High Resolution Melting Curve Analysis Method for Detecting of Carbapenemases Producing Pseudomonas aeruginosa

Background: The, carbapenems have the broadest spectrum of activity among all β-lactam antibiotics, and also, carbapenem resistant P. aeruginosa may be susceptible to other β-lactam antibiotics. There are several methods for detecting to Carbapenems resistance of Pseudomonas aeruginosa (P. aeruginosa) strains. Aim and Objectives: In the current study, High Resolution Melt curve analysis (HRM) is evaluated to detect carbapenem resistant P. aeruginosa. Material and Methods: This experimental study was done on standard isolates of P. aeruginosa and carbapenemase producing strains in Hamadan. 16srRNA of P. aeruginosa and KPC gene of carbapenemase producing strains were applied to detect by HRM. Also, sensitivity and specificity of primers have been evaluated and carbapenemase producing P. aeruginosa were determined based on melt curve temperature range. Finally, melt curve profiles were assessed and analyzed using StepOne Software v2.3 and HRM Software v3.0.1 software. Results: According to dilutions ratios of 108 to 10-4 it was found that the designed primers have the ability to identify 10-3 CFU/ml of bacteria for P. aeruginosa and 10-1 CFU/ml for carbapenemase producing strains. The melt curve was also shown at 80.9±0/5 °C for all DNA dilutions of P. aeruginosa and 82.4±0/5 °C for all DNA dilutions of Carbapenemases strains. Conclusion: The sensitivity and specificity of HRM method indicated that it is highly reliable, rapid and cost-effective to detect carbapenemase producing P. aeruginosa

Comparison of Real-time PCR method and blood culture in diagnosis of septicemia

Background: Bloodstream infections (BSI) have a high incidence and high mortality in the worldwide. The mortality rate is variable between 20-70%. Therefore, early and timely detection of BSI agent in clinical laboratories is necessary. The aim of this study was to determine an efficient diagnostic tool to septicemia in accompany of blood culture method by Real-time PCR (using panbacterial 23S rRNA gene). Methods: This cross-sectional study was conducted in two analytical and clinical stages in Hamadan University of Medical Sciences, Iran, from October 2014 to June 2015. In analytical stage, sensitivity (by serial dilution from 104 to 1 CFU/ml) and specificity of the primer were evaluated with the Staphylococcus aureus (as Gram positive indicator bacteria) and Escherichia coli (as Gram-negative indicator bacteria), human genome (from Hella cell culture), Candida albicans yeast and Aspergillus fumigatus fungus. In clinical stage, 121 blood samples were collected from patients suspected to sepsis in intensive care unit (ICU) from Hamadan University Hospitals. Finally, the results of Real-time PCR and blood culture methods were compared. Results: The Real-time PCR showed a sensitivity ranging from 2 to 10 target copies per reaction to the whole blood for Escherichia coli and Staphylococcus aureus respectively. The specificity of this method was evaluated and no false positive amplification was identified. 57.85% (70 cases) of the samples were positive by Real-time PCR and 13.22% (16 cases) of the samples were positive by blood culture. However, none of the cases that were positive by blood culture were negative in Real-time PCR. As well as, 44.62% (54 cases) of cases were positive by Real-time PCR but blood culture showed no bacteria in the samples, and 42.15% (51 cases) were negative by both methods. Correlation or agreement of Kappa was 0.20, that indicating poor agreement between the two methods. Conclusion: Real-time PCR is more sensitive than blood culture and also, because of high sensitivity of this primer by Real-time PCR, we can use it for screening blood samples from suspected patients of sepsis

Biofilm production among Staphylococcus epidermidis strains isolated from healthy people

Introduction: Staphylococcus epidermidis is well documented as a nosocomial pathogen causing biofilm in patients and healthy people. The aim of this study was to analyze the biofilm formation of S. epidermidis strains isolated from healthy people during 2013-2014. Materials and methods: Totally 200 healthy people were selected and the sampling was carried out from arm, armpit and axillary area using sterile swaps. Swaps were transferred to thiogylcollate broth and then cultured on mannitol salt agar plates. Isolates were identified at the species level using biochemical tests. Potential of biofilm formation of strains was measured using congo red agar plate and microtiter plate tests. Genes involved in biofilm formation, icaA and icaD, were detected using PCR. Results: Totally 104 S. epidermidis strains were isolated from healthy people. Amongst these, 66 (63%) and 38 (37%) strains were positive and negative for biofilm formation, respectively. icaA and icaD genes were detected in 100% of strains. Discussion and conclusion: Prevalence of biofilm producing S. epidermidis isolates among healthy people indicating their colonization with hospital strains. Prevalence of such strains is  urgent for public health

Prevalence and Removal Efficiency of Enterococcal Species and Vancomycin-resistant Enterococci of a Hospital Wastewater Treatment Plant

