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223700.9977Evaluation of Sepsis Flow Chip for identification of Gram-negative bacilli and detection of antimicrobial resistance genes directly from positive blood cultures. Blood stream infections are serious conditions associated with high morbi-mortality. In this study, the new Sepsis Flow Chip (SFC) assay for identification of Gram-negative bacteria and their antimicrobial resistance genes was evaluated in positive blood cultures (BCs). SFC is a microarray with a broad panel comprising the most frequent causative agents of sepsis and antimicrobial resistance genes associated with them. A total of 100 prospective BCs, positive for Gram-negative bacilli, were assessed in the routine of the clinical microbiology laboratory and also applying the SFC assay. Moreover, 19 BCs spiked with well-characterized enterobacterial isolates, harboring antimicrobial resistance genes, were analyzed by the latter. Among the monomicrobial BCs (90), the concordance between SFC identification and the reference method was 94.4%; however, it achieved 100% when SFC was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after 4-h incubation. Regarding polymicrobial BCs (10), 15 out of the 22 bacteria present (68.2%) were correctly identified, including all contained in 50% of the cultures. With regard to antimicrobial resistance genes, 98.8%, 98.9%, and 99% concordance was obtained for bla(CTX-M), bla(OXA-48), and bla(VIM), respectively, in comparison with polymerase chain reaction amplification. SFC assay gives results in only 4 h and showed a high concordance rate with the reference method. Although further evaluation studies are necessary, SFC assay implementation, together with antimicrobial stewardship programs, could contribute to improve the therapeutic approaches and to reduce the morbi-mortality, length of hospital stay, and healthcare-associated costs in patients with sepsis.201829551362
223910.9977The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System. BACKGROUND: Urinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours. METHODS: We designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing. RESULTS: UTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method. CONCLUSIONS: UTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.202133912478
223420.9977Clinical 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
223630.9977Development 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
272540.9977Hygiene practices and antibiotic resistance among dental and medical students: a comparative study. PURPOSE: Healthcare students' hand and smartphone hygiene is critical due to potential pathogenic and antibiotic-resistant bacteria transmission. This study evaluates hygiene practices in medical and dental students at Kuwait University, exploring antibiotic resistance gene prevalence. METHODS: Swab samples were collected from the hands and smartphones of 32 medical and 30 dental students. These samples were cultured on Columbia Blood Agar and McConkey Agar plates to quantify bacterial colony-forming units (CFUs). The extracted DNA from these colonies underwent RT-PCR to identify antibiotic resistance genes, including tem-1, shv, blaZ, and mecA. Additionally, a questionnaire addressing hygiene practices was distributed post-sample collection. RESULTS: Medical students exhibited more frequent hand hygiene compared to dental students (P ≤ 0.0001). Although significantly fewer bacterial CFUs were found on medical students' smartphones (mean = 35 ± 53) than dental students' (mean = 89 ± 129) (P ≤ 0.05), no significant differences were observed in CFU counts on their hands (medical: mean = 17 ± 37; dental: mean = 96 ± 229). Detection of at least one of the targeted antibiotic resistance genes on medical (89% hands, 52% smartphones) and dental students' (79% hands, 63% smartphones) was not statistically significant. However, the prevalence of two genes, tem-1 and shv, was significantly higher on medical students' hands (78% and 65%, respectively) than on dental students' hands (32% and 28%, respectively). CONCLUSION: Clinically significant prevalence of antibiotic resistance genes were found on medical and dental students' hands and smartphones, emphasizing the importance of ongoing education regarding hand hygiene and smartphone disinfection. This continuous reinforcement in the curriculum is crucial to minimizing the risk of cross-contamination.202438514584
