In Vitro antibiotic combinations of Colistin, Meropenem, Amikacin, and Amoxicillin/clavulanate against multidrug-resistant Klebsiella pneumonia isolated from patients with ventilator-associated pneumonia.

BACKGROUND: Hospital infections such as ventilator-associated pneumonia (VAP) due to multidrug-resistant Klebsiella pneumoniae (MDR-KP) strains have increased worldwide. In addition, biofilm production by these resistant isolates has confronted clinicians with higher treatment failure and infection recurrence. Given the paucity of new agents and limited data on combination therapy for MDR-KPs, the present study sought to evaluate the in vitro activity of several antibiotic combinations against planktonic and biofilm MDR-KPs isolated from patients with VAP. RESULTS: All 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates demonstrated multidrug resistance against the tested antibiotics. At planktonic mode, combinations of colistin-meropenem and amoxicillin/clavulanate in combination with meropenem, colistin, or amikacin showed synergism against 60-70% isolates. On the other hand, in the biofilm state, colistin-based combinations exhibited synergism against 50-70% isolates and the most effective combination was colistin-amikacin with 70% synergy. CONCLUSIONS: The results revealed that combinations of amoxicillin/clavulanate with colistin, meropenem, or amikacin in the planktonic mode and colistin with amoxicillin/clavulanate, meropenem, or amikacin in the biofilm mode could effectively inhibit CRKP isolates, and thus could be further explored for the treatment of CRKPs.

Determining effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of clinical strains of Pseudomonas aeruginosa in biofilm-inspired alginate encapsulates.

BACKGROUND: Biofilms play a role in recalcitrance and treatability of bacterial infections, but majority of known antibiotic resistance mechanisms are biofilm-independent. Biofilms of Pseudomonas aeruginosa, especially in cystic fibrosis patients infected with the alginate producing strains in their lungs, are hard to treat. Changes in growth-related bacterial metabolism in biofilm affect their antibiotic recalcitrance which could be considered for new therapies designed based on these changes. In this study, effects of nitrate, arginine, and ferrous were investigated on antibiotic recalcitrance in alginate-encapsulated P. aeruginosa strains isolated from cystic fibrosis patients in the presence of amikacin, tobramycin, and ciprofloxacin. Also, expression of an efflux pump gene, mexY, was analyzed in selected strains in the presence of amikacin and ferrous. METHODS: Clinical P. aeruginosa strains were isolated from cystic fibrosis patients and minimum inhibitory concentration of amikacin, tobramycin, and ciprofloxacin was determined against all the strains. For each antibiotic, a susceptible and a resistant or an intermediate-resistant strain were selected, encapsulated into alginate beads, and subjected to minimal biofilm eradication concentration (MBEC) test. After determining MBECs, sub-MBEC concentrations (antibiotics at concentrations one level below the determined MBEC) for each antibiotic were selected and used to study the effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of encapsulated strains. Effects of ferrous and amikacin on expression of the efflux pump gene, mexY, was studied on amikacin sensitive and intermediate-resistant strains. One-way ANOVA and t test were used as the statistical tests. RESULTS: According to the results, the supplements had a dose-related effect on decreasing the number of viable cells; maximal effect was noted with ferrous, as ferrous supplementation significantly increased biofilm susceptibility to both ciprofloxacin and amikacin in all strains, and to tobramycin in a resistant strain. Also, treating an amikacin-intermediate strain with amikacin increased the expression of mexY gene, which has a role in P. aeruginosa antibiotic recalcitrance, while treating the same strain with ferrous and amikacin significantly decreased the expression of mexY gene, which was a promising result. CONCLUSIONS: Our results support the possibility of using ferrous and arginine as an adjuvant to enhance the efficacy of conventional antimicrobial therapy of P. aeruginosa infections.

Global prevalence and antibiotic resistance in clinical isolates of Stenotrophomonas maltophilia: a systematic review and meta-analysis.