Simultaneous presence of various antibiotics and bacteria in hospital wastewaters creates a suitable environment, in which the bacteria, such as ‎enterococci become resistant to the antibiotics. The aim of this study was to evaluate the performance of different units of the hospital wastewater treatment plant (HWTP) to remove Enterococcus spp and Vancomycin-resistant Enterococcus (VRE). The study was performed on the 27 samples collected from HWTP in Hamedan, Iran during December 2014 to August 2015. Enterococcus spp and VRE were identified by biochemical tests and then the isolates were confirmed by PCR. Finally, the antibiotic susceptibility test was performed using disk diffusion methods. Of the 27 samples examined, 315 a total of enterococcal isolates were obtained. Of the 315 isolates of enterococci investigated, 162 (51.42%) were identified as E. faecium, 87 (27.61%) as E. hirae, 35 (11.11%) as E. faecalis, 11 (3.5%) as E. gallinarum, 7 (2.22%) as E. casseliflavus, 4 (1.26%) E. avium, and 9 (2.85%) isolates VR E. faecium.The results of antibiotic susceptibility testing showed that of the total 315 isolates, 146 (46.34%) were resistance to tetracycline, 9 (2.85%) were resistance to vancomycin and Teicoplanin. Lower antibiotic resistance was seen with Nitrofurantoin 2 (1.26%). This study indicates a high prevalence of multidrug resistance among E. faecium isolated from HWTP, thus, it could be considered as a threat to the health and safety of ‎wastewater workers and even public health

The Study of Antibiotic Resistance and Prevalence of Extended-Spectrum Beta-Lactamase (ESBLs) Encoding Genes in Acinetobacter baumanni Isolates from Raw Foodstuffs

Background and Aims: Pharmaceutical residuals like antibiotics in livestock products and their consumption by humans from food chain can lead to the spread of bacteria resistant to antibiotics. The aim of the current study was to investigate antibiotic resistance and determine the prevalence rate of genes encoding extended spectrum beta-lactamase (ESBLs) in Acinetobacter strains isolated from raw foodstuffs. Materials and Methods: In this study, 300 samples from protein foodstuffs (mutton, beef, chicken, hamburger, hot dog, sausages) and dairy foodstuffs (raw milk and cheese) were prepared and investigated in terms of contamination with Acinetobacter baumannii from July 2015 to November 2016. The isolated bacteria were identified by biochemical tests to species level and confirmed by the PCR technique via blaOXA-51 gene. Antibiotic resistance of the isolates was studied using the diffusion disc method. Also, the presence of ESBLs enzymes in the isolates wa s done phenotypically and genetically through PCR and combined disk tests. Results: The results showed that 43 strains of A. baumannii were isolated from the protein and dairy foodstuffs, 93% from protein foodstuffs and 7%  from dairy foodstuffs. Also, 30% of the isolates had multidrug- resistance (MDR). Further, the findings of PCR illustrated that the prevalence of genes encoding ESBLs in the isolates were: TEM 21%, PER 23.5%, VEB 18.5‌% and SHV35%.  Conclusions: Foodstuffs can act as a food source for A. baumannii that can lead to transferring and spreading genes encoding antibiotic resistance to humans

Diagnostic Value of Melting Curve Analysis Based on Multiplex-Real Time PCR in Identification of Enterococci Species

Background and purpose: Enterococci as a group of bacteria infecting raw foods and dairy products can contaminate different food products. The purpose of this study was to isolate strains of enterococci based on the Real time PCR method using melting curve analysis in food samples. Materials and methods: In this experimental study, 510 samples were collected from which 138 different samples (chicken, meat, milk, and cheese) containing different strains of enterococci were investigated. Then, identification of Enterococcus species was performed by targeting specific sites and specific primers were designed according to Real time PCR-based melting curve analysis. Results: Based on melting curve analysis by Real Time PCR, the Enterococcus species identified were as follows: E.faecalis in 84 isolates (60.86%), E.faecium in 48 ​​ (34.78%), E.gallinarum in 1 (0.7%), E.avium in 4 (2.8%), and E.Caselli flavus in 1 isolate (0.7%). The most frequent isolates were detected in 29 samples of chicken meat (51.44%) and red meat (n= 21, 24.63%). Considering the results of sequencing as a Gold a standard test, the sensitivity and specificity of phenotypic methods for E.faecalis, E.faecium, E.gallinarum, E.avium, and E.Caselli flavus were 94.78% and 90.74%, 89.13 % and 97.77 %, 50% and 98.52%, 66.66% and 98.52%, and 50% and 98.56%, respectively. A significant relationship was observed between the sample and distribution of Enterococcus species (P≤0.05). Conclusion: Due to extensive viability error in identification of Enterococcus species isolated from food by phenotypic methods, using a rapid and sensitive method is necessary