234450.9976Analysis of antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery. PURPOSE: To analyze antibiotic resistance and genetic profile of conjunctival bacteria flora before and after cataract surgery with the focus on coagulase-negative staphylococci (CNS) during cataract surgery and discuss the implications of this colonization as a potential risk of acquiring endophthalmitis. METHODS: After approval of the institutional review board and informed consent from patients had been obtained, conjunctival swabs for culture from 59 patients undergoing cataract surgery were taken of the fellow eye at baseline (C0) and from the eye to be operated before (T0) and after (T1) irrigation with povine-iodine 5%, and at the end of surgery (T2). Genes responsible for virulence (mecA, ica and atlE) and antibiotic profile were determined; strain clonality of persistent colonizing Staphylococcus epidermidis strains was established by the Multi-locus sequence typing (MLST). RESULTS: The frequency of CNS was significantly reduced in T1 (13.6%) from 81.4% in T0 and 86.4% in C0. The frequency of mecA, ica and atlE genes was 34.4%, 37.5% and 61.4%, respectively; and methicillin phenotypic resistance was 35.4%. S. epidermidis was the most frequent species isolated in every time point. MLST revealed in 7 patients 100% coincidence of the seven alleles of the S. epidermidis isolated previous to povine-iodine 5% disinfection and at the end of the surgery. CNS isolates from T1 or T2 corresponded to the same species, antibiotic and virulence profile as those isolates from C0 or T0. CONCLUSION: Povidone-iodine 5% prophylaxis before surgery significantly reduced conjunctival contamination; in those that persisted, the source of contamination was mostly the patient's microbiota confirmed by the MLST system.202335943639
234760.9976Multiple drug resistance of Listeria monocytogenes isolated from aborted women by using serological and molecular techniques in Diwaniyah city/Iraq. BACKGROUND AND OBJECTIVES: The study was sought to detect the effect of Listeria monocytogenes on pregnant Iraqi women at Al-Diwaniya hospitals and determination of virulence genes and antimicrobial susceptibility of isolates. MATERIALS AND METHODS: 360 specimens including blood, urine, vaginal and endocervical were collected from 90 patients with spontaneous abortions. Blood samples were displayed to immunological study and remaining specimens were subjected to bacteriological diagnosis. PCR was used to determine the virulence factors and antimicrobial resistance genes. RESULTS: Fifteen positive samples (16.6%) of patients and thirteen isolates (14.5%) from patients were recognized based on ELISA and PCR assay respectively. The general isolation of L. monocytogenes strains in cases of abortive women was 13/270 (4.8%). L. monocytogenes strains were highly virulent because of presence of virulence factors associated genes, namely actA, hlyA, plcA and prfA in all strains. Multiple drug resistance (MAR) index values of 15.4% of isolates were >0.2. CONCLUSION: It is necessary for conducting susceptibility testing and to select the suitable antibiotics and avoid the effects of these bacteria in pregnant women.202032994901
223370.9976Assessment 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
221080.9976Beyond Culture: Real-Time PCR Performance in Detecting Causative Pathogens and Key Antibiotic Resistance Genes in Hospital-Acquired Pneumonia. Introduction: The rise in hospital-acquired pneumonia (HAP) due to antibiotic-resistant bacteria is increasing morbidity, mortality, and inappropriate empirical antibiotic use. This prospective research aimed to evaluate the performance of a real-time polymerase chain reaction (PCR) assay for detecting causative microorganisms and antibiotic-resistance genes from respiratory specimens compared to traditional methods. Additionally, we aimed to determine the molecular epidemiology of antibiotic resistance genes among HAP patients at The University of Jordan hospital. Methods: Lower respiratory tract samples were collected from HAP patients, including those with ventilator-associated pneumonia (VAP), between May 2024 and October 2024. Clinical data from the medical files were used to collect and analyze demographic and clinical information, including clinical outcomes. Real-time PCR was run to detect causative microbes and antibiotic resistance genes. Results: Among 83 HAP patients (median age 63, 61.45% male), 48.15% died. Culture identified Klebsiella (25.53%), Acinetobacter (22.34%), and Candida (24.47%) as the most common pathogens, while qPCR showed higher detection rates, including for A. baumannii (62.20%, p = 0.02) and K. pneumoniae (45.12%, p < 0.001). Carbapenem resistance was high; A. baumannii showed 100% resistance to most antibiotics except colistin (92.31%). The resistance genes ndm (60%) and oxa-48 (58.46%) were frequently detected and significantly associated with phenotypic resistance (p < 0.001). The qPCR identified resistance genes in all carbapenem-resistant cases. No gene significantly predicted mortality. Conclusions: Real-time PCR diagnostic technique combined with epidemiology of antibiotic resistance genes data may be a rapid and effective tool to improve HAP management. Large, multicenter studies are needed in the future to validate the performance of real-time PCR in HAP diagnosis, and appropriate management is also required.202541009915
