Isolation and Identification of Effective Probiotics on Drug-Resistant Acinetobacter baumannii Strains and Their Biofilms.

Introduction: This study aimed to identify, assess, and isolate strong lactobacilli demonstrating broad antibacterial and anti-biofilm activity against drug-resistant strains of Acinetobacter baumannii. Additionally, the mechanism of inhibition of these organisms was to be determined. Methods: Over a 6-month period (from December 2021 to June 2022), 53 clinical A. baumannii strains were collected from clinical samples. Twenty probiotic strains were isolated from local dairy products. Antibacterial activity of Lactobacillus strains' cell-free supernatant (CFS) was identified using the agar well diffusion method and the microbroth dilution test. Anti-biofilm effect was performed by the microtiter plate assay. The MTT assay was also used to look into the probiotics' cytotoxic effects on the L929 fibroblast cell line. Results: During the 6-month period, 53 clinical A. baumannii strains were obtained and identified. Out of 20 lactobacillus strains, the CFS of a lactobacillus strain (named L9) showed an inhibitory effect against all A. baumannii strains. Using the broth microdilution method, it was shown that the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CFS extracts of L9 strains against A. baumannii strains were both » mg/mL. The result of the anti-biofilm showed that the selected probiotic could inhibit biofilm formation. The most common organic acid produced by all Lactobacillus strains, according to the HPLC method, was lactic acid, which was followed by acetic acid. The L929 fibroblast cell line was used in the cytotoxicity assay, which revealed that 100% of the cells in the L929 fibroblast cell line survived treatment with successive doses of CFSs for a full day. Conclusion: The probiotic strain isolated from local yogurt in this study showed potential anti-biofilm and antimicrobial properties against all drug-resistant Acinetobacter isolates. Given the increasing interest in probiotic microorganisms based on their high health benefits, further studies are recommended on the mechanisms of action between probiotics and A. baumannii strains to find new solutions for biological control and treatment of these infections without the use of antibiotics.

Investigating the Antagonistic Effect of Indigenous Probiotics on Carbapenem-Resistant Pseudomonas aeruginosa Strains.

Introduction: With the increase of hospital infections due to the indiscriminate use of antibiotics, multidrug resistance has increased, decreasing the effectiveness of antibiotics against these infections. For this reason, the identification of alternative agents such as probiotics has been considered. The aim of this study was to isolate and identify effective probiotics against carbapenem-resistant Pseudomonas aeruginosa strains. Material and Methods. During a period of eight months, isolates of P. aeruginosa were collected from patients in three hospitals in Isfahan. The presence of metallo-beta-lactamase enzymes was determined by the combination disc test (CDT). The inhibitory and antimicrobial activities of 20 probiotic bacteria isolated from local dairy products against these strains were investigated by agar dilution. Two probiotic strains that showed broader inhibition results were selected, and the values of the lowest inhibitory concentration (MIC) and the lowest lethal concentration (MBC) and their antibiofilm effect were determined using the microtiter plate method. The concentration of organic acids was done by HPLC. Findings. Of the 100 samples isolated and identified, 61 samples (61%) exhibited multiple drug resistance (MDR) and were selected for further investigation. Phenotypic diagnosis of the presence of metallo-beta-lactamase enzymes revealed that 74.5% of the strains were positive. The results showed that these two probiotics killed P. aeruginosa strains after only one hour, and the inhibition mechanism was due to the presence of lactic acid and acetic acid. The antibiofilm effect of these two probiotics was at concentrations of 1/2 and 1/4. Conclusion: The two Lactobacillus isolates had potential antimicrobial and antibiofilm properties against all carbapenem-resistant P. aeruginosa strains, even at thinner dilutions. Considering the broad activity of this strain, it can potentially be used for biocontrol of these strains.

Emergence of colistin resistant Acinetobacter baumannii clonal complex 2 (CC2) among hospitalized patients in Iran.

