EXTRACTION - Word Related Documents




#
Rank
Similarity
Title + Abs.
Year
PMID
012345
221600.9952Ultrafast detection of β-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing. Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1 h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.202337850345
516110.9950Genomic analysis of contaminant Stenotrophomonas maltophilia, from placental swab culture, carrying antibiotic resistance: a potential hospital laboratory contaminant. Acute chorioamnionitis has been considered as reflective of amniotic fluid infection. Standard microbiological work ups for causative microorganism of intra-amniotic infection is based on microbial identification. However, frequency of positive placental culture is varied depending on placental sampling techniques, contaminations, methods of microbiologic work ups or comprehensive microbiologic work ups. In this report, we performed a hybrid whole genome sequencing of a proven bacterial contaminant obtained from placental culture in a patient with preterm labor and acute chorioamnionitis. This is to unveil genetic characterization of contaminant Stenotrophomonas maltophilia habouring antibiotic resistance genes. Stenotrophomonas maltiphilia was proven to be bacterial contaminant since Ureaplasma urealyticum was subsequently demonstrated in amniotic fluid by 16 S rRNA gene Sanger sequencing. Cultivation results from other sources were no growth. We identified Stenotrophomonas maltiphilia strain RAOG732 which carried several antibiotic resistance genes, including aminoglycoside, fluoroquiolone and beta-lactam. Biofilm production genes were also identified in this genome. We firstly utilized a hybrid sequencing approach to investigate the genome of S. maltiphilia in the patient with preterm and acute chorioamnionitis, a proven bacterial laboratory contaminant. The analysis provided several antibiotic resistance-associated and genes biofilm-associated genes. The detection of S. maltiphilia raised the awareness of the colonization of biofilm-producing bacteria in hospitals, where surveillance for decontamination is necessary.202540594762
519320.9949Antibiotic resistance genes prediction via whole genome sequence analysis of Stenotrophomonas maltophilia. BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is the first dominant ubiquitous bacterial species identified from the genus Stenotrophomonas in 1943 from a human source. S. maltophilia clinical strains are resistance to several therapies, this study is designed to investigate the whole genome sequence and antimicrobial resistance genes prediction in Stenotrophomonas maltophilia (S. maltophilia) SARC-5 and SARC-6 strains, isolated from the nasopharyngeal samples of an immunocompromised patient. METHODS: These bacterial strains were obtained from Pakistan Institute of Medical Sciences (PIMS) Hospital, Pakistan. The bacterial genome was sequenced using a whole-genome shotgun via a commercial service that used an NGS (Next Generation Sequencing) technology called as Illumina Hiseq 2000 system for genomic sequencing. Moreover, detailed in-silico analyses were done to predict the presence of antibiotic resistance genes in S. maltophilia. RESULTS: Results showed that S. maltophilia is a rare gram negative, rod-shaped, non sporulating bacteria. The genome assembly results in 24 contigs (>500 bp) having a size of 4668,850 bp with 65.8% GC contents. Phylogenetic analysis showed that SARC-5 and SARC-6 were closely related to S. maltophilia B111, S. maltophilia BAB-5317, S. maltophilia AHL, S. maltophilia BAB-5307, S. maltophilia RD-AZPVI_04, S. maltophilia JFZ2, S. maltophilia RD_MAAMIB_06 and lastly with S. maltophilia sp ROi7. Moreover, the whole genome sequence analysis of both SARC-5 and SARC-6 revealed the presence of four resistance genes adeF, qacG, adeF, and smeR. CONCLUSION: Our study confirmed that S. maltophilia SARC-5 and SARC-6 are one of the leading causes of nosocomial infection which carry multiple antibiotic resistance genes.202438128408
223830.9949Rapid detection of carbapenem resistance among gram-negative organisms directly from positive blood culture bottles. BACKGROUND: Carbapenemase producing gram-negative bacteria (GNB) has become a huge problem in majority of tertiary care centers worldwide. They are associated with very high morbidity and mortality rates, especially when they cause invasive infections. Therefore, rapid detection of these organisms is very important for prompt and adequate antibiotic therapy as well as infection control. The aim of this study was rapid detection of carbapenemase genes and thereby likely carbapenem resistance, 24-48 hours in advance, directly from the positive-flagged blood culture bottles using CHROMagar and Xpert® Carba-R. METHODS: Aspirate from positively flagged blood culture bottles was subjected to differential centrifuge. All gram-negative bacilli on gram stain from the deposit were processed in Xpert® Carba-R and inoculated on CHROMagar. The presence of genes and growth on CHROMagar was compared with carbapenem resistance on VITEK-2 Compact. RESULTS: A total of 119 GNB isolates were processed. One or more of the carbapenemase genes were detected in 80 isolates. On comparison with VITEK-2 result, 92 samples showed concordance for carbapenem resistance 48 hours in advance. There was discordance in 21 isolates with 12 major errors and 09 minor errors. The sensitivity of direct Xpert® Carba-R test for rapid detection of carbapenem resistance, 48 hours in advance, was 81.42%. The sensitivity of direct CHROMagar test for accurate detection of carbapenem resistance, 24 hours in advance, was 92.06%. CONCLUSION: The ability to detect carbapenem resistance with very high accuracy, 48 hours in advance, helps in appropriate antibiotic therapy and implementation of effective infection control practices.202337193528
