Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria. - Related Documents




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577901.0000Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria. The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.202439452724
577810.9998A Simple and Rapid Low-Cost Procedure for Detection of Vancomycin-Resistance Genes in Enterococci Reveals an Outbreak of Vancomycin-Variable Enterococcus faecium. The detection of resistance to vancomycin in enterococci cultured from patients is important for the treatment of individual patients and for the prevention of hospital transmission. Phenotypic antimicrobial resistance tests may fail to detect potential vancomycin-resistant enterococci. We have developed and tested a PCR based procedure for routine screening for vancomycin-resistance genes in clinical samples with enterococci. Primary cultures from diagnostic samples reported with growth of Enterococcus faecium or E. facalis were tested for vanA and vanB genes by real-time PCR without the isolation of specific bacteria. Up to ten samples were pooled and tested in each real-time PCR reaction, with subsequent individual testing of cultures from positive pools. In a one-month test period in 2017 vanA gene was detected in one out of 340 urine samples with vancomycin-susceptible enterococci reported from diagnostic culture. A second test period in 2018 included 357 urine samples, and vanA gene was detected in samples from eight patients. Subsequently, all urine samples reported with growth of E. faecium during a period of one year were tested. Fifty-eight individuals were identified with enterococci, carrying the vanA gene not previously detected. Routine molecular testing of primary culture material from patient samples may improve the detection of hospitalized patients carrying E. faecium with resistance genes to vancomycin.202236140520
224720.9998Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing. Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.202338192400
223530.9998Nanosphere's Verigene(®) Blood Culture Assay to Detect Multidrug-Resistant Gram-Negative Bacterial Outbreak: A Prospective Study on 79 Hematological Patients in a Country with High Prevalence of Antimicrobial Resistance. Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene(®)) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.201934595420
579740.9998PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis. Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.201323447637
578350.9998Molecular Investigation and Virulence Determination of Methicillin and Vancomycin Resistant Clinical Staphylococcus Aureus Isolates. Staphylococcus aureus is an opportunistic pathogen that provides conditions for host invasion due to various virulence factors and plays a role in causing various infections. The pathogenicity of these bacteria may vary depending on the host's susceptibility. This study investigates the sensitivity of S. aureus strains isolated from clinical samples to methicillin and vancomycin, and it evaluates the presence of resistance, virulence and toxin-producing genes, and their expression level in the methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-intermediate S. aureus (VISA) isolates. A cross-sectional study was conducted, encompassing 502 S. aureus isolates obtained from diverse infections over the course of a year. The methicillin and vancomycin sensitivities of the isolates were ascertained by disk diffusion and microdilution broth methods, respectively. The presence of genes associated with resistance, adhesion, and toxin production was subsequently investigated through the implementation of multiplex polymerase chain reaction (PCR) methodology. The expression levels of virulence and resistance genes were detected in resistant and sensitive isolates using real-time quantitative PCR (qPCR). Among the 502 S. aureus isolates, 168 (33.6%) were identified as MRSA. Furthermore, a total of six isolates (1.2%) were identified as VRSA, and two isolates (0.4%) were identified as VISA. The distribution of virulence and resistance-related genes varied among the isolates. The results of the gene expression study demonstrated that the expression levels of the majority of the studied genes were significantly higher in resistant isolates (MRSA and VRSA) compared to sensitive isolates. It is imperative to acknowledge that VRSA and MRSA are regarded as grave hazards to human health. The present study underscores the necessity for enhanced sanitary measures to more effectively control this hospital pathogen, particularly in light of the presence and expression of genes encoding virulence factors in S. aureus isolates.202540980455