272690.9976Associations of HIV status and the abundance of blaCTX-M and vanB resistance genes in stool samples of Ghanaian individuals. BACKGROUND: A cross-sectional study was performed to investigate associations of enteric colonization with resistant bacteria in Ghanaian individuals who were tested positive and negative for the human immunodeficiency virus (HIV). METHODS: Abundance of the ESBL-(extended spectrum beta-lactamase-)type resistance-mediating gene blaCTX-M and the vancomycin resistant enterococci-(VRE-)associated genes vanA and vanB genes was associated with available clinical and epidemiological data on the study participants. RESULTS: In terms of enteric carriage of ESBL-positive bacteria with CTX-M-type beta-lactam resistance genes, being HIV-positive (93.3% vs. 83.3%, P = 0.003) and having low CD4+ T-lymphocyte counts <200 cells/µL (microliter) (96.8% vs. 91.2%, P = 0.009) were identified as risk factors. Enteric carriage of ESBL-positive bacteria with CTX-M-type resistance genes was associated with poor immunological status in terms of lower CD4+ T-leukocyte counts, lower CD4+/CD8+ ratios, higher viral replication, as well as with immune activation. For VRE, a non-significant trend for more VRE in control individuals without known HIV infection (6% vs. 2.5%, P = 0.089) was observed. CONCLUSIONS: An association of ESBL colonization and immunological status was recorded. No such association was detected for VRE, suggesting different determinants of local VRE epidemiology.202541091553
2136100.9976Antibiotic profiling of multidrug resistant pathogens in one-day-old chicks imported from Belgium to benin. BACKGROUND: Little data exist on the presence of resistant pathogens in day-old chicks imported into Benin. The occurrence of pathogenic bacteria was assessed in 180 one-day-old chicks imported from Belgium and received at the Cardinal Bernardin Gantin International Airport in Cotonou (Benin). The samples included swabbing the blisters of 180 chicks, followed by 18 pools of 10 swabs for bacterial isolation. Classic bacteriological methods based on Gram staining, culture on specific media and biochemical characterization were used. Antibacterial susceptibility screening to antibiotics was conducted using the Kirby-Bauer disc diffusion method, and the results were interpreted according to guidelines from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). DNA extraction was performed by the heat treatment method. Resistance genes were screened by real-time PCR. RESULTS: We isolated 32 bacteria, including Escherichia coli (50%), Enterococcus spp. (28%), and coagulase-negative staphylococci (10%). The isolates were investigated for antibiotic resistance against antibiotics using the disk diffusion method and showed that in the Escherichia coli strains isolated, the highest rate of resistance was obtained against ciprofloxacin (81%), followed by trimethoprim + sulfamethoxazole (62%). Enterobacter cloacae was sensitive to all the antibiotics tested. Pseudomonas spp. resistant to amoxicillin and trimethoprim + sulfamethoxazole was noted. The SulII gene was found in all cloacal samples, while the SulI and bla(TEM) genes were present at 44.44% and 16.67%, respectively. CONCLUSION: This study confirms that imported day-old chicks can be a potential source of dissemination of resistant bacteria in poultry production. A system for immediate detection of resistant bacteria in chicks upon arrival in the country is thus needed.202336670436
2421110.9975Antimicrobial susceptibility test and antimicrobial resistance gene detection of extracellular enzyme bacteria isolated from tilapia (Oreochromis niloticus) for probiotic candidates. BACKGROUND AND AIM: Antimicrobial resistance (AMR) is a global problem that can increase mortality and morbidity rates and adversely affect health. Therefore, AMR control must be carried out in various sectors, including the fisheries sector, using probiotics. Bacteria can become resistant to antibiotics, including bacteria used for probiotics. This study aimed to isolate bacteria as potential producers of extracellular enzymes, phenotypic characterization, and antibiotic-resistant gene patterns. MATERIALS AND METHODS: In this study, 459 bacterial isolates were isolated from the stomach of tilapia in Indonesia. Tilapia was obtained from Sukabumi, Ciamis, Serang, Banjarnegara, Jayapura, Sorong, Manokwari Selatan, Takalar, Lampung, Batam, and Mandiangin. Enzymatic bacteria were identified. An antimicrobial susceptibility test was conducted by agar disk diffusion, and genotypic detection of encoding genes was performed using a molecular method. RESULTS: This study obtained 137 isolates (29.84%) that can produce extracellular enzymes. The highest number of E-sensitive isolates was found, including 130 isolates (94.89%). Six isolates (6/137) can produce four enzymes (amylase, protease, cellulose, and lipase), and they were sensitive to antibiotics. A total of 99 isolates can produce extracellular enzymes, and they were sensitive to antibiotics. Such isolates serve as a consortium of probiotic candidates. The isolates that are resistant to oxytetracycline (OT), erythromycin (E), tetracycline (TE), and enrofloxacin (ENR) included 15 isolates (10.95%), seven isolates (5.11%), three isolates (2.19%), and one isolate (0.73%), respectively. In addition, four isolates (2.92%) were detected as multidrug-resistant. The tet(A) gene obtained the highest result of detection of resistance genes in isolates that were intermediate and resistant to TE and OT. Isolates that serve as ENR intermediates have a high qnr(S) resistance gene. CONCLUSION: The data in this study provide the latest update that bacteria can serve as a consortium of potential probiotics with antibiotic-resistant genes for the treatment of fish. Bacteria that are intermediate to antibiotics may contain resistance genes. The results of this study will improve the policy of probiotic standards in Indonesia.202337042005
