad (21.42%) emerged as the most frequently contaminated food items. Among the 100 isolates of S. aureus, 94% were characterized as MSSA, with the remaining 6% identified as MRSA. The highest resistance was observed for penicillin (12%). MRSA isolates exhibited significantly higher resistance rates. Seventy-nine percent of the isolates were positive for sea, 14% for seb, 8% for a sec, and 0% for sed enterotoxin-encoding genes. Sixteen percent of isolates harbored two or more staphylococcal enterotoxin genes, simultaneously. Moreover, 97%, 94%, 24%, and 22% of isolates were positive for hla, hld, tst, and pvl virulence-encoding genes, respectively. No isolate was positive for the exfoliative toxins encoding eta and etb genes. MRSA isolates belonged to CC8 (n = 4) and CC22 (n = 2). Isolates in CC8 belonged to lineage ST239-MRSA-III and spa type t030; the isolates in CC22 belonged to ST22-MRSA-IV and spa types t310 and t223. In conclusion, a relatively high proportion of our retail food samples were contaminated with S. aureus. The high incidence of isolates with toxigenic genes raises serious health concerns. Furthermore, the presence of MRSA lineages linked to humans suggests that retail foods may be contaminated with human origin.

Prevalence and mechanisms of ciprofloxacin resistance in Escherichia coli isolated from hospitalized patients, healthy carriers, and wastewaters in Iran.

BACKGROUND: This study was aimed to evaluate the prevalence and molecular characteristics of ciprofloxacin resistance among 346 Escherichia coli isolates collected from clinical specimens (n = 82), healthy children (n = 176), municipal wastewater (n = 34), hospital wastewater (n = 33), poultry slaughterhouse wastewater (n = 12) and livestock (n = 9) slaughterhouse wastewater in Iran. METHODS: Ciprofloxacin minimum inhibitory concentration (MIC) was determined by agar dilution assay. Phylogroups and plasmid-mediated quinolone resistance (PMQR) genes were identified using PCR. Mutations in gyrA, gyrB, parC, and parE genes and amino acid alterations were screened through sequencing assay. The effect of efflux pump inhibitor (PAßN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method. RESULTS: In total, 28.03% of E. coli isolates were phenotypically resistant to ciprofloxacin. Based on sources of isolation, 64.63%, 51.51%, 33.33%, 14.70%, 10.22% and 8.33% of isolates from clinical specimens, hospital wastewater, livestock wastewater, municipal wastewater, healthy children and poultry wastewater were ciprofloxacin-resistant, respectively. Eighty-one point eighty-one percent (Ser-83 → Leu + Asp-87 → Asn; 78.78% and Ser-83 → Leu only; 3.03% (of ciprofloxacin-resistant E. coli isolates showed missense mutation in GyrA subunit of DNA gyrase, while no amino-acid substitution was noted in the GyrB subunit. DNA sequence analyses of the ParC and ParE subunits of topoisomerase IV exhibited amino-acid changes in 30.30% (Ser-80 → Ile + Glu-84 → Val; 18.18%, Ser-80 → Ile only; 9.10% and Glu-84 → Val only; 3.03%0 (and 15.38% (Ser-458 → Ala) of ciprofloxacin-resistant E. coli isolates, respectively. The PMQR genes, aac(6')-Ib-cr, qnrS, qnrB, oqxA, oqxB, and qepA were detected in 43.29%, 74.22%, 9.27%, 14.43%, 30.92% and 1.03% of ciprofloxacin-resistant isolates, respectively. No isolate was found to be positive for qnrA and qnrD genes. In isolates harboring the OqxA/B efflux pump, the MIC of ciprofloxacin was reduced twofold in the presence of PAßN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates. CONCLUSIONS: This study proved the high incidence of ciprofloxacin-resistant E. coli isolates in both clinical and non-clinical settings in Iran. Chromosomal gene mutations and PMQR genes were identified in ciprofloxacin resistance among E. coli population.