Introduction: Stenotrophomonas maltophilia is a little-known environmental opportunistic bacterium that can cause broad-spectrum infections. Despite the importance of this bacterium as an emerging drug-resistant opportunistic pathogen, a comprehensive analysis of its prevalence and resistance to antibiotics has not yet been conducted. Methods: A systematic search was performed using four electronic databases (MEDLINE via PubMed, Embase, Scopus, and Web of Science) up to October 2019. Out of 6,770 records, 179 were documented in the current meta-analysis according to our inclusion and exclusion criteria, and 95 studies were enrolled in the meta-analysis. Results: Present analysis revealed that the global pooled prevalence of S. maltophilia was 5.3 % [95% CI, 4.1-6.7%], with a higher prevalence in the Western Pacific Region [10.5%; 95% CI, 5.7-18.6%] and a lower prevalence in the American regions [4.3%; 95% CI, 3.2-5.7%]. Based on our meta-analysis, the highest antibiotic resistance rate was against cefuroxime [99.1%; 95% CI, 97.3-99.7%], while the lowest resistance was correlated with minocycline [4·8%; 95% CI, 2.6-8.8%]. Discussion: The results of this study indicated that the prevalence of S. maltophilia infections has been increasing over time. A comparison of the antibiotic resistance of S. maltophilia before and after 2010 suggested there was an increasing trend in the resistance to some antibiotics, such as tigecycline and ticarcillin-clavulanic acid. However, trimethoprim-sulfamethoxazole is still considered an effective antibiotic for treating S. maltophilia infections.

Molecular Characteristics of Staphylococcus aureus Strains Isolated from Nasal Cavity and Wound Infections Among Diabetic Patients.

Staphylococcus aureus is the most common pathogen contributing to diabetic foot infections (DFI). Nasal transmission of S. aureus potentially increases the risk of endogenous infection. The aim of this study was to determine the genetic diversity and antibiotic resistance profile of S. aureus isolates in nasal and wound samples from diabetic patients. A cross-sectional study was conducted from July 2018 to September 2019. S. aureus was isolated from the anterior nares and wounds of diabetic patients. All S. aureus isolates were characterized by detection of resistance and virulence genes (mecA, ermA, ermC, hla, hlb, hlg, sea, lukDE, pvl), staphylococcal cassette chromosome mec (SCCmec)-typing and staphylococcal protein A (spa)-typing. A total of 34 S. aureus were isolated from the wounds of 115 diabetic patients with DFI. Twenty-four S. aureus isolates were collected from the anterior nares of patients, and thirteen patients had concurrent S. aureus in nasal and wound specimens. The prevalence of methicillin-resistant S. aureus (MRSA) in nasal specimens was noticeable (41.7%), and the most common spa-type in nasal and wound specimens was t14870. Nearly half of the patients with concurrent S. aureus in wound and nasal specimens had similar isolates from both sites. Our data suggest that detection and screening of S. aureus colonization in the nasal cavity may prevent subsequent endogenous infections, particularly with MRSA strains.

Antimicrobial resistance pattern, virulence determinants and molecular analysis of carbapenem-resistant Klebsiella pneumoniae isolated from clinical samples in Iran.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as an important global threat in recent years. The objective of the present study was to characterize the molecular characteristics, antibiotic resistance pattern and the distribution of virulence factors in CRKP isolated from different clinical specimens. A total of 60 clinical CRKP isolates were collected from clinical samples. Based on Clinical Laboratory Standards Institute guidelines, antimicrobial susceptibility testing was assessed by the disk diffusion method. Carbapenem and aminoglycoside resistance determinants in addition to virulence genes were inspected by PCR. Molecular characteristics of CRKP isolates were analyzed by random amplified polymorphic DNA (RAPD) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR. All isolates were resistant to imipenem, meropenem, cefoxitin, levofloxacin, cefotaxime, ceftazidime and ciprofloxacin. Resistance to tetracycline, gentamicin and kanamycin were detected in 53%, 75% and 72% of isolates, respectively. The most common carbapenem resistance genes were OXA-48 (28.5%) and NDM (22%). The most common aminoglycosides resistance genes were aac6´Ib (57%) and aac(3)-IVa (28%). The most prevalent virulence genes were mrkD (82%), entB (62%) and ybts (58%). ERIC and RAPD analyses revealed 55 and 53 different patterns of CRKP isolates, respectively. We conclude that CRKP infections have been associated with different genotypes and that the carbapenemase type (OXA-48) and AME gene (aac6´-Ib) were widely distributed in CRKP isolates in our hospital. Continued compliance with existing phenotypes and genotypes, and strict enforcement of infection control guidelines, are recommended approaches for the prevention and dissemination of these strains.