Acinetobacter baumannii is a major causative agent of serious nosocomial infections. This study was carried out to investigate the molecular characterization of colistin resistant isolates of A. baumannii from hospitalized patients, based on multilocus sequence typing (MLST). A cross-sectional study was conducted to collect A. baumannii from clinical samples in Isfahan from 2021 to 2022. Isolates were identified as A. baumannii using biochemical tests and PCR of blaOXA-51. Antibiotic susceptibility testing was carried out using the Kirby-Bauer method and minimum inhibitory concentration (MIC) values were determined for colistin. Additionally, MLST was performed according to the Pasteur scheme to assess the relationship between colistin resistant A. baumannii. A total of 70 non-repetitive A. baumannii isolates were obtained from different clinical samples. MIC results showed that seven A. baumannii isolates were resistant to colistin. The antibiotic susceptibility pattern revealed that all seven colistin resistant strains were resistant to all tested antibiotics. Based on MLST analysis, the colistin resistant isolates were assigned to five unique STs namely, ST2 (3; 42.9%) followed by ST78 (1; 14.3%), ST1077 (1; 14.3%), ST415 (1; 14.3%) and ST391 (1; 14.3%). Among them ST2, ST391 and ST415 belong to clonal complex 2. Colistin resistant A. baumannii ST2 is the main circulating clone in clinical settings in Iran, but additionally ST415, ST391, and ST1077 are found for the first time in our country. Intensive control procedures and strict adherence to surveillance programs are recommended to decrease the spread of carbapenem and colistin resistant A. baumannii strain.

Determination of Capsular Serotypes, Antibiotic Susceptibility Pattern, and Molecular Mechanism of Erythromycin Resistance among Clinical Isolates of Group B Streptococcus in Isfahan, Iran.

BACKGROUND: Documented streptococcal resistance to erythromycin has recently been raised. The aim of this study is to identify the molecular mechanism of erythromycin resistance among group B Streptococcus (GBS) strains and to correlate with the clinical origin of strains. MATERIALS AND METHODS: A total number of 134 colonizing (n = 36), invasive (n = 36), noninvasive (n = 46), and asymptomatic (n = 16) GBS isolates were characterized by the detection of dltS gene, capsular serotyping, antibiotic susceptibility profiles using disc diffusion method, and screening of the ermB, ermTR, and mefA resistance genes. RESULTS: The distribution of capsular serotypes was as follow: serotype III (24.6%), Ia (21.6%), V (17.9%), Ib (14.9%), II (8.9%), IV (8.9%), VI (1.5%), and VII (1.5%). From 134 GBS isolates, 51 (38%) isolates were resistant to erythromycin. The constitutive macrolide lincosamide streptogrmin B (MLSB) was the most common resistance phenotype (62.7%), followed by inducible MLSB (27.4%) and M phenotype (9.8%). Erythromycin resistance rate was higher among asymptomatic GBS strains (13/16, 81.2%). Serotype III was the most prevalent type among resistant isolates (41.1%). The ermB gene highly distributed among resistant strains (64.7%), followed by ermTR (21.5%) and mefA (9.8%). The ermB gene was related to constitutive MLSB phenotype (84.3%, P < 0.05) and serotypes III (61.9%), Ib (87.5%), and V (83.3%). All M phenotype strains harbored mefA gene and were in association with serotype Ia (90%). CONCLUSION: The current study suggests that ribosomal modification with erm genes is the main mechanism of erythromycin resistance. Because of relatively high prevalence of erythromycin resistance, double disc test highly recommended for GBS disease treatment and intrapartum prophylaxis among penicillin intolerant patients in our region.

Prevalence, population structure, distribution of serotypes, pilus islands and resistance genes among erythromycin-resistant colonizing and invasive Streptococcus agalactiae isolates recovered from pregnant and non-pregnant women in Isfahan, Iran.

BACKGROUND: The information on antibiotic resistance and molecular features of Group B Streptococcus (GBS) are essential for epidemiological purposes as well as vaccine development. Therefore, we aimed to assess the antimicrobial resistance profiles and molecular characteristics of GBS isolates in Isfahan, Iran. A total number of 72 colonizing and invasive GBS were collected from pregnant and non-pregnant women. The GBS isolates were analyzed for resistance profiles, capsular genotyping, and detection of PI-1, PI-2a, PI-2b, hvgA, ermB, ermTR, lnuB and, mefA genes. Besides, erythromycin-resistant strains were subjected to multilocus sequence typing (MLST). RESULTS: The prevalence of colonizing and invasive GBS were 11 and 0.05%, respectively. The frequency of capsular serotypes was as follows: III (26.3%), Ia (20.83%), Ib and V (each 15.2%), IV (9.7%), II (8.3%), VII (2.7%), and VI (1.3%). Overall frequencies of PIs were as follows: PI-1, 37.5%, PI-1 + PI-2a, 30.5%, PI-1 + PI-2b, 29.1% and PI-2b, 2.7%. Two maternal colonizing GBS (2.6%) were hvgA positive and were belonged to ST-17/CPS-III/PI-1 + PI-2b lineage. Among 30(41.6%) erythromycin resistant GBS, 21 isolates (70%) harbored ermB gene, followed by ermTR (23.3%) and mefA (10%). One clindamycin-resistant isolate harbored the lnuB gene. MLST analysis revealed the following five clonal complexes (CCs) and nine STs: (CC-19/ST-335, ST-19, and ST-197), (CC-12/ST-43, ST-12), (CC-23/ST-163, ST-23), (CC-17/ST-17) and (CC-4/ST-16). CONCLUSION: The study shows an alarmingly high prevalence of erythromycin-resistant GBS in Iran. In addition, we report dissemination of ST-335/CPS-III clone associated with tetracycline and erythromycin resistance in our region. The distribution of capsular and pilus genotypes varies between invasive and colonizing GBS that could be helpful for vaccine development.