Molecular identification of multiple drug resistance (MDR) strain of Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVES: Isoniazid and rifampin are the first -line drugs against Mycobacterium tuberculosis. Resistance to these important drugs is a serious threat to human public health. Therefore, this study aimed at molecular detection of resistance to these valuable drugs. MATERIALS AND METHODS: In this descriptive cross-sectional study, 111 non - duplicated clinical samples including sputum and Bronchoalveolar lavage (BAL) samples were collected from patients referred to the Ardabil Health Center between 2017 and 2020. The samples were first examined by microscopic method, then their DNA was extracted using the boiling method. Specific primers and MAS-PCR method were employed for the detection resistance to isoniazid and rifampin drugs and identification of MDR strain. RESULTS: of 111 specimens, 15.3% belonged to NTM. In total, the resistance rate to isoniazid and rifampin was 17% and 27% respectively while the resistance rate to isoniazid and rifampin among NTM was 61.54% and 38.46%. CONCLUSION: In our study, the prevalence of resistance to isoniazid and rifampin among Mycobacterium tuberculosis complex(MTC) and non-tuberculous mycobacteria(NTM) was investigated using the MAS-PCR method. This work highlighted the high anti- tuberculosis resistance rate among NTM compared to MTC strains.

Antibiotic resistance and virulence potentials of E. faecalis and E. faecium in hospital wastewater: a case study in Ardabil, Iran.

Hospital wastewater can contaminate the environment with antibiotic-resistant and virulent bacteria. We analyzed wastewater samples from four hospitals in Ardabil province, Iran for Enterococcus faecium and Enterococcus faecalis using culture and molecular methods. We also performed antimicrobial susceptibility testing and polymerase chain reaction testing for resistance and virulence genes. Out of 141 enterococci isolates, 68.8% were E. faecium and 23.4% were E. faecalis. Ciprofloxacin and rifampicin showed the highest level of resistance against E. faecalis and E. faecium isolates at 65%. High-level gentamicin resistance (HLGR), high-level streptomycin resistance (HLSR), ampicillin, and vancomycin resistance were observed in 25, 5, 10, and 5.15% of E. faecium, and 15, 6, 15, and 3.03% of E. faecalis isolates, respectively. The ant(6')-Ia and ant(3')-Ia genes that were responsible for streptomycin resistance were observed in HLSR isolates and aph(3')-IIIa and aac(6') Ie-aph(2″)-Ia genes accounting for gentamicin resistance were detected in HLGR isolates. vanA was the predominant gene detected in vancomycin-resistant isolates. The majority of isolates were positive for gelE, asa1, esp, cylA, and hyl virulence genes. We found that drug-resistant and virulent E. faecalis and E. faecium isolates were prevalent in hospital wastewater. Proper treatment strategies are required to prevent their dissemination into the environment.

Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran.

Antimicrobial disinfectants have been extensively used to control hospital-acquired infections worldwide. Prolonged exposure to bacteria could promote resistance to antimicrobial disinfectants. This study evaluated the antimicrobial activity of four commonly used disinfectants; triclosan, chlorhexidine digluconate, benzalkonium chloride, and formaldehyde against Acinetobacter baumannii clinical isolates. This study also determined the prevalence and association of efflux pumps encoding genes qacE, qacED1, emrA, and aceI with tolerance to disinfectants. A total of 100 A. baumannii isolates were included in the current study. The antimicrobial disinfectants' minimum inhibitory concentration (MIC) was determined using an agar dilution method. Genes involved in resistance to disinfectants were investigated by PCR method. The benzalkonium chloride MICs ranged between 32 and 128 µg mL-1, chlorhexidine digluconate 8-64 µg mL-1, triclosan 1-32 µg mL-1, and formaldehyde 128 µg mL-1. Overall, the highest MIC90 value was identified for formaldehyde (128 µg mL-1), followed by benzalkonium chloride and chlorhexidine digluconate (64 µg mL-1, each one) and triclosan (4 µg mL-1). In the present study, the qacE, qacED1, emrA, and aceI genes were found in 91%, 55%, 100%, and 88% of isolates, respectively. The qacG gene was not identified in our A. baumannii isolates. The qacED1 gene was associated with higher MICs for all disinfectants tested (P < 0.05), while the qacE and aceI genes were associated with higher MICs for benzalkonium chloride and chlorhexidine. This study indicated that triclosan is the most effective disinfectant against A. baumannii isolates.