2343120.9975Investigation of Virulence Genes of the Predominant Bacteria Associated with Renal Stones and their Correlation with Postoperative Septic Complications. PURPOSE: Nephrolithiasis is a worldwide disease, and 4.7% of the patients may develop postoperative sepsis. Characterization of virulence genes of bacteria associated with renal stones is still lacking in the literature. The study aimed to investigate the virulence genes of the predominant stone bacterial isolate and their association with postoperative septic complications in patients treated with percutaneous nephrolithotomy (PCNL). METHODS: Stone and midstream urine samples were collected from 200 nephrolithiasis patients who underwent PCNL. Microbiological examination and virulence profile were studied for the common bacteria isolated from the stones. RESULTS: Microbiological analysis revealed that Staphylococcus aureus was the predominant organism in stone samples (42.8%), while Escherichia coli (56.6%) was the dominant pathogen in midstream urine. Eight patients (4%) developed septic complications; stone culture was positive for S. aureus in seven and E. coli in one patient, while all but one had negative midstream urine. The patient with positive midstream urine culture had also S. aureus infection. Detection of virulence genes in S. aureus isolated from stones showed a high positivity of the hemolysine gene hla (93.3%) and adhesion gene fnbA (73.3%), whereas enterotoxin genes (sec and sea) were negative in all S. aureus stone cultures. Moreover, the adhesion genes (fnbB and can), hemolysine gene (hlb), panton-valentine leukocidin (pvl) gene and the enterotoxin gene (seb) were significantly higher in septic patients compared to the non-septic ones (p< 0.05). Interestingly, there was a significant relation between the existence of virulence genes and the resistance of antibiotics (p < 0.05). CONCLUSION: There has been a notable shift toward gram-positive organisms (S. aureus) in the stone culture. Moreover, S. aureus virulence genes were significantly attributed to the resistance of some antibiotics and postoperative septic complications, suggesting that the stone culture could be more informative than urine culture, especially in predicting the risk of postoperative sepsis.202235844358
1476130.9975Evaluation of the BioFire FilmArray Pneumonia Panel for rapid detection of respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate specimens. OBJECTIVES: The performance of the investigational-use-only version of the BioFire FilmArray Pneumonia Panel (FA-Pneumo), a high-order nested multiplex PCR, was evaluated for the detection of typical respiratory bacterial pathogens and antibiotic resistance genes in sputa and endotracheal aspirate (ETA) specimens. METHODS: Thirty-one sputa and 69 ETA specimens were analyzed. The diagnostic performance of FA-Pneumo was assessed using routine microbiological methods as the reference standard. RESULTS: Overall sensitivity and specificity for organism detection using FA-Pneumo were 98.5% and 76.5%, respectively. The sensitivity for each pathogen was 100%, except for Klebsiella aerogenes, and the range of specificity was 83.3-99.0%. FA-Pneumo detected antimicrobial resistance genes in 17 out of 18 specimens (94.4%) that were resistant by antimicrobial susceptibility testing. FA-Pneumo additionally detected 25 resistance genes in 22 specimens, and sequencing for the presence of resistance genes confirmed the majority of these results (20/25, 80%). Semi-quantitative analysis of bacterial nucleic acid amounts by FA-Pneumo revealed that 88.2% of the identified bacteria (67/76) with ≥10(6) copies/ml also gave culture-positive results with significant amounts of bacteria. CONCLUSIONS: FA-Pneumo is a rapid test with high sensitivity for the detection of bacteria and antimicrobial resistance genes in sputum and ETA specimens and could aid in determining antibiotic therapy.202032179139