223360.9998Assessment of the multiplex PCR-based assay Unyvero pneumonia application for detection of bacterial pathogens and antibiotic resistance genes in children and neonates. BACKGROUND: Pneumonia is a major healthcare problem. Rapid pathogen identification is critical, but often delayed due to the duration of culturing. Early, broad antibacterial therapy might lead to false-negative culture findings and eventually to the development of antibiotic resistances. We aimed to assess the accuracy of the new application Unyvero P50 based on multiplex PCR to detect bacterial pathogens in respiratory specimens from children and neonates. METHODS: In this prospective study, bronchoalveolar lavage fluids, tracheal aspirates, or pleural fluids from neonates and children were analyzed by both traditional culture methods and Unyvero multiplex PCR. RESULTS: We analyzed specimens from 79 patients with a median age of 1.8 (range 0.01-20.1). Overall, Unyvero yielded a sensitivity of 73.1% and a specificity of 97.9% compared to culture methods. Best results were observed for non-fermenting bacteria, for which sensitivity of Unyvero was 90% and specificity 97.3%, while rates were lower for Gram-positive bacteria (46.2 and 93.9%, respectively). For resistance genes, we observed a concordance with antibiogram of 75% for those specimens in which there was a cultural correlate. CONCLUSIONS: Unyvero is a fast and easy-to-use tool that might provide additional information for clinical decision making, especially in neonates and in the setting of nosocomial pneumonia. Sensitivity of the PCR for Gram-positive bacteria and important resistance genes must be improved before this application can be widely recommended.201829086343
220970.9997Concordance Between Antibiotic Resistance Genes and Susceptibility in Symptomatic Urinary Tract Infections. PURPOSE: Studies have shown that multiple genes influence antibiotic susceptibility, but the relationship between genotypic and phenotypic antibiotic susceptibility is unclear. We sought to analyze the concordance between the presence of antibiotic resistance (ABR) genes and antibiotic susceptibility results in urine samples collected from patients with symptomatic urinary tract infection (UTI). PATIENTS AND METHODS: Urine samples were collected from patients presenting to 37 geographically disparate urology clinics across the United States from July 2018 to February 2019. Multiplex polymerase chain reaction was used to detect 27 ABR genes. In samples containing at least one culturable organism at a concentration of ≥ 10(4) cells per mL, pooled antibiotic susceptibility testing (P-AST), which involves simultaneous growing all detected bacteria together in the presence of antibiotic and then measure susceptibility, was performed against 14 antibiotics. The concordance rate between the ABR genes and the P-AST results was generated for the overall group. The concordance rates for each antibiotic between monomicrobial and polymicrobial infection were compared using chi-square test. RESULTS: Results from ABR gene detection and P-AST of urine samples from 1155 patients were included in the concordance analysis. Overall, there was a 60% concordance between the presence or absence of ABR genes and corresponding antimicrobial susceptibility with a range of 49-78% across antibiotic classes. Vancomycin, meropenem, and piperacillin/tazobactam showed significantly lower concordance rates in polymicrobial infections than in monomicrobial infections. CONCLUSION: Given the 40% discordance rate, the detection of ABR genes alone may not provide reliable data to make informed clinical decisions in UTI management. However, when used in conjunction with susceptibility testing, ABR gene data can offer valuable clinical information for antibiotic stewardship.202134447256
223680.9997Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples. The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including bla(KPC)-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.201931799215
583390.9997Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing. Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.200717141897
5777100.9997Rapid Detection of Antimicrobial Resistance Genes in Critically Ill Children Using a Custom TaqMan Array Card. Bacteria are identified in only 22% of critically ill children with respiratory infections treated with antimicrobial therapy. Once an organism is isolated, antimicrobial susceptibility results (phenotypic testing) can take another day. A rapid diagnostic test identifying antimicrobial resistance (AMR) genes could help clinicians make earlier, informed antimicrobial decisions. Here we aimed to validate a custom AMR gene TaqMan Array Card (AMR-TAC) for the first time and assess its feasibility as a screening tool in critically ill children. An AMR-TAC was developed using a combination of commercial and bespoke targets capable of detecting 23 AMR genes. This was validated using isolates with known phenotypic resistance. The card was then tested on lower respiratory tract and faecal samples obtained from mechanically ventilated children in a single-centre observational study of respiratory infection. There were 82 children with samples available, with a median age of 1.2 years. Major comorbidity was present in 29 (35%) children. A bacterial respiratory pathogen was identified in 13/82 (16%) of children, of which 4/13 (31%) had phenotypic AMR. One AMR gene was detected in 49/82 (60%), and multiple AMR genes were detected in 14/82 (17%) children. Most AMR gene detections were not associated with the identification of phenotypic AMR. AMR genes are commonly detected in samples collected from mechanically ventilated children with suspected respiratory infections. AMR-TAC may have a role as an adjunct test in selected children in whom there is a high suspicion of antimicrobial treatment failure.202338136735