Phenotypic and molecular characterization of extended-spectrum ß-lactamase/AmpC- and carbapenemase-producing Klebsiella pneumoniae in Iran.

BACKGROUND: The objective of the current study is to evaluate the phenotypic and molecular characterization of ESBL/AmpC- and carbapenemase-producing K. pneumoniae isolates in Iran. METHODS: From October 2018 until the end of April 2020, different clinical samples were collected and K. pneumoniae isolates were identified using conventional biochemical tests and PCR assay. Antibiotic susceptibility pattern was determined using the Kirby-Bauer disk diffusion method. Modified Hedge Test (MHT) was applied to the identification of carbapenemase-producing K. pneumoniae. ESBL and AmpC-producing K. pneumoniae were detected using Double Disc Test (DDT) and Disc Potentiation Test (DPT), respectively. The presence of carbapenemase, ESBL, and AmpC encoding genes was screened by Polymerase Chain Reaction (PCR) assay. RESULTS: A total of 100 K. pneumoniae isolates were collected. K. pneumoniae isolates had the highest resistance rate to cefazolin (66%) and cefotaxime (66%). Meropenem and amikacin with sensitivity rates of 76% and 69% were the most effective antimicrobial agents on K. pneumoniae isolates. It was found that 12 (12%), 27 (27%), and 9 (9%) K. pneumoniae isolates were positive in MHT, DDT, and DPT tests, respectively. Among the carbapenemase-encoding genes, blaOXA-48 (24%) and blaIMP (13%) genes had the highest frequency, while blaKPC and blaGIM genes were not detected among K. pneumoniae isolates. blaTEM (48%) and blaCMY (8%) genes had the highest frequency among ESBL and AmpC ß-lactamase-encoding genes, respectively. CONCLUSIONS: It is vital to adopt effective control strategies for K. pneumoniae infections and ensure rapid identification of antibiotic resistance profile.

Prevalence of resistance genes to biocides in antibiotic-resistant Pseudomonas aeruginosa clinical isolates.

BACKGROUND: Biocides are frequently used as preservative, disinfectant and sterilizer against many microorganisms in hospitals, industry and home. However, the reduced susceptibility rate of Pseudomonas aeruginosa (P. aeruginosa) strains to biocides is increasing. The aim of this study was to evaluate the antimicrobial activity of four frequently used biocides against P. aeruginosa and to determine the prevalence of genes involved in biocide resistance. METHODS: A total of 76 clinical isolates of P. aeruginosa strains were used in the present study. The minimum inhibitory concentrations (MICs) of four biocides, i.e. chlorhexidine digluconate, benzalkonium chloride, triclosan and formaldehyde, against P. aeruginosa strains were determined using agar dilution method. In addition, the prevalence of biocide resistance genes was determined using the polymerase chain reaction (PCR) method. RESULTS: In the present study, the highest MIC90 and MIC95 (epidemiological cut-off) values were observed for benzalkonium chloride (1024 µg/mL), followed by formaldehyde (512 µg/mL), triclosan (512 µg/mL) and chlorhexidine digluconate (64 µg/mL). Furthermore, the prevalence of qacEΔ1, qacE, qacG, fabV, cepA and fabI genes were 73.7% (n = 56), 26.3% (n = 20), 11.8% (n = 9), 84.2% (n = 64), 81.5% (n = 62) and 0% (n = 0), respectively. A significant association was observed between the presence of biocide resistance genes and MICs (p < 0.05). Furthermore, there was no significant association between the presence of biocide resistance genes and antibiotic resistance (p > 0.05), except for levofloxacin and norfloxacin antibiotics and qacE and qacG genes (p < 0.05). CONCLUSION: Our results revealed that chlorhexidine digluconate is the most effective biocide against P. aeruginosa isolates in Ardabil hospitals. However, we recommend continuous monitoring of the antimicrobial activity of biocides and the prevalence of biocide-associated resistance genes for a better prevention of microorganism dissemination and infection control in hospitals.