1475140.9975Evaluation of the FilmArray(®) Pneumonia Plus Panel for Rapid Diagnosis of Hospital-Acquired Pneumonia in Intensive Care Unit Patients. The FilmArray(®) Pneumonia plus Panel (FAPP) is a new multiplex molecular test for hospital-acquired pneumonia (HAP), which can rapidly detect 18 bacteria, 9 viruses, and 7 resistance genes. We aimed to compare the diagnosis performance of FAPP with conventional testing in 100 intensive care unit (ICU) patients who required mechanical ventilation, with clinically suspected HAP. A total of 237 samples [76 bronchoalveolar lavages (BAL(DS)) and 82 endotracheal aspirates (ETA(DS)) obtained at HAP diagnosis, and 79 ETA obtained during follow-up (ETA(TT))], were analyzed independently by routine microbiology testing and FAPP. 58 patients had paired BAL(DS) and ETA(DS). The positivity thresholds of semi-quantified bacteria were 10(3)-10(4) CFUs/mL or 10(4) copies/mL for BAL, and 10(5) CFUs/mL or copies/mL for ETA. Respiratory commensals (H. influenzae, S. aureus, E. coli, S. pneumoniae) were the most common pathogens. Discordant results for bacterial identification were observed in 33/76 (43.4%) BAL(DS) and 36/82 (43.9%) ETA(DS), and in most cases, FAPP identified one supplemental bacteria (23/33 BAL(DS) and 21/36 ETA(DS)). An absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. No linear relationship was observed between bin and CFUs/mL variables. Concordant results between paired BAL(DS) and ETA(DS) were obtained in 46/58 (79.3%) patients with FAPP. One of the 17 resistance genes detected with FAPP (mecA/C and MREJ) was not confirmed by conventional testing. Overall, FAPP enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. Implementing this strategy may allow clinicians to make more timely and informed decisions.202032983057
2209150.9975Concordance 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
2221160.9975Rapid detection of blaKPC carbapenemase genes by real-time PCR. Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla(KPC)) enzymes are among the most common beta-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla(KPC) genes using TaqMan chemistry. The q-PCR amplification of bla(KPC) DNA was linear over 7 log dilutions (r(2) = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla(KPC) genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla(KPC) genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla(KPC) genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla(KPC) q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla(KPC) detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.200818614657
5284170.9975Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study. Routine postoperative antibiotic prophylaxis is not recommended for third molar extractions. However, amoxicillin still continues to be used customarily in several clinical practices worldwide to prevent infections. A prospective cohort study was conducted in cohorts who underwent third molar extractions with (group EA, n = 20) or without (group E, n = 20) amoxicillin (250 mg three times daily for 5 days). Further, a control group without amoxicillin and extractions (group C, n = 17) was included. Salivary samples were collected at baseline, 1-, 2-, 3-, 4-weeks and 3 months to assess the bacterial shift and antibiotic resistance gene changes employing 16S rRNA gene sequencing (Illumina-Miseq) and quantitative polymerase chain reaction. A further 6-month follow-up was performed for groups E and EA. Seven operational taxonomic units reported a significant change from baseline to 3 months for group EA (adjusted p < 0.05). No significant change in relative abundance of bacteria and β-lactamase resistance genes (TEM-1) was observed over 6 months for any group (adjusted p > 0.05). In conclusion, the salivary microbiome is resilient to an antibiotic challenge by a low-dose regimen of amoxicillin. Further studies evaluating the effect of routinely used higher dose regimens of amoxicillin on gram-negative bacteria and antibiotic resistance genes are warranted.201931822712
2211180.9975The molecular analysis of antibiotic resistance and identification of the aerobic bacteria isolated from pleural fluids obtained from patients. OBJECTIVE: Pleural effusion is a common clinical condition due to various etiological causes. Infectious pleural effusion can be seen in 20-40% of patients. In this study, follow-up of patients admitted to our hospital and diagnosed with pleural effusion are reported. It was aimed to investigate the prevalence of bacteria isolated from patients with pleural effusion and their antibiotic resistance profiles. MATERIALS AND METHODS: The pleural fluids obtained from the patients during surgical operations were analyzed microbiologically. Conventional culture, Vitek 2, 16S rRNA, and single Polymerase Chain Reaction (sPCR) were used for microbiological analysis. RESULTS: Twenty-two (12.2%) bacteria were isolated from 180 patients. The most prominent of them were 16 (8.8%) Klebsiella pneumoniae strains. As for the antibiotic sensitivity, gram-negative bacteria showed the highest sensitivity to colistin, while Gram-positive bacteria showed sensitivity to different antibiotics. In 16S rRNA PCR, 22 samples were found to be positive. In the analysis of antibiotic resistance genes, the OXA-48 gene was determined as the highest. CONCLUSIONS: In our region, it is essential to perform a microbiological analysis of the sample in patients with pleural effusion. It was thought that revealing both the phenotype and genotype of the antibiotic resistance of the patients was important in terms of treatment. In hospital surveillance, it was considered important to reveal and record the resistance gene profiles of the patients.202236263534
5779190.9975Development 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