Multiplex detection of five common respiratory pathogens from bronchoalveolar lavages using high resolution melting curve analysis.

BACKGROUND: The study describes the application of the multiplex high-resolution melting curve (MHRM) assay for the simultaneous detection of five common bacterial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli) directly from bronchoalveolar lavage samples. RESULTS: Our MHRM assay successfully identified all five respiratory pathogens in less than 5 h, with five separate melting curves with specific melt peak temperatures (Tm). The different Tm were characterized by peaks of 78.1 ± 0.4 °C for S. aureus, 83.3 ± 0.1 °C for A. baumannii, 86.7 ± 0.2 °C for E. coli, 90.5 ± 0.1 °C for K. pneumoniae, 94.5 ± 0.2 °C for P. aeruginosa. The overall sensitivity and specificity of MHRM were 100% and 88.8-100%, respectively. CONCLUSIONS: Our MHRM assay offers a simple and fast alternative to culture approach for simultaneous detection of five major bacterial lower respiratory tract infection pathogens. Utilization of this assay can help clinicians initiate prompt and appropriate antimicrobial treatment, towards reducing the morbidity and mortality of severe respiratory infections.

Multiple-locus variable-number tandem repeat analysis for genotyping of erythromycin-resistant group B streptococci in Iran.

BACKGROUND: Group B Streptococcus (GBS or S. agalactiae) is an important pathogen causing severe invasive diseases in neonates, pregnant women, and adults with underlying medical conditions. METHODS: To investigate the incidence of resistance to macrolide, lincosamide and streptogramin type B (MLSB) antibiotics, macrolide and tetracycline resistance determinants and genetic relationships, a total of 146 clinical isolates of GBS were collected from Tehran, Iran. The genetic relationships between erythromycin-resistant strains were determined by multilocus variable tandem repeat analysis (MLVA). RESULTS: All isolates were susceptible to penicillin, vancomycin, linezolid, and quinupristin-dalfopristin, but were resistant to tetracycline (96.6%, 141/146), erythromycin (28.1%, 41/146) and clindamycin (16.4%, 24/146). Among the 41 erythromycin-resistant GBS (ERGBS), the most common antimicrobial resistance gene was tetM detected in 92.7% (38/41) of the isolates followed by ermTR and ermB found in 65.8% (27/41) and 29.3% (12/41) of isolates, respectively. Of the 41 ERGBS, 95% (39/41) exhibited the constitutive MLSB phenotype, 2.4% (1/41) displayed inducible MLSB and 2.4% (1/41) had M phenotype. The erm methylase genes were widely related to MLSB phenotype isolates, while the mefA gene was associated with M phenotype. MLVA analysis performed on the 41 ERGBS revealed that 34 MLVA types (MTs). MLVA analysis showed that infections due to ERGBS have been caused by a variety of genotypes, suggesting that ERGBS were clonally unrelated and dissemination of these isolates was not due to a clonal outbreak. CONCLUSION: Careful usage of macrolide antibiotics in therapy, continued surveillance of resistance rate and appropriate infection control measures can help to reduce spreading of resistance isolates.

Multiplex high-resolution melting assay for simultaneous detection of five key bacterial pathogens in urinary tract infections: A pilot study.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.

Distribution and Characteristics of Bacteria Isolated from Cystic Fibrosis Patients with Pulmonary Exacerbation.

Background: Cystic fibrosis (CF) is an inherited recessive disorder characterized by recurrent and persistent pulmonary infections, resulting in lung function deterioration and early mortality. Methods: A cross-sectional study was conducted on the bacterial profile and antibiotic resistance pattern of 103 respiratory specimens from CF patients with signs of pulmonary exacerbation. Antibiotic susceptibility testing and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa isolates were performed by the Kirby-Bauer disc diffusion method and microtiter plate assay, respectively. Molecular typing of S. aureus and P. aeruginosa isolates was carried out by spa typing and repetitive extragenic palindromic element PCR. Results: In a total of 129 isolates, the most prevalent organisms were S. aureus (55.3%) and P. aeruginosa (41.7%). Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. The highest rate of resistance for S. aureus was observed to azithromycin and erythromycin (80%), ciprofloxacin (52.3%), clindamycin (44.6%) and tetracycline (43%). Twenty percent of S. aureus isolates were methicillin-resistant S. aureus (MRSA) and 47.6% were MDR S. aureus. For P. aeruginosa isolates the highest resistance was to cefepime (38.3%) and levofloxacin (33.3%) and 20% showed MDR phenotype. Conclusion: Our study demonstrated a significant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa showed a shift in prevalence toward older ages. Molecular typing methods showed great diversity between isolates.