Antimicrobial Biocides Susceptibility and Tolerance-Associated Genes in Enterococcus faecalis and Enterococcus faecium Isolates Collected from Human and Environmental Sources.

Enterococci are among the most common causes of nosocomial infections worldwide. Antimicrobial biocides are extensively used to control the growth of microorganisms on different surfaces. The purpose of this study was to determine the susceptibility of Enterococcus faecalis and Enterococcus faecium isolates collected in Iran to biocide agents, formaldehyde (FOR), benzalkonium chloride (BZC), triclosan (TRE), and chlorhexidine digluconate (CHDG). Additionally, the frequency of biocide tolerance-associated (BTA) genes, qacA/B, qacED1, emeA, sigV and gasp65 were investigated. In this study, 222 isolates of E. faecalis and 425 isolates of E. faecium from clinical and non-clinical sources were investigated. Minimum inhibitory concentration (MIC) of biocide agents was determined using agar dilution method. Biocides epidemiological cutoff values (ECOFFs) were determined using 95% rule. BTA genes were identified using PCR testing. ECOFFs for CHDG, BZC, TRE and FOR were 8 µg/mL, 16 µg/mL, 32 µg/mL and 512 µg/mL for both species, respectively. MIC values showed that the distribution of isolates with high level of tolerance to antimicrobial biocides was clearly different, depending on ecological niches. The BTA genes, qacA/B, qacED1, emeA, sigV and gasp65 were detected in 19.4% (43), 19.8% (44), 42.8% (95), 89.6% (199) and 70.2% (156) of E. faecalis and 10.3% (44), 17.2% (73), 27.8% (118), 42.2% (188) and 82.8% (352) of E. faecium isolates, respectively. Based on the distribution pattern of BTA genes 14 and 18 different profiles were identified for E. faecalis and E. faecium isolates respectively. Generally, the isolates carrying at least a single BTA gene showed higher MIC90 against all biocides compared to isolates with no BTA genes. However, there were no clear association between MIC90 values and carrying particular BTA genes profile. The results of this study showed that CHDG was the most effective biocide against E. faecalis and E. faecium isolates. The data presented in current study can be used to define the biocides resistance breakpoints.

Allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheter in a bladder model in vitro.

Stone formation and catheter blockage are major complications of Proteus UTIs. In this study, we investigated the ability of allicin to inhibit P. mirabilis-induced struvite crystallization and catheter blockage using a synthetic bladder model. Struvite crystallization inhibition study was carried out using P. mirabilis lysate as urease enzyme source in synthetic urine (SU). Struvite productions were monitored by phase contrast light microscopy and measurements of pH, Mg2+ and Ca2+ precipitation and turbidity. A catheter blockage study was performed in a synthetic bladder model mimicking natural UTI in the presence of allicin at sub-MIC concentrations (MIC = 64 µg/ml). The results of crystallization study showed that allicin inhibited pH rise and consequently turbidity and precipitation of ions in a dose-dependent manner. The results of catheter blockage study showed that allicin at sub-MIC concentrations (2, 4, 8 µg/ml) significantly increased the time for catheter blockage to occur to 61, 74 and 92 h respectively compared to allicin-free control (48 h). In a similar way, the results showed that allicin delayed the increase of SU pH level in bladder model in a dose-dependent manner compared to allicin-free control. The results also showed that following the increase of allicin concentration, Mg2+ and Ca2+ deposition in catheters were much lower compared to allicin-free control, further confirmed by direct observation of the catheters' eyehole and cross sections. We conclude that allicin prevents the formation of Proteus-induced urinary crystals and the blockage of catheters by delaying pH increase and lowering Mg2+ and Ca2+ deposition in a dose-dependent manner.

Prevalence of bacterial vaginosis in pregnant and non-pregnant Iranian women: a systematic review and meta-analysis.