5788110.9997Shifting of Distribution and Changing of Antibiotic Resistance in Gram-Positive Bacteria from Bile of Patients with Acute Cholangitis. BACKGROUND: Gram-negative bacteria are the predominant pathogens responsible for biliary infections; however, the prevalence of Gram-positive bacteria is currently increasing. Investigating the bacterial spectrum and evolving antibiotic resistance patterns of Gram-positive bacteria is crucial for optimizing the management of acute cholangitis, particularly in the context of the global rise in antibiotic resistance. METHODS: This retrospective analysis focused on Gram-positive bacteria isolated from the bile of patients undergoing biliary drainage with acute cholangitis at our hospital from January 1, 2018, to March 31, 2024. In total, 342 strains of Gram-positive bacteria were examined. RESULTS: The main Gram-positive bacteria detected included Enterococcus (57.23%), Staphylococcus (23.41%), and Streptococcus (13.01%). The most common species detected were Enterococcus faecium (36.42%), Enterococcus faecalis (14.16%), and Staphylococcus epidermidis (7.80%). Trend analysis revealed a decrease in the proportion of Enterococcus and an increase in Streptococcus. Additionally, the detection rate of methicillin-resistant Staphylococcus (MRS) showed a significant rise. Gram-positive bacteria exhibited high resistance to erythromycin and penicillin but remained highly susceptible to linezolid and vancomycin. Further, resistance to quinolones among Gram-positive bacteria was notably elevated. CONCLUSION: The bacterial spectrum and antibiotic resistance patterns of Gram-positive bacteria in acute cholangitis have undergone significant changes. Penicillin is not recommended for the treatment of Gram-positive bacterial infections. Antibiotic resistance should be closely monitored when using quinolones. Particular attention is warranted regarding the markedly increasing antibiotic resistance of Enterococcus faecium.202540034266
5539120.9997Staphylococcus aureus from Subclinical Cases of Mastitis in Dairy Cattle in Poland, What Are They Hiding? Antibiotic Resistance and Virulence Profile. Bovine mastitis is a common disease worldwide, and staphylococci are one of the most important etiological factors of this disease. Staphylococcus aureus show adaptability to new conditions, by which monitoring their virulence and antibiotic resistance mechanisms is extremely important, as it can lead to the development of new therapies and prevention programs. In this study, we analyzed Staphylococcus aureus (n = 28) obtained from dairy cattle with subclinical mastitis in Poland. The sensitivity of the isolated strains to antibiotics were confirmed by the disc diffusion method. Additionally, minimum inhibitory concentration values were determined for vancomycin, cefoxitin and oxacillin. Genotyping was performed by two methods: PCR melting profile and MLVF-PCR (multiple-locus variable-number tandem-repeat fingerprinting). Furthermore, the presence of antibiotic resistance and virulence genes were checked using PCR reactions. The analyzed strains showed the greatest resistance to penicillin (57%), oxytetracycline (25%) and tetracycline (18%). Among the analyzed staphylococci, the presence of 9 of 15 selected virulence-related genes was confirmed, of which the icaD, clfB and sea genes were confirmed in all staphylococci. Biofilm was observed in the great majority of the analyzed bacteria (at least 70%). In the case of genotyping among the analyzed staphylococci (combined analysis of results from two methods), 14 patterns were distinguished, of which type 2 was the dominant one (n = 10). This study provides new data that highlights the importance of the dominance of biofilm over antibiotic resistance among the analyzed strains.202236558738
2362130.9997Distribution of pathogenic bacteria and antimicrobial sensitivity of eye infections in Suzhou. AIM: To investigate the types of bacteria in patients with eye infections in Suzhou and their drug resistance to commonly used antibacterial drugs. METHODS: The clinical data of 155 patients were retrospectively collected in this study, and the pathogenic bacteria species and drug resistance of each pathogenic bacteria were analyzed. RESULTS: Among the 155 patients (age from 12 to 87 years old, with an average age of 57, 99 males and 56 females) with eye infections (160 eyes: 74 in the left eye, 76 in the right eye and 5 in both eyes, all of which were exogenous), 71 (45.81%) strains were gram-positive bacteria, 23 (14.84%) strains were gram-negative bacteria and 61 (39.35%) strains were fungi. Gram-positive bacteria were highly resistant to penicillin and erythromycin (78.87% and 46.48% respectively), but least resistant to vancomycin at 0. Gram-negative bacteria were highly resistant to cefoxitin and compound sulfamethoxazole (100% and 95.65% respectively), but least resistant to meropenem at 0. Comparison of the resistance of gram-positive and gram-negative bacteria to some drugs revealed statistically significant differences (P<0.05) in the resistance of both to cefoxitin, cotrimoxazole, levofloxacin, cefuroxime, ceftriaxone and ceftazidime, and both had higher rates of resistance to gram-negative bacteria than to gram-positive bacteria. The distribution of bacterial infection strains showed that Staphylococcus epidermidis was the most common strain in the conjunctiva, cornea, aqueous humor or vitreous body and other eye parts. Besides, Fusarium and Pseudomonas aeruginosa were also among the most common strains of conjunctival and corneal infections. CONCLUSION: Gram-positive bacteria are the dominant bacteria in eye infections, followed by gram-negative bacteria and fungi. Considering the resistance of gram-negative bacteria to multiple drugs, monitoring of bacteria should be strengthened in eye bacterial infections for effective prevention and control to reduce complications caused by eye infections.202438638249