Antimicrobial Resistance in Hospitalized Patients with Community Acquired Urinary Tract Infection in Isfahan, Iran.

BACKGROUND: Antibiotic resistance against uro-pathogens is a worldwide health concern. The aim of this study was to determine the causative bacteria and antibiotic susceptibility patterns among hospitalized patients with community acquired urinary tract infection (UTI). METHODS: This cross-sectional study was performed in 2016-2018 in Isfahan, Iran. Urine samples were examined for strain identification and antimicrobial resistance pattern using standard tests. Stratification was done based on gender and age (<20 and >20 years) groups. Chi-square and Fisher exact tests were applied to assess differences in etiology and susceptibility rates between groups. RESULTS: Among 1180 patients, Escherichia coli was the commonest pathogen (68.1%) followed by Enterococcus spp. (8.8%) and Klebsiella pneumonia (8.0 %). Non-E. coli pathogens were more frequent among males (41.8% versus 24.8% in females, P<0.01) and in those aged under 20 years (61.0% versus 22.2% in older than 20 years, P<0.01). Isolated bacteria revealed high susceptibility to imipenem (94.9%), meropenem (92.2%), and amikacin (91.9%); moderate sensitivity to gentamicin (64.4%), cefepime (52.6%) and ceftazidime (47.2%); and low susceptibility to ceftriaxone (41.8%), cefotaxime (40.0%), ciprofloxacin (38.6%) and trimethoprim-sulfamethoxazol (31.3%). The sensitivity of isolates to ceftriaxone, ceftazidime, cefepime, imipenem, meropenem, amikacin and ciprofloxacin was significantly higher in females. Compared to the older age group, uro-pathogens were more susceptible to ciprofloxacin, ceftazidime and gentamicin in patients aged under 20 years. CONCLUSION: We found that imipenem, meropenem and amikacin were good choices for empiric therapy of complicated or severe hospitalized patients with community acquired UTI; and gentamicin, cefepime and ceftazidime were acceptable as initial choices in non-severe infections in the area.

Methodology and Early Results of the First Surveillance Program on Prevention and Control of Antimicrobial Resistance in Isfahan, Iran: The IAS-I Study.

BACKGROUND: Isfahan Antibiotic Resistance Surveillance System-1 has been instituted in Isfahan, Iran to construct a project for surveillance of clinically significant bacteria, and to help raise a logic regional stewardship program for prevention and control of disseminating-resistant organisms. METHODS: During March 2016 to March 2018, an antibiotic resistance surveillance system was designed and implemented by Isfahan Infectious Diseases and Tropical Medicine Research Center. The surveillance program was implemented in three general hospitals in Isfahan. In addition to the routine microbiology data, clinical data (differentiation between true infections and contamination, healthcare-associated infections (HCAI) and community-acquired infections (CAI), as well as determination of the infection site) were obtained and analyzed by WHONET software. RESULTS: During a 2-year period, from 7056 samples that revealed growth of bacteria, 3632 (51.5%) isolates were detected as contamination and 3424 (48.5%) true bacterial isolates were identified. Of these, about 32% of isolates were recognized as HCAI. Totally, the most recognized infections were urinary tract infection, bloodstream infection and skin and soft tissue infections. In patients with HCAIs, 70% of isolates were gram negative and in patients with CAIs 73% isolates were gram negative bacteria. CONCLUSIONS: The strength of the project is gathering enough clinical information in addition to microbiologic data, which would increase application of the results for empiric treatment and prevention of the infectious diseases in clinical settings.

Is There a Correlation between Helicobacter Pylori and Enterohepatic Helicobacter Species and Gallstone Cholecystitis?