PURPOSE: Bacterial vaginosis (BV) is a vaginal disorder which occurs either symptomatic or asymptomatic because of an imbalance between H2O2-producing Lactobacillus and Gardnerella vaginalis in the vagina. This systematic review and meta-analysis is the first to determine the prevalence of BV in pregnant and non-pregnant women in Iran. METHODS: We used national (SID, Irandoc, Iranmedex and Magiran) and international (PubMed, Scopus, Google Scholar and ISI web of knowledge) electronic databases to systematically search and collect available studies using related keywords (up to 1 December 2017). Inclusion and exclusion criteria were defined to select eligible studies. RESULTS: The overall prevalence of BV among Iranian women was 18.9% (95% CI 14-25). Gardnerella vaginalis was the most prevalent isolated bacteria. The prevalence of BV in non-pregnant women was 28% (95% CI 15.1-45.9) which was higher compared with pregnant women who had a prevalence of 16.5% (95% CI 12.5-21.6). CONCLUSION: The present review revealed a high prevalence of BV in non-pregnant women. Given that BV is associated with a series of reproductive complications such as infertility, taking preventive measures such as awareness of patients as well as monitoring and controlling of syndrome are essential.

In vitro antibacterial activity of poly (amidoamine)-G7 dendrimer.

BACKGROUND: Nano-scale dendrimers are synthetic macromolecules that frequently used in medical and health field. Traditional anibiotics are induce bacterial resistence so there is an urgent need for novel antibacterial drug invention. In the present study seventh generation poly (amidoamine) (PAMAM-G7) dendrimer was synthesized and its antibacterial activities were evaluated against representative Gram- negative and Gram-positive bacteria. METHODS: PAMAM-G7 was synthesized with divergent growth method. The structural and surface of PAMAM-G7 were investigated by transmission electron microscopy, scanning electron microscope and fourier transform infrared. Pseudomonas. aeruginosa (n = 15), E. coli (n = 15), Acinetobacter baumanni (n = 15), Shigella dysenteriae (n = 15), Klebsiella pneumoniae (n = 10), Proteus mirabilis (n = 15), Staphylococcus aureus (n = 15) and Bacillus subtilis (n = 10) have been used for antibacterial activity assay. Additionally, representative standard strains for each bacterium were included. Minimum Inhibitory Concentration (MIC) was determined using microdilution method. Subsequently, Minimum Bactericidal Concentration (MBC) was determined by sub-culturing each of the no growth wells onto Mueller Hinton agar medium. The cytotoxicity of PAMAM-G7 dendrimer were evaluated in HCT116 and NIH 3 T3 cells by MTT assay. RESULTS: The average size of each particle was approximately 20 nm. PAMAM-G7 was potentially to inhibit both Gram positive and gram negative growth. The MIC50 and MIC90 values were determined to be 2-4 µg/ml and 4-8 µg/ml, respectively. The MBC50 and MBC90 values were found to be 64-256 µg/ml and 128-256 µg/ml, respectively. The cytotoxity effect of dendrimer on HCT116 and NIH 3 T3 cells is dependent upon exposure time to and concentration of dendrimers. The most reduction (44.63 and 43%) in cell viability for HCT116 and NIH 3 T3 cells was observed at the highest concentration, 0.85 µM after 72 h treatmentm, respectively. CONCLUSIONS: This study we conclude that PAMAM-G7 dendrimer could be a potential candidate as a novel antibacterial agent.

Inhibition of streptococcal pyrogenic exotoxin B using allicin from garlic.

Streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) and inactivation of SpeB results in the significantly decreased virulence of the bacterium. The protein is secreted as an inactive zymogen of 40 KDa (SpeBz) and undergoes proteolytic truncation to result in a 28 KDa mature active protease (SpeBm). In this study the effect of allicin on the proteolytic activity of SpeBm was evaluated using azocasein assay. Allicin neutralized the SpeBm proteolytic activity in a concentration dependent manner (IC50 = 15.71 ± 0.45 µg/ml). The loss of activity was completely reversed by subsequent treatment with a reducing agent, dithiothreitol (DTT; 10 mM final concentration), suggesting that allicin likely inhibits the SpeBm by forming a disulfide linkage with an active thiol group in its active site. This mechanism of action was further confirmed with the fact that DTT did not reverse the SpeBm activity in the presence of E-64, a cysteine protease-specific inhibitor, which works specially by forming a thioether linkage with free sulfhydryl groups in enzymes active site. The MIC of allicin against GAS was found to be 32 µg/ml. Exposure of GAS culture to allicin (25 µg/ml) inhibited maturation of SpeBz to the SpeBm. In conclusion, the results of this study suggest that allicin inhibits the maturation of SpeBz and proteolytic activity of SpeBm and could be a potential therapeutic agent for the treatment of GAS infections.