5688140.9997Isolation and molecular identification of bacteria from sheep with eye infections. BACKGROUND: Ocular disease in sheep is a severe concern for the health and welfare of livestock animals, as well as losses of productivity and value to the livestock industry. AIM: This study aimed to isolate and characterize bacteria in sheep with eye disease on the molecular level. METHODS: One hundred fifty sheep with eye infections were treated, and tissue samples were taken for microbiological studies. We isolated bacteria from traditional cultures and discovered molecules by polymerase chain reaction (PCR) of single bacterial genes. RESULTS: A total of 150 ocular samples were collected from sheep, with bacterial growth observed in 120 samples, resulting in an isolation rate of 80%. Staphylococcus aureus was the most bacteria isolated in this study, which PCR also confirmed. We found antibiotic-resistant bacteria such as S. aureus, Escherichia coli, and Pasteurella multocida. These results reveal that preventing sheep ocular infections requires the effective use of antibiotics. CONCLUSION: This study suggests the prevalence of bacterial infection in sheep eyes and argues the utility of molecular methods in veterinary diagnosis. Record levels of antibiotic resistance must be maintained in animal husbandry and the use of antibiotic stewardship programs.202439927373
2363150.9997Analysis of distribution and antibiotic resistance of Gram-positive bacteria isolated from a tertiary-care hospital in southern China: an 8-year retrospective study. OBJECTIVE: Due to the severe drug resistance situation of Gram-negative bacteria, especially Gram-negative enterobacter, relatively little attention has been paid to the changes in Gram-positive bacteria species and drug resistance. Therefore, this study analyzed the prevalence and drug resistance of Gram-positive bacteria in a general tertiary-care hospital from 2014 to 2021, in order to discover the changes in Gram-positive bacteria distribution and drug resistance that cannot be easily identified, inform clinicians in their respective regions when selecting antimicrobial agents, and to provide the basis for the diagnosis of Gram-positive bacterial infection, and for the comprehensive and multi-pronged prevention and control of drug-resistant bacteria. METHODS: A retrospective study was conducted on Gram-positive bacteria isolated from patients presented to a general tertiary-care hospital from January 2014 to December 2021. A total of 15,217 Gram-positive strains were analyzed. RESULTS: During the 8-year period, the total number and the species of Gram-positive bacteria isolated from clinic increased continuously. The seven most common species were Streptococcus pneumoniae (21.2%), Staphylococcus aureus (15.9%), Enterococcus faecium (20.6%), Enterococcus faecalis (14.0%), and Staphylococcus epidermidis (7.8%), Staphylococcus haemolyticus (4.8%), Streptococcus agalactiae (3.6%). The isolation rates of Staphylococcus aureus and Streptococcus agalactiae increased, and the isolation rate of Enterococcus faecium decreased. The resistance rates of Staphylococcus aureus to erythromycin, clindamycin, tetracycline, rifampicin and furantoin decreased obviously. The resistance rates of Streptococcus pneumoniae to cefepime (non-meningitis) and ceftriaxone (meningitis) decreased significantly. The resistance rates of Enterococcus faecium to penicillin, ampicillin, erythromycin, levofloxacin, ciprofloxacin and furantoin rose rapidly from 50.3, 47.6, 71.5, 44.9, 52.3, and 37.5% in 2014 to 93.1, 91.6, 84.9, 86.8, 86.8, and 60.0% in 2021, respectively. CONCLUSION: The total number and the species of Gram-positive bacteria isolated during the 8-year period increased continuously. Streptococcus pneumoniae and Staphylococcus aureus are the main causes of positive bacterial infections in this hospital. The resistance rates of Enterococcus faecium to a variety of commonly used antibiotics increased significantly. Therefore, it is very important to monitor the distribution of bacteria and their resistance to antibiotics to timely evaluate and identify changes in drug resistance that are not easily detected.202337840716
2987160.9997A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae. BACKGROUND: The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates. RESULTS: In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3″)-I,aph(3')-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively. CONCLUSION: This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.201323497180