Identification of major sequence types among aminoglycoside resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical samples.

Background and Objectives: Aminoglycosides have been widely used for treating severe staphylococcal infections. Production aminoglycoside modifying enzymes (AMEs) is the main mechanism of resistance to this antibiotic. The aim of this study was to determine the prevalence of AME genes and molecular characterization of aminoglycoside-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical specimens in Iran. Materials and Methods: A total of 42 clinical isolates of Gram-positive cocci (20 S. aureus and 22 S. epidermidis) with resistance to gentamicin were tested for antimicrobial resistance and differentiated by multilocus sequence typing (MLST). Results: All 42 isolates were resistant to methicillin, kanamycin, and most of them were also resistant to amikacin (98%), tobramycin (98%) and netilmycin (78.5%). Overall, aac(6')-Ie-aph(2")-Ia was the dominant AME gene found in 100% of isolates, followed by aph(3')IIIa found in 90% of isolates. MLST classified S. aureus and S. epidermidis into 5 and 9 distinct sequence types (ST), respectively. The majority of the strains belonged to ST239 (50%) for S. aureus and ST2 (36%) for S. epidermidis. Conclusion: The resistance to aminoglycosides was mainly due to the presence of the aac(6')-Ie-aph(2")-Ia and aph(3') IIIa genes as well as the ST239 for S. aureus and ST2 for S. epidermidis have become the predominant clones in the selected university hospital of Tehran, Iran. Thus, it is critical that clinicians and healthcare workers are aware of the population of S. aureus and S. epidermidis present in order to make decisions for appropriate treatment and infection control practices.

A More Positive Culture by Resin-containing Media Usage after Suspicious Arthroscopic Infections in Patients Receiving Antimicrobial Therapy.

BACKGROUND: Although infections following arthroscopic surgery of cruciate ligament and meniscus are uncommon, they have potentially serious consequences for the graft and articular cartilage. This study aimed to investigate the efficacy of correct sampling and appropriate media, especially resin-containing media, for the detection of infections in patients receiving antibiotics under suspicion of joint infection after arthroscopic anterior cruciate ligament (ACL) and meniscal surgery. In such cases, proper sampling and the use of suitable culture media that cause the neutralization of antibiotics are very effective in isolating microorganisms from the patient samples and positive cultures. METHODS: In total, 10 patients who had received antibiotics with suspected knee infection after arthroscopic ACL and meniscal surgery were identified after referral to surgeons during a period of 10 months and investigated in this study. The sample collection, culture on various media (i.e., resin-containing culture media), microbiological tests, and antibiotic susceptibility tests were performed in this study. The amplification of the mecA gene using PCR assay was accomplished for methicillin-resistant staphylococcus strains. RESULTS: This study was conducted on 10 patients who underwent arthroscopic procedures and had received antibiotics. Overall, joint fluid and tissue culture were positive in 60% of the patients. The resin-containing media revealed a trend toward increased detection of bacteria. Coagulase-negative staphylococcus strains were the most frequently isolated bacteria in arthroscopic ACL surgery infections. Out of five methicillin-resistant staphylococcus strains, four strains were found that were resistant to cefoxitin and positive-mecA designated as methicillin-resistant strains. Except for one case, the rest of the staphylococcal strains were resistant to methicillin but susceptible to vancomycin. CONCLUSION: Despite uncommon and low percentage of infections after arthroscopic ACL and meniscal surgery, the results of our study showed that correct sampling, appropriate cultures, especially aerobic and anaerobic resin-containing media, and microbiological testing remained useful and valuable for diagnosing bacterial infections.

Efficacy of 16S rRNA variable regions high-resolution melt analysis for bacterial pathogens identification in periprosthetic joint infections.