Serotyping and molecular profiles of virulence-associated genes among Klebsiella pneumoniae isolates from teaching hospitals of Ardabil, Iran: A cross-sectional study.

Background and Aims: Klebsiella pneumoniae is a Gram-negative bacterium that colonized various organs. This bacterium is associated with different community-acquired and hospital-acquired infections. The present study aims to assess the capsular serotypes and frequency of virulence-associated genes in K. pneumoniae isolates from teaching hospitals in Ardabil, Iran. Methods: From October 1, 2019, to November 31, 2021, different clinical samples were collected and K. pneumoniae isolates were diagnosed using conventional biochemical tests. The final identification of K. pneumoniae was performed through the polymerase chain reaction (PCR) method using a specific primer targeting the khe gene. The PCR method was employed to confirm the presence of virulence-associated genes and aerobactin, and the main capsular serotypes based on the specific primers. Results: Of all 100 K. pneumoniae isolates, 4% and 2% were typeable with K5 and K2 primers, respectively. In addition, entB (94%), fimH (91%), and wcaG (87%) had the highest frequency among the virulence-associated genes. 24% of K. pneumoniae isolates harbored the entB-wcaG-fimH genes simultaneously. Moreover, 50% of capsular serotype 5 harbored the ybts-mrkD-entB-wcaG-fimH genes simultaneously. Conclusion: The findings revealed that 6% of all K. pneumoniae isolates were typeable, distributed in the two serotypes K5 and K2. Most K. pneumoniae isolates were positive for multiple types of virulence genes. Identifying bacterial virulence genes aids in molecular detection, assay development, and therapeutic pathways.

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

Background and Objectives: he occurrence and characteristics of Extended Spectrum- and AmpC-ß-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. Materials and Methods: A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Results: Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). Conclusion: ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

Macrolide-Lincosamide Resistance and Virulence Genes in Staphylococcus aureus Isolated from Clinical Specimens in Ardabil, Iran.

Background & Objective: Staphylococcus aureus causes various hospital- and community-acquired infections. This study aimed to investigate the phenotypic and genotypic characteristics of erythromycin and inducible clindamycin resistance, virulence gene profiles, and spa types of S. aureus isolates collected from patients in Ardabil Province, Iran. Methods: A total of 118 clinical S. aureus isolates, including 50 (42.4%) methicillin-resistant S. aureus (MRSA) and 68 (57.6%) methicillin-susceptible S. aureus (MSSA) strains, were investigated. Resistance patterns were determined by the disk diffusion method and minimum inhibitory concentration (MIC) test. Inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance was detected using D-test method. The polymerase chain reaction (PCR) was used to identify the virulence and resistance-encoding genes. Additionally, the spa types of the isolates were determined using the PCR, followed by sequencing. Results: In total, 49.1% (58/118) and 44% (52/118) of the isolates were resistant to erythromycin and clindamycin, respectively. Overall, 13.5% (16/118) of the isolates showed the iMLSB resistance phenotype. The ermC gene (72.4% [42]) was the most frequent erythromycin resistance-encoding gene, followed by ermA (60.3% [35]), ermB (60.3% [35]), ermTR (51.7% [30]), and msrA (15.5% [9]) genes among erythromycin-resistant isolates. The virulence genes hla, hld, sea, LukS PV, tst, seb, sed, eta, sec, and etb were detected in 93.2%, 74.5%, 70.3%, 32.2%, 29.6%, 17%, 8.5%, 8.5%, 5.9%, and 4.2% of the isolates, respectively. Ten different spa types were identified for 58 erythromycin-resistant S. aureus strains, of which t030 and t078 types were the most common types. Conclusion: A high frequency of macrolide- and lincosamide-resistant S. aureus isolates with different genetic backgrounds of resistance and virulence may be found in patients in Ardabil Province, Iran.

