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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

High-level resistance to aminoglycosides and ampicillin among clinical isolates of Enterococcus species in an Iranian referral hospital.

BACKGROUND AND OBJECTIVES: Nowadays, high-level aminoglycosides and ampicillin resistant Enterococcus species are among the most common causes of nosocomial infections. The present study was conducted to determine the prevalence of high-level resistance to aminoglycosides and ampicillin among clinical isolates of Enterococcus species in Ardabil, Iran. MATERIALS AND METHODS: In this cross-sectional study, a total of 111 Enterococcus species were collected from different clinical specimens between 2013 and 2015. Enterococcus species were identified using standard phenotypic and genotypic methods. BHI agar screen and agar dilution methods were used for detection of high-level gentamicin and streptomycin resistance (HLGR and HLSR) and minimal inhibitory concentration (MIC) of ampicillin, respectively. RESULTS: Of 111 clinical isolates, 59 (53.2%) and 25 (22.5%) isolates were E. faecalis and E. faecium, respectively, based on the PCR results. Totally, 60.3% and 56.7% of isolates were HLGR and HLSR, respectively, as well as 51.35% were HLGR plus HLSR. Among HLGR isolates, 36 (61.01%), 18 (72%) and 13 (48.14%) were E. faecium, E. faecalis and non-faecalis non-faecium species, respectively. Among HLSR isolates, 33 (55.93%), 16 (64%) and 14 (51.85%) were E. faecalis, E. faecium and non-faecalis non-faecium species, respectively. All HLGR isolates contained aac(6')Ie-aph(2″)Ia gene. Overall, the prevalence of high-level ampicillin resistance among Enterococcus species was 17.1%. For E. faecalis, E. faecium and non-faecalis non-faecium species, ampicillin resistance rates were as follows: 11 (40.74%), 7 (28%) and 1 (1.69%), respectively. For aminoglycoside antibiotics, the resistance rate was significantly higher in E. faecium isolates and for ampicillin it was higher in E. faecalis isolates. CONCLUSION: The frequency of high-level aminoglycoside resistant enterococcal isolates in our hospital was high and significant ampicillin resistance was noticed. This would require routine testing of enterococcal isolates for HLAR and ampicillin susceptibility.

Evaluating the efficiency of TaqMan real-time PCR and serological methods in the detection of Brucella spp. in clinical specimens collected from suspected patients in Ardabil, Iran.

BACKGROUND: This study aims to evaluate the efficiency of the TaqMan real-time PCR and serological methods in detecting Brucella spp. in clinical specimens that have been collected from suspected patients in Ardabil, Iran. METHODS: In this cross-sectional study, a total of 113 consecutive patients suspected of brucellosis who were referred to the three hospitals in Ardabil province were selected. In the first step, the diagnosis of brucellosis was performed by serological methods including the Rose Bengal slide agglutination test, Wright test, 2-ME test, and BrucellaCapt test. In the next step, TaqMan real-time PCR with primer and probe targeting the bcsp31 gene was used for the detection of Brucella spp. Specificity, sensitivity, and positive and negative predictive values of the TaqMan real-time PCR assay were calculated. RESULTS: Among 113 suspected patients with different clinical manifestations, the Rose Bengal slide agglutination test, Wright test, and 2-ME test were positive in 60 cases; however, the BrucellaCapt test titer was 1:160 for one patient. Six patients had high initial serum antibody titers; 2-ME titers of ≥1:640; STA titers of ≥1:1280; BrucellaCapt titers of ≥ 1:2560. Among positive cases, no correlation was observed among gender, age, and life (residence) in urban or rural areas. The TaqMan real-time PCR was positive in 35% of all 60 positive cases. The comparison of the results of the BrucellaCapt and TaqMan real-time PCR methods revealed that 19 out of 54 (35.2%) and 2 out of 6 (33.4%) BrucellaCapt positive cases with titers of >1:320 and ≤ 1:320 were positive, respectively. The sensitivities and specificities of the TaqMan real-time PCR assay were 49.1% and 100% respectively. CONCLUSION: The sensitivity of the TaqMan real-time PCR assay was low in the diagnosis of brucellosis, while the BrucellaCapt test turned out to be a very valuable, sensitive, and specific test for the diagnosis of brucellosis in suspected patients and, thus, can provide reliable results in medical laboratories.