BACKGROUND Cholecystitis is a common surgical condition. Recently, several authors have reported that DNA of bile tolerant Helicobacter spp. has been found in the human bile colonizing the biliary tract. The aim of this study was to evaluate the association between the presence of Helicobacter spp. and gallstone cholecystitis. METHODS In this case-control study, gallstones, bile, and gallbladder mucosa were collected from 25 patients without gallstone disease, 24 with acute cholecystitis, and 28 with chronic cholecystitis. The presence of Helicobacter pylori (H. pylori), Helicobacter bilis (H. bilis), Helicobacter hepaticus (H. hepaticus) , and Helicobacter pullorum (H. pullorum) were investigated by polymerase chain reaction (PCR) using species-specific primers. RESULTS In this study, 77 subjects with acute and chronic cholecystitis and control groups with a mean age of 46.85 ± 14.53 years, including 58 (67.25%) women and 19 (32.75%) men were included. DNA of 10 Helicobacter spp. was detected in the bile of the patients with cholecystitis including eight H. pylori and two H. bilis. However, we could not detect H. hepaticus and H. pullorum DNA in the samples. Moreover, there was an association between H. pylori and acute cholecystitis (p = 0.048), which was found to be stronger in 31-40-year-olds group (p = 0.003). CONCLUSION We found an association between the presence of H. pylori DNA and acute gallstone cholecystitis. There is not statistically significant correlation between three enterohepatic Helicobacter spp. ( H. bilis, H. hepaticus , and H. pullorum) and cholelithiasis. Given the low sample size of the patients, more studies are required to clear the clinical role of Helicobacter spp. in the gallstone disease and cholecystitis.

The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of Pseudomonas aeruginosa.

The emergence of antibiotic-resistant and food-spoilage microorganisms has renewed efforts to identify safe and natural alternative agents of antibiotics such as probiotics. The aim of this study was the isolation of lactobacilli as potential probiotics from local dairy products with broad antibacterial and anti-biofilm activities against antibiotic-resistant strains of Pseudomonas aeruginosa and determination of their inhibition mechanism. Antibiotic susceptibility and classification of acquired resistance profiles of 80 P. aeruginosa strains were determined based on Centers for Disease Control and Prevention (CDC) new definition as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) followed by antibacterial assessment of lactobacilli against them by different methods. Among the 80 P. aeruginosa strains, 1 (1.3%), 50 (62.5%), and 78 (97.5%) were PDR, XDR, and MDR, respectively, and effective antibiotics against them were fosfomycin and polymyxins. Among 57 isolated lactobacillus strains, two strains which were identified as Lactobacillus fermentum using biochemical and 16S rDNA methods showed broad inhibition/killing and anti-biofilm effects against all P. aeruginosa strains. They formed strong biofilms and had bile salts and low pH tolerance. Although investigation of inhibition mechanism of these strains showed no bacteriocin production, results obtained by high-performance liquid chromatography (HPLC) analysis indicated that their inhibitory effect was the result of production of three main organic acids including lactic acid, acetic acid, and formic acid. Considering the broad activity of these two L. fermentum strains, they can potentially be used in bio-control of drug-resistant strains of P. aeruginosa.

Molecular characteristics and antibiotic resistance pattern of Staphylococcus aureus nasal carriage in tertiary care hospitals of Isfahan, Iran.

Carriage of S. aureus in the anterior nares seems to play a significant role in the pathogenesis of infection. This study aimed to determine the molecular characteristics and antibiotic susceptibility pattern of S. aureus isolates obtained from the nasal carriage of health care workers (HCWs). This study was performed during July 2014 to July 2015 at three tertiary care hospitals. Nasal samples were collected from the nasal cavity of HCWs. Standard microbiological methods were used for identification of S. aureus isolates. Antibiotic susceptibility pattern was determined by the disc diffusion method. Determination of SCCmec typing and virulence genes was performed by the PCR method. From the isolates of 340 nasal swab samples of HCWs, 65 S. aureus strains (19%) including 22 (33.8%) MRSA were isolated. The highest sensitivity for MRSA isolates was towards vancomycin and rifampicin, each with 90.9%. Overall, 17% (11/65) and 92.3% (60/65) of S. aureus isolates were positive for pvl and hla genes, respectively. The rates of SCCmec types II, III, IV, V and I among MRSA isolates were 36.4 %, 22.7 %, 22.7 %, 9.1% and 4.5% respectively. The results of the present study indicate that S. aureus nasal carriage with potential virulence ability still remains a significant healthcare problem, especially in hospital environments.

