PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis. - Related Documents




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579701.0000PCR-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
223510.9998Nanosphere'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
577920.9998Development 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
579830.9998Rapid identification of bacteria, mecA and van genes from blood cultures. The Genotype technology, a quick molecular genetic assay based on DNA multiplex amplification with biotinylated primers followed by hybridization to membrane bound probes, complies with the requirements for a fast diagnosis of sepsis. We evaluated the new Genotype BC Gram-negative and Gram-positive test kits (Hain Life Science, Germany) which respectively allow for the identification of 15 species of Gram-negative (GN) rods, and the identification of 17 Gram-positive (GP) bacteria species together with the determination of methicillin and vancomycin resistance (mecA and van genes). The study was performed on 60 positive blood cultures from BacT/ALERT bottles (aerobic, anaerobic and pediatric bottles). First, a Gram stain was carried out to select between Genotype BC GP or GN test, then identification were performed by the Genotype BC tests and by biochemical conventional tests after subculture and phenotypic susceptibility determination. The operating procedure was very easy to carry out and required a small amount of starting material (5 to 10 microL of blood culture). The results were available within 4.5 hours. For all the blood cultures, the Genotype BC results correlated with the biochemical identification and phenotypic antibiotics susceptibility. According to our results, this DNA strip technology based assay can easily be incorporated into routine diagnosis.200717913394
583340.9998Rapid 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
577850.9997A 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
221360.9997The distribution and resistance of pathogens causing blood stream infections following liver transplantation: a clinical analysis of 69 patients. BACKGROUND/AIMS: To study the distribution and drug resistance of pathogens causing blood stream infections (BSIs) and provide the evidence for clinical therapy after liver transplantation. METHODOLOGY: Blood samples were processed by the BACTEC 9120 blood culture system. Species identification was performed using the Vitek-2 system. The drug susceptibility of pathogens was performed using the ATB FUNGUS 3 system. RESULTS: One hundred and twenty six episodes of BSIs occurred in 69 patients between January 31, 2003 and January 31, 2014. The gram-positive bacteria emerged as major pathogens and constituted 48.4% of all pathogens (61/126). The most common bacilli were Enterobacter spp and Enterococcus spp followed by S. aureus. The gram-negative bacteria were relatively sensitive to carbapenems and the gram-positive bacteria were relatively sensitive to glycopeptides and oxazolidone antibiotics. The drug resistance of fungi to amphotericin B, flucytosine, voriconazole and caspofungin was not found. CONCLUSION: In liver transplantation, gram-positive bacteria caused BSls more frequently than gram-negative bacteria. The resistance rate of bacteria to antibiotics was high while the rate was low in fungi.201425699372
223470.9997Clinical relevance of molecular identification of microorganisms and detection of antimicrobial resistance genes in bloodstream infections of paediatric cancer patients. BACKGROUND: Bloodstream infections (BSIs) are the major cause of mortality in cancer patients. Molecular techniques are used for rapid diagnosis of BSI, allowing early therapy and improving survival. We aimed to establish whether real-time quantitative polymerase chain reaction (qPCR) could improve early diagnosis and therapy in paediatric cancer patients, and describe the predominant pathogens of BSI and their antimicrobial susceptibility. METHODS: Blood samples were processed by the BACTEC system and microbial identification and susceptibility tests were performed by the Phoenix system. All samples were screened by multiplex 16 s rDNA qPCR. Seventeen species were evaluated using sex-specific TaqMan probes and resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were screened by SYBR Green reactions. Therapeutic efficacy was evaluated at the time of positive blood culture and at final phenotypic identification and antimicrobial susceptibility results. RESULTS: We analyzed 69 episodes of BSI from 64 patients. Gram-positive bacteria were identified in 61 % of the samples, Gram-negative bacteria in 32 % and fungi in 7 %. There was 78.2 % of agreement between the phenotypic and molecular methods in final species identification. The mecA gene was detected in 81.4 % of Staphylococcus spp., and 91.6 % were concordant with the phenotypic method. Detection of vanA gene was 100 % concordant. The concordance for Gram-negative susceptibilities was 71.4 % for Enterobacteriaceae and 50 % for Pseudomonas aeruginosa. Therapy was more frequently inadequate in patients who died, and the molecular test was concordant with the phenotypic susceptibility test in 50 %. CONCLUSIONS: qPCR has potential indication for early identification of pathogens and antimicrobial resistance genes from BSI in paediatric cancer patients and may improve antimicrobial therapy.201627585633
583480.9997Real-Time PCR to Identify Staphylococci and Assay for Virulence from Blood. The genus Staphylococcus includes pathogenic and non-pathogenic facultative anaerobes. Due to the plethora of virulence factors encoded in its genome, the species Staphylococcus aureus is known to be the most pathogenic. S. aureus strains harboring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, however, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbor mecA, the genetic driver for staphylococcal methicillin-resistance. In this chapter, the detailed practical procedure for operating a real-time pentaplex PCR assay in blood cultures is described. The pentaplex real-time PCR assay simultaneously detects markers for the presence of bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl), and methicillin resistance (mecA).201728600770
231890.9997Distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analysis of integron resistance genes in respiratory tract isolates of uninfected patients. BACKGROUND: We studied the distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analyzed the integron resistance genes in respiratory tract isolates of uninfected patients. METHODS: Retrospective analysis was used to select sputum samples from 400 lung cancer patients after chemotherapy admitted in Fuyang People's Hospital from July 2017 to July 2019. Culture, isolation and identification of strains were conducted in accordance with the national clinical examination operating procedures. RESULTS: A total of 134 strains were identified. In 120 patients with pulmonary infection, 114 strains were cultured. Twenty strains of klebsiella pneumoniae were cultured in 280 patients without pulmonary infection. Among the 134 strains, the detection rate of gram-negative bacteria was 79.10%. The first four strains were Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Haemophilus influenzae. The gram-positive bacteria detection rate was 4.47%, mainly Staphylococcus aureus and Streptococcus. The fungus detection rate was 16.42%. The drug sensitivity results showed that the resistance rate of gram-negative bacillus to penicillin and cephalosporin was higher, and were more sensitive to carbapenem, piperacillin tazobactam and cefoperazone sulbactam. Gram-positive cocci were resistant to penicillin, macrolide and clindamycin, and sensitive to linezolid, vancomycin and rifampicin. All strains of fungal culture were candida albicans, which were sensitive to common antifungal drugs. Among the 20 strains of klebsiella pneumoniae cultured in sputum specimens of non-infected patients with lung cancer undergoing chemotherapy, 2 strains were integron-positive strains, and all of them were class I integrons. CONCLUSIONS: Lung cancer patients after chemotherapy have a high resistance to commonly used antimicrobial drugs, so it is necessary to detect the resistance of pathogenic microorganisms in clinical practice. The strains carried by patients with lung cancer without pulmonary infection during chemotherapy can isolate type I integrons, suggesting that the spread of drug resistance at gene level should be closely detected.202032944333
5796100.9997Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures. Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.201626712265
2236110.9997Development 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
2233120.9997Assessment 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
2349130.9996DETECTION 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
2247140.9996Metagenomic 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
2350150.9996Antibiotic 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
2362160.9996Distribution 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
2363170.9996Analysis 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
5968180.9996A PCR assay for rapid detection of vancomycin-resistant enterococci. Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.200212007446
5974190.9996Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus. As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.201021083822