Phenotypic and Molecular Characterization of Carbapenems Resistant Escherichia coli Isolated from Patients with Urinary Tract Infections in Ardabil Province, Iran.

Background & Objective: Carbapenem-resistant is Gram-negative bacteria representing a worldwide public health problem. The present study aims to survey the phenotypic and genotypic characteristics of carbapenem-resistant Escherichia coli isolates collected from hospitalized patients and outpatients in Ardabil province, Iran. Methods: Two hundred samples were collected from the patients who had already been referred to the hospitals in Ardabil, Iran, from January to June 2017. Each patient's social and demographic data were recorded in the first step. The resistance profile of all E. coli isolates against imipenem and meropenem antibiotics were determined using the Kirby-Bauer disk diffusion method. Moreover, the broth microdilution method determined the Minimum Inhibitory Concentration (MIC) of E. coli isolates to imipenem. The Carbapenem Inactivation Method (CIM) and Carba NP test were employed for screening carbapenem-resistant strains. The frequency of carbapenem-encoding genes was determined using Polymerase Chain Reaction (PCR) method. The Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR analysis was used to evaluate the genetic relatedness of E. coli isolates. Results: Out of 200 urine samples, 66% (n = 132) of the samples were collected from women. The patients' age varied from 1 month to 93 years. Results of the disk diffusion method revealed that 33% (n=66/200) of E. coli isolates were resistant to imipenem. However, imipenem resistance was detected in 37% (n = 74/200) of the E. coli isolates using broth microdilution method. All E. coli isolates were negative in CIM and Carba NP tests. Moreover, we could not detect any carbapenemase encoding genes among E. coli isolates. The ERIC-PCR method revealed the E. coli strains were classified into 39 clusters with 80% similarity. Conclusion: It appears that E. coli is the most common cause of urinary tract infection in Ardabil province.

Fecal Carriage of Extended-Spectrum ß-Lactamases and pAmpC Producing Enterobacterales in an Iranian Community: Prevalence, Risk Factors, Molecular Epidemiology, and Antibiotic Resistance.

This study aimed to determine the prevalence and risk factors associated with intestinal carriage of extended-spectrum ß-lactamases producing Enterobacterales (ESBL-PE) and plasmid-mediated AmpC ß-lactamase producing Enterobacterales (AmpC-PE) in healthy children in Ardabil, Iran. A total of 305 fecal samples were collected. Isolates underwent antimicrobial susceptibility testing, phenotypic and genotypic identification of ß-lactamase production, and epidemiologic molecular typing. In total, 21.5%, 1.5%, and 1.2% of volunteers were extended-spectrum ß-lactamase (ESBL)-, AmpC-, and simultaneous ESBL/AmpC-PE carriers, respectively. Escherichia coli was the predominant ESBL producing bacterium (70.2%) found in ESBL-PE colonized subjects. Beyond ESBL positive isolates, bla CTX-M group genes were the most common type (75.6%) and bla TEM (non-bla TEM-1 and non- bla TEM-2) were in the second place (25.6%). Among bla CTX-M genes, bla CTX-M-1 (55.3%) and bla CTX-M-15 (55.3%) were the most predominant types with equal prevalence. Some isolates were multi-enzyme producers. bla CIT and bla DHA genes were common AmpC type enzyme encoding genes found in AmpC-PE isolates. Most isolates produced both enzymes at the same time. The number of students in the classes was statistically associated with ESBL-PE intestinal carriage (p < 0.05). Moreover, 46 (65.7%), 3 (60%), 4 (100%), and 98 (39.8%) ESBL-, AmpC-, ESBL/AmpC, and non-ESBL/AmpC-PE isolates were multidrug-resistant, respectively. Overall, regardless of ß-lactam antibiotics, 62% and 59.5% of isolates were resistant to co-trimoxazole and tetracycline, respectively. The majority of ESBL producing E. coli isolates (69.2%) belonged to phylogroup A. According to Enterobacterial repetitive intergenic consensus polymerase chain reaction, there was no clonal relatedness between isolates. This study showed a high rate of multi-resistant ESBL-PE intestinal carriage among healthy individuals in Iran.