Resistotyping, phenotyping and genotyping of New Delhi metallo-ß-lactamase (NDM) among Gram-negative bacilli from Iranian patients.

PURPOSE: The purpose of this study was to investigate New Delhi metallo-ß-lactamase (NDM) production among Gram-negative bacilli. METHODOLOGY: Antibiogram-resistotyping and detection of New Delhi metallo-ß-lactamase (NDM) in clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and comparative evaluation of the diagnostic performance of three phenotypic methods for NDM detection, with PCR considered as the gold standard, were performed. Minimum inhibitory concentration (MIC) of antibiotics against NDM-positive strains using E-tests and clonal relationship analysis using enterobacterial repetitive intergenic consensus (ERIC)-PCR in these strains were determined. RESULTS: The most effective antibiotics against strains of the species K. pneumoniae were Colistin, Chloramphenicol and Tigecycline; against P. aeruginosa were Fosfomycin and Polymyxins, and against A. baumannii were Polymyxins, Ampicillin/Sulbactam and Minocycline. Overall, 66, 31 and 40 different resistotypes were observed among K. pneumoniae, A. baumannii and P. aeruginosa strains, respectively. The blaNDM-1 gene was detected in 28 (8.5 %) strains of the bacteria investigated. The sensitivities and specificities of the Meropenem-EDTA combined disk test, the meropenem-dipicolinic acid combined disk test and the modified Hodge test methods for NDM detection were 96.43, 55.15; 96.43, 54.85; and 89.29, 35.15, respectively. Additionally, in spite of the low positive predictive values of these tests, their negative predictive values were high. ERIC-PCR results revealed two main clusters in NDM-positive strains of each of the species P. aeruginosa and A. baumannii, and ten main clusters in K. pneumoniae. In all the NDM-positive strains maximum MIC rates (>256) were observed for all beta-lactam antibiotics. CONCLUSION: There were high levels of antibiotic resistance and a high frequency of multi-drug resistance and extensive-drug resistance profiles, as well as highly prevalent blaNDM-1 genes in the bacteria investigated.

Determination of Acquired Resistance Profiles of Pseudomonas aeruginosa Isolates and Characterization of an Effective Bacteriocin-Like Inhibitory Substance (BLIS) Against These Isolates.

BACKGROUND: The emergence of pan-drug resistant strains (PDR) of Pseudomonas aeruginosa has led to renewed efforts to identify alternative agents, such as bacteriocins and bacteriocin-like inhibitory substances (BLISs). OBJECTIVES: The aims of this study were to determine the acquired resistance profiles of multidrug-resistant (MDR), extensively drug-resistant (XDR), and PDR P. aeruginosa isolates based on the revised definitions of the CDC and ECDC and to screen and characterize effective BLISs against these isolates. PATIENTS AND MATERIALS: In a cross-sectional study, 96 P. aeruginosa strains were isolated during a 12-month period. The resistance profiles of these isolates were determined as MDR, XDR, and PDR, and the data were analyzed using WHONET5.6 software. A BLIS against the P. aeruginosa strains was characterized based on its physicochemical properties, size, growth curves, and production profiles. RESULTS: Among the 96 isolates of P. aeruginosa, 2 (2.1%), 94 (97.9%), and 63 (65.6%) were non-MDR, MDR, and XDR, respectively, and 1 (1.1%) was PDR. The most effective antibiotics against these isolates were polymyxins and fosfomycin. A BLIS isolated from the P. aeruginosa DSH22 strain had potent activity against 92 (95.8%) of the 96 isolates. The BLIS was heat stable, (up to 100°C for 10 min), UV stable, and active within a pH range of 3 - 9. The activity of BLIS disappeared when treated with trypsin, proteinase K, and pepsin, indicating its proteinous nature. Based on its size (25 kDa), the BLIS may belong to the large colicin-like bacteriocin family. BLIS production started in the midexponential phase of growth, and the maximum level (2700 AU/mL) occurred in the late-stationary phase after 25 hours of incubation at 30°C. CONCLUSIONS: This BLIS with broad-spectrum activity may be a potential agent for the treatment or control of drug-resistant strains of P. aeruginosa infection.

Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016.

Frequency assessment of ß-lactamase enzymes in Escherichia coli and Klebsiella isolates in patients with urinary tract infection.