582840.9948Target-enriched sequencing enables accurate identification of bloodstream infections in whole blood. Bloodstream infections are within the top ten causes of death globally, with a mortality rate of up to 70%. Gold standard blood culture testing is time-consuming, resulting in delayed, but accurate, treatment. Molecular methods, such as RT-qPCR, have limited targets in one run. We present a new Ampliseq detection system (ADS) combining target amplification and next-generation sequencing for accurate identification of bacteria, fungi, and antimicrobial resistance determinants directly from blood samples. In this study, we included removal of human genomic DNA during nucleic acid extraction, optimized the target sequence set and drug resistance genes, performed antimicrobial resistance profiling of clinical isolates, and evaluated mock specimens and clinical samples by ADS. ADS successfully identified pathogens at the species-level in 36 h, from nucleic acid extraction to results. Besides pathogen identification, ADS can also present drug resistance profiles. ADS enabled detection of all bacteria and accurate identification of 47 pathogens. In 20 spiked samples and 8 clinical specimens, ADS detected at least 92.81% of reads mapped to pathogens. ADS also showed consistency with the three culture-negative samples, and correctly identified pathogens in four of five culture-positive clinical blood specimens. This Ampliseq-based technology promises broad coverage and accurate pathogen identification, helping clinicians to accurately diagnose and treat bloodstream infections.202234915067
223450.9948Clinical 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
224060.9948Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines. BACKGROUND: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. OBJECTIVES: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. METHODS: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. RESULTS: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common β-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3 h. CONCLUSIONS: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.201930476137
579870.9948Rapid 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
224180.9948Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collections of bacteria. Two multiplex PCR (mPCR) methods were developed to screen large collections of trimethoprim-resistant Escherichia coli isolates for the most prevalent resistance determinants. Five common integron-carried genes (dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17) were selected as PCR targets. Primers and conditions for standard mPCRs and real-time mPCRs were selected and tested. Two protocols using essentially the same primer pairs were established. The standard mPCR protocol also included an internal control targeting the E. coli 16S rRNA gene. Both protocols proved to be sensitive and specific for detection of the five selected genes. Screening of three different collections of clinical urinary and blood isolates (n = 368) with the two multiplex methods revealed that the five dfr genes accounted for 75-86% of trimethoprim resistance. The standard mPCR is useful and accessible for most laboratories, while the real-time mPCR requires additional equipment and expensive reagents, but is very convenient for high-throughput screening of large collections of bacterial isolates.200717725650
221790.9947MALDI-TOF MS based carbapenemase detection from culture isolates and from positive blood culture vials. BACKGROUND: Antibiotic resistance in bacteria leads to massive health problems. Incidence of carbapenem and multidrug resistance in Gram-negative bacteria are increasing globally and turn out to be a very urgent challenge in health care. Resistant bacteria play an important clinical role during hospital outbreaks as well as in sepsis. Rapid diagnostic tests are necessary to provide immediate information for antimicrobial treatment and infection control measures. METHODS: Our mass spectrometry-based assay was validated with 63 carbapenemase-producing Gram-negative bacterial isolates, and 35 carbapenem-resistant Gram-negative species with no carbapenemase production. These were analyzed from solid culture media and positive blood culture vials. After 4 h of incubation the carbapenemase products were analyzed with the MALDI-TOF MS. All the isolates were genotyped for carbapenemase genes by PCR and sequencing. RESULTS: For culture isolates the concordance of hydrolysis assay to genetic results was 98 % for OXA variants, KPC, VIM, IMP, GIM, and NDM. In contrast, only 14 of 29 Acinetobacter baumannii isolates carrying the OXA and NDM genes could be identified from blood culture. However, from blood culture vials our method allowed the detection of carbapenemases in 98 % of Pseudomonas and Enterobacteriaceae isolates harboring different genes. CONCLUSIONS: This MALDI-TOF MS-based assay permitted the detection of carbapenemases either from solid culture media (98-100 %) or blood culture vials (96 %) for all non-A. baumannii isolates within 4 h. In case of A. baumannii isolates the assay was highly sensitive for the detection of carbapenemases directly from solid culture media.201626839024