Prevalence of fluoroquinolone-resistant Salmonella serotypes in Iran: a meta-analysis.

The present study was conducted to investigate the antimicrobial susceptibility profiles of Salmonella serotypes, especially fluoroquinolone-resistant strains, recovered from clinical samples in Iran. A full electronic search using related keywords was conducted in Persian and English languages in ISI Web of Knowledge, PubMed, Scopus, Google Scholar and the Scientific Information Database (SID) search engines to find papers published between 1983 and 1 July 2019. According to the inclusion and exclusion criteria, 46 eligible articles were selected for the final analysis out of the initial 13,186 studies retrieved. The pooled prevalence of quinolone-resistant Salmonella serotypes in clinical specimens in Iran was 2.9% to ciprofloxacin and 48.1% to nalidixic acid. Additional data on antibiotic resistance was as follows: 54.3% to tetracycline, 50.6% to ceftizoxime, 50.2% to streptomycin, 37.9% to ampicillin, 36.5% to kanamycin, 33.5% to trimethoprim-sulfamethoxazole, 27.2% to chloramphenicol, 19.1% to cephalothin, 8.8% to ceftriaxone, 7.6% to cefotaxime, 7.4% to aztreonam, 7.2% to gentamicin, 7% to cefepime, 6.8% to ceftazidime, 5.8% to cefixime, 2.7% to imipenem and 2.2% to meropenem. Findings of the present study showed a rising trend of resistance to the drugs of choice for the treatment of Salmonella infections, i.e. ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole in Iran. However, ciprofloxacin, third-generation cephalosporins and carbapenems are still effective antibiotics especially against multi-drug resistant strains in Iran.

Faecal carriage of high-level aminoglycoside-resistant and ampicillin-resistant Enterococcus species in healthy Iranian children.

OBJECTIVES: High-level aminoglycoside, ampicillin and vancomycin resistance and virulence genes among enterococcal isolates collected from healthy middle-school children in Ardabil, Iran, during 2016 were investigated. METHODS: Totally, 305 faecal specimens were collected. Isolates underwent antimicrobial susceptibility testing, virulence gene detection and molecular typing. RESULTS: Totally, 409 enterococcal isolates were collected, comprising Enterococcus faecium (235; 57.5%), Enterococcus faecalis (56; 13.7%) and other Enterococcus spp. (118; 28.9%). Overall, 71 (17.4%), 11 (2.7%) and 10 (2.4%) isolates were identified as high-level streptomycin-resistant (HLSR), high-level gentamicin-resistant (HLGR) and ampicillin-resistant (AR), respectively. Among HLSR isolates, 40 (56.3%), 5 (7.0%) and 26 (36.6%) were E. faecium, E. faecalis and other Enterococcus spp., respectively. Among HLGR isolates 4 (36.4%) and 7 (63.6%) and among AR isolates 7 (70.0%) and 3 (30.0%) were E. faecium and other Enterococcus spp., respectively. Accordingly, 21.6%, 3.6% and 3.3% of subjects were colonised with HLSR, HLGR and AR Enterococcus spp. Carriage of HLGR, HLSR and AR isolates was associated with prior antibiotic consumption (P≤0.05). Additionally, male sex and antacid consumption were associated with AR enterococcal carriage. Moreover, 69 (97.2%), 10 (90.9%) and 9 (90.0%) of HLSR, HLGR and AR isolates were multidrug-resistant, respectively. No vancomycin-resistant enterococci were detected. ERIC-PCR revealed high genetic diversity among isolates. gelE and asa1 were major virulence genes both in E. faecalis and E. faecium. Presence of gelE was associated with HLSR and HLGR phenotypes (P≤0.05). CONCLUSION: Community intestinal carriage of HLSR enterococci was high; however, carriage of HLGR and AR enterococci was low.

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.