BACKGROUND: Accurate and rapid identification of microorganisms causing periprosthetic joint infections (PJIs) are necessary for choosing an appropriate antibiotic therapy. Therefore, molecular techniques are suggested for diagnosis in suspected PJIs. The Broad-range PCR and High-Resolution Melt Analysis (HRMA) were evaluated for the identification of causative organisms of PJIs in this study. RESULTS: For 47 of 63 specimens, both the culture and broad-range PCR were positive. The culture was found to be able of organism's detection in 74.6% (47/63) of patients. Of 47 positive cultures, 11 (23.4%) were polymicrobial and 36 (76.59%) were monomicrobial cultures, in which 34 (91.89%) cases were detected by HRM assay. The sensitivity, specificity of HRMA vs monomicrobial culture were 91.89, 93.75%, respectively. The sensitivity, specificity of total HRMA (mono + poly) vs culture were 82.92, 93.75%. CONCLUSIONS: HRM assay coupled with broad-range PCR are effective screening, rapid, and relatively cost-effective methods for discrimination of PJIs especially in aiding culture method. Using computer programs such as the Matlab-2018b program for HRM data analysis is also valuable and helpful in diagnosis.

Antibiotic resistance pattern of Bacteroides fragilis isolated from clinical and colorectal specimens.

BACKGROUND: Bacteroides fragilis is a part of the normal gastrointestinal flora, but it is also the most common anaerobic bacteria causing the infection. It is highly resistant to antibiotics and contains abundant antibiotic resistance mechanisms. METHODS: The antibiotic resistance pattern of 78 isolates of B. fragilis (22 strains from clinical samples and 56 strains from the colorectal tissue) was investigated using agar dilution method. The gene encoding Bacteroides fargilis toxin bft, and antibiotic resistance genes were targeted by PCR assay. RESULTS: The highest rate of resistance was observed for penicillin G (100%) followed by tetracycline (74.4%), clindamycin (41%) and cefoxitin (38.5%). Only a single isolate showed resistance to imipenem which contained cfiA and IS1186 genes. All isolates were susceptible to metronidazole. Accordingly, tetQ (87.2%), cepA (73.1%) and ermF (64.1%) were the most abundant antibiotic-resistant genes identified in this study. MIC values for penicillin, cefoxitin and clindamycin were significantly different among isolates with the cepA, cfxA and ermF in compare with those lacking such genes. In addition, 22.7 and 17.8% of clinical and GIT isolates had the bft gene, respectively. CONCLUSIONS: The finding of this study shows that metronidazole is highly in vitro active agent against all of B. fragilis isolates and remain the first-line antimicrobial for empirical therapy.

Phenotypic and Genotypic Prevalence of Extended-Spectrum ß-Lactamase-Producing Escherichia coli: A Systematic Review and Meta-Analysis in Iran.

Background: Despite the existence of discrete and varied studies regarding extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) in Iran, a comprehensive analysis on the prevalence of ESBL-EC has not yet been carried out. The current study analyzed published data regarding ESBL-EC in different regions of Iran to gain insight into this significant subject. Methods: A meta-analysis was performed using Comprehensive Meta-Analysis Software (version 2.2; Biostat) to determine the prevalence of ESBL-EC in Iran. A web-based search was conducted in electronic databases, including PubMed, Scopus, and Web of Sciences. The eligibility of articles published between 2008 and 2018 was assessed, and relevant data were extracted for statistical analysis. A random-effects model was used based on the heterogeneity test. Publication bias was determined using Begg's rank correlation and Egger's weighted regression methods. Results: Among 31,135 studies examined, 61 met inclusion criteria and were included for review. Iran's overall pooled proportion of ESBL-EC was 43.2% (confidence interval [95% CI] 39.2-47.3), and the overall heterogeneity (I2) between studies was significantly high (93.5%, p = 0.00). The most prevalent of ESBLs in E. coli was CTX-M and TEM, with prevalence of 31.2% (95% CI 25.4-37.6), 27.6% (95% CI 22.7-33.2), respectively. Conclusion: The available studies show a high rate of ESBL-EC in Iran. This result highlights a need for appropriate and rapid methods for estimating ESBL infection, which can help our understanding of the actual epidemiology of ESBL and provide protocols for the prevention and control of infection.