BACKGROUND: Production of ß-lactamase enzymes is the most common and important mechanism of resistance in Gram-negative bacteria. The objective of this study was to assess frequency of three main ß-lactamase enzymes, including extended spectrum ß-lactamases (ESBLs), metallo-ß-lactamase (MBL), and Klebsiella pneumoniae carbapenemase (KPC) enzymes in Escherichia coli and Klebsiella spp. isolated from nosocomial and community urinary tract infections (UTI). MATERIALS AND METHODS: In a cross-sectional study from March to December 2012, midstream urine samples were obtained from patients suspicious of UTI who were hospitalized or referred to Al-Zahra Hospital, Isfahan, Iran. Samples were cultured and E. coli and Klebsiella spp. were isolated. Prevalence of ESBLs, KPC, and MBLs producing E. coli and Klebsiella spp. were studied by double-disk (combined-disk), the modified Hodge test and imipenem-ethylenediaminetetraacetic acid combined disc methods respectively. In addition, their antimicrobial susceptibility patterns determined and resistant to carbapenem drugs confirmed by minimum inhibitory concentrations based on E-test method. RESULTS: A total of 1080 E. coli and 484 Klebsiella strains were isolated during study period. Among 720 E. coli and 384 Klebsiella isolates from hospitalized patients, 300 (41.7%) and 198 (51.5%) were ESBLs producers, respectively. In out-patients samples, the rate of ESBLs production was 25% (90/360) and 40% (40/100) in E. coli and Klebsiella isolates, respectively. Prevalence of MBLs producing in hospital E. coli and Klebsiella isolates were 0.3% (2/720) and 2.6% (10/384), and for KPC data were 1.4% (10/720) and 48.4% (186/384), respectively. No MBLs and KPC producing isolate was seen in non-hospital E. coli and Klebsiella isolates except for one non-hospital KPC producing Klebsiella isolate. CONCLUSION: The result of our study showed high prevalence of ESBLs and KPC, but low prevalence of MBLs in cultured bacteria from urine samples of patients with acute UTI. In addition, KPC was the main carbapenem resistance mechanism in Klebsiella and E. coli isolates.

Antimicrobial susceptibility pattern of extended-spectrum ß-lactamase-producing bacteria causing nosocomial urinary tract infections in an Iranian referral teaching hospital.

OBJECTIVE: Gram-negative bacilli are the most important cause of nosocomial urinary tract infections (UTIs). The production of extended-spectrum ß-lactamase (ESBL) enzymes is a common mechanism of resistance among these bacteria. The aim of this study was to determine the rate of ESBL producing Gram-negative bacteria causing nosocomial UTI in a referral hospital as well as their susceptibility pattern to the most commonly used antibiotics. METHODS: In a prospective cross-sectional study performed over a 6-month period, urinary specimens obtained from hospitalized patients with documented culture-proved nosocomial UTI (age range of 1-87 years). Isolated aerobic Gram-negative bacteria underwent further microbiologic tests for detection of ESBL, as well as antimicrobial susceptibility test using Kirby-Bauer (disk diffusion) and E-test methods. FINDINGS: During the study period, 213 urine samples were detected to have growth of Gram-negative organism. Escherichia coli was the most frequently isolated organism (61%). ESBL was detected in 102 isolates including 38.5% of E. coli, 39.5% of Klebsiella pneumonia, 88.5% of Pseudomonas aeruginosa, and 100% of Acinetobacter baumannii strains. Imipenem and meropenem were the most effective antibiotics on E. coli and K. pneumoniae strains. P. aeruginosa and A. baumannii strains showed high resistance to all tested antibiotics. CONCLUSION: Large numbers of Gram-negative bacteria causing nosocomial UTIs produce ESBL with most being multidrug-resistant. Therefore, routine ESBL detection testing and subsequent antibiogram with disk diffusion method could be useful to determine the best treatment options for UTI.

Antimicrobial activity of a UV-stable bacteriocin-like inhibitory substance (BLIS) produced by Enterococcus faecium strain DSH20 against vancomycin-resistant Enterococcus (VRE) strains.