5790170.9997Activity Assessment of Antibiotics Used Against Different Bacterial Etiological Agents of UTI in Najaf, Iraq. BACKGROUND & OBJECTIVE: Antibiotic resistance in urinary tract infection (UTI) is increasing nowadays, therefore, the aim of this study was to evaluate the resistance patterns of many pathogens toward several antibiotics that are in common use in our hospitals. METHODS: Subculture and identification of pathogenic bacteria were performed on 1148 hospitals' bacterial primary cultures which were considered positive for UTI. An antibiotic sensitivity test was performed by using the disc diffusion method. The rates of resistance were statistically analyzed and correlated with the types of antibiotics and bacteria. RESULTS: It was found that 1148 out of 2087 urine samples were UTI positive, the majority of cases (76%) were from females (P<0.0001). Escherichia coli and Klebsiella were the most isolated Gram-negative bacteria, while Staphylococcus spp. was the most isolated Gram-positive pathogen. E. coli showed the highest resistance rate among all bacteria, while Streptococcus spp. was the most sensitive. The highest resistance was noticed to be against gentamicin and ampicillin, while the most effective drugs were imipenem and amikacin. There was a significant difference in resistance rates among the different bacterial categories (P<0.0001), while no significant difference was noticed in resistance rates among antibiotics categories (P>0.05). CONCLUSION: Elevated rates of antibiotic resistance were noticed in this study in UTI-causing bacteria; therefore, it is highly important at least to every general hospital to investigate the antibiotic resistance rates occasionally to determine the proper antimicrobial treatment as well as re-evaluate antibiotics which were considered as empirical.202439687449
2349180.9997DETECTION OF MECA AND NUC GENES OF MULTI-DRUG RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM DIFFERENT CLINICAL SAMPLES. BACKGROUND: During this study, six isolates of multiple antibiotic resistant Staphylococcus aureus bacteria were obtained from different clinical specimens (burn swabs, urinary tract infections, wound swabs): three isolates from burns, two isolates from urinary tract infections, and one isolate from wound swabs. They were obtained from private laboratories in Baghdad from 1/1/2023 to 3/15/2023. METHOD: The diagnosis of these isolates was confirmed using the Vitek2 device. A susceptibility test was conducted on ten antibiotics, and S. aureus bacteria showed resistance to most antibiotics, polymerase chain reaction was done to mecA and Nuc gene by conventional PCR. RESULTS: The results of the molecular detection of the MecA gene showed that all isolates of multi-drug-resistant S. aureus possess this gene. In contrast, the results of the molecular detection of the nuc gene showed that only isolates No. 1 and No. 4 carry this gene, while the rest of the isolates do not carry this gene. CONCLUSION: S. aureus are resistant to antibiotics because they possess resistance genes such as the mecA gene.202439724880
2350190.9997Antibiotic Resistance Profiles and MLST Typing of Staphylococcus Aureus Clone Associated with Skin and Soft Tissue Infections in a Hospital of China. OBJECTIVE: To analyze the antibiotic resistance profile, virulence genes, and molecular typing of Staphylococcus aureus (S. aureus) strains isolated in skin and soft tissue infections at the First Affiliated Hospital, Gannan Medical University, to better understand the molecular epidemiological characteristics of S. aureus. METHODS: In 2023, 65 S. aureus strains were isolated from patients with skin and soft tissue infections. Strain identification and susceptibility tests were performed using VITEK 2 and gram-positive bacteria identification cards. DNA was extracted using a DNA extraction kit, and all genes were amplified using polymerase chain reaction. Multilocus sequence typing (MLST) was used for molecular typing. RESULTS: In this study, of the 65 S. aureus strains were tested for their susceptibility to 16 antibiotics, the highest resistance rate to penicillin G was 95.4%. None of the staphylococcal isolates showed resistance to ceftaroline, daptomycin, linezolid, tigecycline, teicoplanin, or vancomycin. fnbA was the most prevalent virulence gene (100%) in S. aureus strains isolated in skin and soft tissue infections, followed by arcA (98.5%). Statistical analyses showed that the resistance rates of methicillin-resistant S. aureus isolates to various antibiotics were significantly higher than those of methicillin-susceptible S. aureus isolates. Fifty sequence types (STs), including 44 new ones, were identified by MLST. CONCLUSION: In this study, the high resistance rate to penicillin G and the high carrying rate of virulence gene fnbA and arcA of S.aureus were determine, and 44 new STs were identified, which may be associated with the geographical location of southern Jiangxi and local trends in antibiotic use. The study of the clonal lineage and evolutionary relationships of S. aureus in these regions may help in understanding the molecular epidemiology and provide the experimental basis for pathogenic bacteria prevention and treatment.202438933775