Determination of Antimicrobial Resistance Pattern in Methicillin-Resistant Staphylococcus saprophyticus and Staphylococcus epidermidis and Detection of Resistance Genes to Clindamycin and Erythromycin

Background and Aims: Clindamycin is one of the selective drugs for treatment of staphylococcal infections. Molecular methods can complete phenotypic methods to diagnosis induction resistance to clindamycin. The aim of this study was to identify the genes responsible for the resistance to clindamycin and erythromycin, and determine their antibiotic resistance pattern. Materials and Methods: 100 isolates of Staphylococcus epidermidis and Staphylococcus saprophyticus were isolated from 466 different clinical specimens using biochemical tests. Using the disc diffusion method, Antibiogram susceptibility test was conducted to determinate lincosamides and tetracycline resistance pattern. Then  ermA, ermB, ermC and msrA genes were identified and investigated by PCR method. Results: Out of 100 strains of coagulase-negative staphylococci isolated from clinical specimens, 5 isolates were identified as S. saprophyticus (5%) and 55 isolates of S. epidermidis (55%), respectively. Out of the 5 isolated of S. saprophyticus, 2 (40%) isolates were resistant to methicillin and one (20%) isolate had D phenotype. In addition, 1 isolate had ermA gene and 1 isolate had ermB. Out of the 55 isolates of S. epidermidis, 25 (45.45%) isolates were resistant to methicillin, of which nine (36%) isolates had D phenotype. Also, 4 (16%) isolates had ermA gene, 3 (12%) isolates had ermB, 6 (24%) isolates had ermC and 1 (4%) isolate was carrying the msrA.  Conclusions: The phenotypic pattern of resistance to macrolide- lincosamides groups does not have a high degree of accuracy in detecting methicillin-resistant MLSB strains

A New Approach to Identify and Determine the Relationship between MecA Gene Mutations Based on HRM with Clinical Species in Staphylococcus aureus Isolates

Background and Aim: Gene mutation in Staphylococcus aureus is one of the most important causes of antibiotic-resistant strains. The High Resolution Melting Curve (HRM) analysis of DNA method can detect these mutations very high quality. The purpose of this study was to evaluate the role of clinical sample type in the occurrence of nucleotide mutations in the mecA gene of S. aureus by HRM method. Materials and Methods: In this experimental study, 43 clinical isolates of S. aureus were used. To detect possible mutations, isolates with mecA gene were replicated and sequenced. Then, analysis was performed using StepOne Software v2.3 and HRM v3.0.1 software. Sequencing results were used as gold-standard. Ethical Considerations: This study with research ethics code IR.UMSHA.REC.1396.637 has been approved by research ethics committee at Hamadan University of Medical Sciences. Findings: Of 43 clinical isolates of S. aureus, 11 isolates (25.58%) had mecA gene and 32 isolates (47.41%) lacked the mecA gene. According to different clinical samples, 3 isolates (27.27%) were resistant to methicillin from blood samples, 2 isolates (18.18%) from urine specimens, 2 isolates (18.18%) from wound samples, 2 isolates (18.18%) of the catheter samples, 1 isolate (9.09%) of the abscess and 1 isolate (9.09%) were separated from the nose swab. In the meanwhile, isolates from the wound and urine had the highest mutation in the adenine amino acid as A → T, A → G, A → C, and A → X. Isolates taken from blood have mutations in Guanine amino acid as G → A. Conclusion: There was a significant relationship between type of mutation and type of clinical specimen in methicillin-resistant Staphylococcus aureus isolates

Association between the accessory gene regulator (agr) locus and the presence of superantigen genes in clinical isolates of methicillin-resistant Staphylococcus aureus

Abstract Objective Methicillin-resistant Staphylococcus aureus cause to a variety of hard to cure infections. MRSA isolates also, produce an arsenal of virulence factors contribute to severe infections. The aim of this study was to find out the relationship between agr locus and presence of S. aureus superantigens (SAgs). Results Clinical isolates in two groups from two different states of Iran were collected. Antibiotic resistance patterns, agr typing, and virulence factor genes prevalence were identified and relationship between them was analyzed using SPSS software version16. Most of the samples were collected from wound 39 isolates in Group 1 and 61 isolates in Group 2. Frequency of MRSA strains was 38.1% in Group 1 and 52.1% in Group 2. Also, the most common resistance among both groups was to penicillin. agr positive isolates were detected in 132 isolates of Group 1 and 104 isolates of Group 2. In Conclusion, a significant relationship between the SAgs frequency and agr locus in both groups has been indicated. The production of superantigens in S. aureus plays an important role in the classification of agr locus, and this locus can affect differently in methicillin-resistant strains