BACKGROUND/PURPOSE: The narrow spectrum of action of most bacteriocins is an important limitation for their application as antimicrobial agents. The current study describes a novel bacteriocin-like inhibitory substance (BLIS) that display extended spectrum antimicrobial activity against vancomycin-resistant Enterococcus (VRE) strains. METHODS: Acquired resistance profiles of Enterococcus isolates determined based on the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) definition as multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug resistant (PDR). BLIS activity of Enterococcus isolates was investigated against Enterococcus faecalis (E. faecalis) ATCC 29212 as the indicator strain and clinical isolates including VRE, methicillin resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria containing Pseudomonas aeruginosa (P. aeruginosa), Klebsiella, Acinetobacter, and Escherichia coli (E. coli). RESULTS: Among 273 Enterococcus isolates, 27 and 2 VRE isolates, respectively, were XDR and PDR and eight isolates had BLIS activity against the indicator strain. One of these isolates, identified as E. faecium strain DSH20 based on its phenotypical and biochemical properties, as well as its 16S rRNA gene sequence, had potent BLIS production against all 29 VRE strains, but had no activity against MRSA, P. aeruginosa, Klebsiella, Acinetobacter, and E. coli strains. It was heat stable up to 121°C for 15 minutes (autoclave condition), active within the pH range of 3-9 and had UV stability, but its activity disappeared by treatment with proteinase K, pepsin, and trypsin, demonstrating its proteinaceous nature. It was designated as an approximately 35kDa peptide using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. CONCLUSION: This peptide is a potential agent for use as an alternative antibacterial agent for the treatment of drug-resistant strains of VRE infection.

Phenotypic and Molecular Characterization of Extended-Spectrum ß-Lactamase Produced by Escherichia coli, and Klebsiella pneumoniae Isolates in an Educational Hospital.

BACKGROUND: Extended-spectrum beta-lactamases (ESBLs) are a group of enzymes that hydrolyze antibiotics, including those containing new cephalosporins, and they are found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains. With the widespread use of antibiotics, difficulties with infection therapy caused by drug resistant organisms, especially those that have acquired resistance to beta-lactams, such as broad-spectrum cephalosporins, have amplified the above-mentioned organisms. OBJECTIVES: This study was conducted to characterize ESBLs among E. coli and K. pneumonia isolates by molecular and phenotypic methods. MATERIALS AND METHODS: Different strains of E. coli and K. pneumonia were collected from patients with urinary tract infections. The ESBL phenotype was determined by a double disk diffusion test (DDDT). In addition, polymerase chain reaction (PCR) analysis specific for ß-lactamase genes of the TEM and SHV family was carried out. The PCR products were run on agarose and examined for DNA bands. RESULTS: A total of 245 E. coli and 55 K. pneumonia strains were isolated from different samples. In total, 128 of the 300 isolates were confirmed as potential ESBLs producers as follows: 107 (43.67%) E. coli and 21 (38.18%) K. pneumonia. ESBLs genes were found in 24 isolates (18.75%): 21 E. coli and 3 K. pneumonia isolates. The TEM gene was present in 13 (12.14%) E. coli strains, but it was not detected in K. pneumonia. In addition, the SHV gene was present in 8 (7.47%) E. coli and 3 (14.28%) K. pneumonia isolates. Five (4.67%) of the E. coli isolates harbored both TEM and SHV genes. All isolates (100%) were susceptible to imipenem. The lowest rates of resistance to other antibiotics were observed for; piperacillin-tazobactam (6.25%), amikacin (12.5%) and gentamicin (14.84%). The rates of resistance to other antibiotics were as follow: nitrofurantoin (16.4%), nalidixic acid (23.43), co-trimoxazole (25%), cefepime (32%), ciprofloxacin (55.46%), ampicillin (69.53%), ceftazidime (100%), and cefotaxime (100%). CONCLUSIONS: The results of this study indicate the widespread prevalence of ESBLs and multiple antibiotic resistance in E. coli and K. pneumoniae. Therefore, beta-lactam antibiotics and beta-lactamase inhibitors or carbapenems should be prescribed based on an antibacterial susceptibility test.

Deodorant effects of a sage extract stick: Antibacterial activity and sensory evaluation of axillary deodorancy.