Molecular characterization, antibiotic resistance pattern and capsular types of invasive Streptococcus pneumoniae isolated from clinical samples in Tehran, Iran.

BACKGROUND: Streptococcus pneumoniae causes serious infections worldwide. The aim of this study was to determine the molecular characteristic, antibiotic resistance pattern and capsular types of invasive S. pneumoniae in Tehran, Iran. RESULTS: Of the 44 pneumococcal invasive isolates, 39 (89%) were isolated from children and 5 (11%) from adults. The results show that all pneumococcal isolates were susceptible to linezolid but had varying resistance to trimethoprim-sulfamethoxazole (86%), erythromycin (73%), tetracycline (66%), clindamycin (43%), penicillin (16%), chloramphenicol (14%) and levofloxacin (2%). The range of erythromycin, tetracycline and penicillin MICs were 2 - ≥ 256 µg/mL, 4 - ≥ 48 µg/mL, and 0.047 - ≥ 256 respectively. All of the penicillin resistant isolates were multidrug resistant (MDR) and in addition to penicillin were resistant to tetracycline, erythromycin and trimethoprim-sulfamethoxazole. The most common capsular types detected in 64% of the pneumococcal isolates was 6A/B, 19A, 15A, 23F. The multilocus sequence typing (MLST) of 10 pneumococcal isolates revealed 9 different sequence types (STs), including ST 15139 (capsular type 19A) and ST 15140 (capsular type 23F), which have not previously been reported. CONCLUSIONS: The study revealed that the S. pneumoniae isolates belonged to diverse capsular types and clones with high rate of resistance to erythromycin, tetracycline, and penicillin.

Combinatorial effects of antibiotics and enzymes against dual-species Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the wound-like medium.

Bacterial biofilms are one of the major issues in the treatment of chronic infections such as chronic wounds, where biofilms are typically polymicrobial. The synergy between species can occur during most polymicrobial infections, where antimicrobial resistance enhances as a result. Furthermore, self-produced extracellular polymeric substance (EPS) in biofilms results in a high tolerance to antibiotics that complicates wound healing. Since most antibiotics fail to remove biofilms in chronic infections, new therapeutic modalities may be required. Disruption of EPS is one of the effective approaches for biofilm eradication. Therefore, degradation of EPS using enzymes may result in improved chronic wounds healing. In the current study, we investigated the efficacy of trypsin, ß-glucosidase, and DNase I enzymes on the degradation of dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in a wound-like medium. These species are the two most common bacteria associated with biofilm formation in chronic wounds. Moreover, the reduction of minimum biofilm eradication concentration (MBEC) of meropenem and amikacin was evaluated when combined with enzymes. The minimum effective concentrations of trypsin, ß-glucosidase, and DNase I enzymes to degrade biofilms were 1 µg/ml, 8 U/ml, and 150 U/ml, respectively. Combination of 0.15 µg/ml trypsin and 50 U/ml DNase I had a significant effect on S. aureus-P. aeruginosa biofilms which resulted in the dispersal and dissolution of all biofilms. In the presence of the enzymatic mixture, MBECs of antibiotics showed a significant decrease (p < 0.05), at least 2.5 fold. We found that trypsin/DNase I mixture can be used as an anti-biofilm agent against dual-species biofilms of S. aureus-P. aeruginosa.

Toxigenic and non-toxigenic patterns I, II and III and biofilm-forming ability in Bacteroides fragilis strains isolated from patients diagnosed with colorectal cancer.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) associated with the initiation and progression of colorectal cancer (CRC) has been alarmingly reported all over the world. In this study, simultaneous investigation of toxigenic and non-toxigenic patterns I, II and III and biofilm formation ability of Bacteroides fragilis isolated from patients with colorectal cancer was performed. METHODS: Thirty-one patients diagnosed with CRC and thirty-one control subjects were recruited in this study. Specimens were cultured on BBE and BBA culture media. Classical phenotypic identification tests and PCR was performed to verify Bacteroides fragilis presence. Also, biofilm-forming ability and expression of bft gene were assessed under biofilm and planktonic forms. RESULTS: A total of 68 B.fragilis was isolated from all colorectal tissue, of which 13 isolates (19.1%) (11 isolates from CRC and 2 from normal tissue) were positive for bft gene. The abundance patterns of I, II and III were as follow in descending order; pattern I > pattern III > pattern II in CRC subjects and pattern II > pattern III > pattern I in normal tissues. Also, pattern I showed higher biofilm formation ability compared to other patterns. Toxin expression was significantly reduced in biofilm form comparing with planktonic form. CONCLUSIONS: Based on our findings, there was a difference between the abundance of patterns I, II, and III and biofilm formation in isolates obtained from CRC and normal tissues. Biofilm formation ability and toxin encoding gene (bft) are two main virulence factors in B. fragilis pathogenicity which require more investigation to treat B. fragilis infections effectively.

Prevalence of Genes Encoding Aminoglycoside-Modifying Enzymes in Clinical Isolates of Gram-Positive Cocci in Iran: A Systematic Review and Meta-Analysis.

Introduction: Several studies have investigated the genes encoding aminoglycoside-modifying enzymes (AMEs) among gram-positive cocci (GPC) such as Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), coagulase-negative staphylococci (CoNS), and Enterococcus spp. in Iran; however, a comprehensive analysis has not yet been performed. Thus, the present systematic review and meta-analysis was conducted to determine the prevalence of genes encoding AMEs among GPC in Iran. Methods: A systematic review of the data published in the English and Persian languages from January 2000 to October 2018 was performed by searching different electronic databases (Medline, Embase, Web of Science, and the Iranian Database). Meta-analysis was performed by using the Comprehensive Meta-Analysis (Biostat V2.2) software. Cochran's Q and I2 statistics were used to test heterogeneity, and publication bias was assessed by using funnel plot and Begg's and Egger's tests. Results: Out of 117 studies, 28 were considered eligible for inclusion in the current meta-analysis. The most prevalent AMEs gene among GPC was aac(6')-Ie-aph(2'')-Ia, with a prevalence of 97.7% (95% CI; 94.4-99) in high-level gentamicin-resistant enterococci and 67.7% (95% CI; 59.2-75.2) in MRSA. The second most common gene was ant(4')Ia, with a prevalence of 45.3% (95% CI; 23.9-68.6) in MRSA. Conclusions: It was ultimately determined that the prevalence of AMEs genes among GPC had reached alarming levels in Iran; therefore, aminoglycosides should be prescribed with caution by clinicians. The implementation of a regional and nationwide surveillance system to monitor antimicrobial resistance, especially aminoglycosides, and increasing the awareness of AMEs genes among clinicians are essential to guiding empirical and pathogen-specific therapy.

The efficacy of lyticase and ß-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles.

BACKGROUND: Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and ß-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. RESULTS: This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, ß-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines. CONCLUSION: Considering the characteristics of the ß-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

Molecular analysis and antimicrobial resistance pattern of distinct strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Iran.

BACKGROUND AND OBJECTIVES: Colonization of Pseudomonas aeruginosa in Cystic Fibrosis (CF) patients may lead to severe pulmonary disease and death. Different characteristics of P. aeruginosa from these patients were determined in the present study. MATERIALS AND METHODS: Antimicrobial susceptibility and AmpC-overproduction were determined. The ß-lactamase genes were detected by PCR and the oprD gene was sequenced in some of the carbapenem resistance isolates. Distribution of exo genes was determined by PCR. Cytotoxicity of Exo effector proteins was measured using A549 cells. Biofilm production was determined by microtiter plate assay. Random amplified polymorphic DNA (RAPD) -PCR was performed for molecular analysis. RESULTS: Polymyxin B, piperacillin/tazobactam and meropenem were the most active antibiotics and 9.6% of isolates were ampC overproducers. The prevalence of bla VEB, bla OXA, bla VIM, and bla PER genes were as follow: 22.7%, 3.75%, 6.25% and 3.75%, respectively. A high proportion (83.5%) of isolates was able to produce biofilm. The exoT gene was present in all isolates while exoU was present in about 35% of them. RAPD-PCR revealed 49 patterns among 78 tested isolates in which 34 patterns were detected once. CONCLUSION: Biofilm formation ability and relatively high frequency of exoS may contribute to the persistence of bacteria within lungs of CF patients. Some characteristics of isolates recovered from a single patient after several sampling procedures were similar, while others lacked resemblance.