BACKGROUND: Deodorant products prevent the growth and activity of the degrading apocrine gland bacteria living in the armpit. Common antibacterial agents in the market like triclosan and aluminum salts, in spite of their suitable antibacterial effects, increase the risk of Alzheimer's disease, breast and prostate cancers or induce contact dermatitis. Therefore, plant extracts possessing antibacterial effects are of interest. The aim of the present study was to verify the in vitro antimicrobial effects of different sage extracts against two major bacteria responsible for axillary odor, and to evaluate the deodorant effect of a silicon-based stick containing sage extracts in different densities in humans. MATERIALS AND METHODS: Different fractions of methanolic extract of Salvia officinalis (sage) were evaluated on a culture of armpit skin surface of volunteers through agar microdilution antimicrobial assay. Then, randomized, double-blind placebo-controlled clinical trial with the best antibacterial fraction was conducted on 45 female healthy volunteers. Participants were treated with a single dose in four groups, each containing 15 individuals: Group 1 (200 µg/mL), 2 (400 µg/mL), 3 (600 µg/mL) of dichloromethane sage extract, and placebo (without extract). A standard sensory evaluation method for the evaluation of deodorant efficacy was used before, and two hours, four hours, and eight hours after single application of a deodorant or placebo (ASTM method E 1207-87 Standard Practice for the Sensory Evaluation of Axillary Deodorancy). RESULTS: The data were analyzed with two factors relating to densities and time. In 45 participants with a mean [± standard deviation (SD)] age of 61.5±11.8 years, statistically significant within-group differences were observed before and two, four, and eight hours after deodorant treatment for groups 1, 2, and 3. Groups 1, 2, and 3 had a significantly smaller odor score than placebo after two, four, and eight hours (P < 0.001). In a comparison of different deodorant densities, the interaction effect was not significant between deodorant 200 and 400 µg/mL, but was significant between 200 and 600 and between 400 and 600 µg/mL sage extract sticks (P < 0.001). Before running the sensory evaluation of the deodorant sticks on the subjects, a rabbit skin patch test was used to demonstrate that the formulation had no irritants. CONCLUSION: A single treatment with a stick deodorant containing dichloromethane sage extract of 200, 400, or 600 µg/mL concentrations was effective in reducing the axillary malodor level compared with the control, in healthy subjects.

Ammonium production during the nitrogen-fixing process by wild Paenibacillus strains and cell-free extract adsorbed on nano TiO2 particles.

During the nitrogen-fixing process, ammonia (NH3) is incorporated into glutamate to yield glutamine and is generally not secreted. However, in this study, NH3- excreting strains of nitrogen-fixing Paenibacillus were isolated from soil. The ammonium production by the Paenibacillus strains was assayed in different experiments (dry biomass, wet biomass, cell-free extract, and cell-free extract adsorbed on nano TiO2 particles) inside an innovative bioreactor containing capsules of N2 and H2. In addition, the effects of different N2 and H2 treatments on the formation of NH3 were assayed. The results showed that the dry biomass of the strains produced the most NH3. The dry biomass of the Paenibacillus strain E produced the most NH3 at 1.50, 0.34, and 0.27 micrometer NH3/mg biomass/h in the presence of N2 + H2, N2, and H2, respectively, indicating that a combined effluent of N2 and H2 was vital for NH3 production. Notwithstanding, a cell-free extract (CFE) adsorbed on nano TiO2 particles produced the most NH3 and preserved the enzyme activities for a longer period of time, where the NH3 production was 2.45 micrometer/mg CFE/h over 17 h. Therefore, the present study provides a new, simple, and inexpensive method of NH3 production.

Indole-3-acetic acid (IAA) production in symbiotic and non-symbiotic nitrogen-fixing bacteria and its optimization by Taguchi design.

Production of Indole-3-acetic acid (IAA) in 35 different symbiotic and non-symbiotic nitrogen-fixing bacteria strains isolated from soil and plant roots was studied and assayed by chromatography and colorimetric methods. These bacteria included Agrobacterium, Paenibacillus, Rhizobium, Klebsiella oxytoca, and Azotobacter. The best general medium and synergism effects of isolates for IAA production were investigated. Effects of different variables containing physical parameters and key media components and optimization of condition for IAA production were performed using the Design of Experiments. Qualitek-4 (W32b) software for automatic design and analysis of the experiments, both based on Taguchi method was used. The results showed that Rhizobium strains, symbiotic, and Paenibacillus non-symbiotic bacteria yielded the highest concentrations of IAA (in the range of 5.23-0.27 and 4.90-0.19 ppm IAA/mg biomass, respectively) and IAA production was increased by synergism effect of them. Yeast Extract Mannitol medium supplemented with L-tryptophan was the best general medium for IAA production. The analysis of experimental data using Taguchi method indicated that nitrogen source is very prominent variable in affecting the yield and mannitol as carbon source, potassium nitrate (1%), and L-tryptophan (3 g/l) as nitrogen sources after 72-h incubation at 30 degrees C were the optimum conditions for production of IAA. 5.89 ppm IAA/mg biomass was produced under these optimal conditions.