4939100.9947Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.201727667325
2225110.9947Evaluation of the DNA microarray "AMR Direct Flow Chip Kit" for detection of antimicrobial resistance genes from Gram-positive and Gram-negative bacterial isolated colonies. INTRODUCTION: The AMR Direct Flow Chip assay allows the simultaneous detection of a large variety of antibiotic resistance genetic markers. To assess this kit's performance, we use isolated colonies as starting material. The assay has been approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD) using clinical specimens. METHODS: A total of 210 bacterial isolates harbouring either one or more antimicrobial resistance genes including plasmid-encoded extended-spectrum β-lactamases (SHV, CTX-M) and carbapenemases (GES, SME, KPC, NMC/IMI, SIM, GIM, SPM, NDM, VIM, IMP, and OXA), mecA, vanA and vanB, and 30 controls were included. RESULTS: The assay displayed a sensitivity and specificity of 100% for all target genes included in the array. CONCLUSION: The AMR Direct Flow Chip Kit is an accurate assay for detecting genes which commonly confer resistance to β-lactams and vancomycin from isolated colonies in culture of Gram-positive and Gram-negative bacteria.201930857832
5073120.9947Parallel Detection of the Unamplified Carbapenem Resistance Genes bla(NDM-1) and bla(OXA-1) Using a Plasmonic Nano-Biosensor with a Field-Portable DNA Extraction Method. Antimicrobial resistance (AMR) is a rapidly growing global concern resulting from the overuse of antibiotics in agricultural and clinical settings. The challenge is exacerbated by the lack of rapid surveillance for resistant bacteria in clinical, environmental, and food supply settings. The increasing resistance to carbapenems, an important sub-class of beta-lactam antibiotics, is a major concern in the healthcare community. Carbapenem resistance (CR) has been found in the environment and food supply chain, where it has the potential to spread to pathogens, animals, and humans through direct or indirect contact. Rapid detection for preventative and control measures should be developed. This study utilized a gold nanoparticle-based plasmonic biosensor for the parallel detection of the CR genes bla(NDM-1) and bla(OXA-1). To explore the field portability, DNA was extracted using two methods: a commercial extraction kit and a boiling method. The results were compared between the two methods using a spectrophotometer and a cellphone application for RGB values to quantify the visual results. The results showed that the boiling method of extraction was more effective than extraction with a commercial kit for this analysis. The parallel detection of unamplified genes extracted via the boiling method is novel. When combined with other portable testing equipment, the approach has the potential to be an inexpensive, rapid, and simple on-site CR gene detection protocol.202539997014
2236130.9947Development 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
5122140.9947Clinical long-read metagenomic sequencing of culture-negative infective endocarditis reveals genomic features and antimicrobial resistance. BACKGROUND: Infective endocarditis (IE) poses significant diagnostic challenges, particularly in blood culture-negative cases where fastidious bacteria evade detection. Metagenomic-based nanopore sequencing enables rapid pathogen detection and provides a new approach for the diagnosis of IE. METHOD: Two cases of blood culture-negative infective endocarditis (IE) were analyzed using nanopore sequencing with an in silico host-depletion approach. Complete genome reconstruction and antimicrobial resistance gene annotation were successfully performed. RESULTS: Within an hour of sequencing, EPI2ME classified nanopore reads, identifying Corynebacterium striatum in IE patient 1 and Granulicatella adiacens in IE patient 2. After 18 h, long-read sequencing successfully reconstructed a single circular genome of C. striatum in IE patient 1, whereas short-read sequencing was used to compare but produced fragmented assemblies. Based on these results, long-read sequencing was exclusively used for IE patient 2, allowing for the complete and accurate assembly of G. adiacens, confirming the presence of these bacteria in the clinical samples. In addition to pathogen identification, antimicrobial resistance (AMR) genes were detected in both genomes. Notably, in C. striatum, regions containing a class 1 integron and multiple novel mobile genetic elements (ISCost1, ISCost2, Tn7838 and Tn7839) were identified, collectively harbouring six AMR genes. This is the first report of such elements in C. striatum, highlighting the potential of nanopore long-read sequencing for comprehensive pathogen characterization in IE cases. CONCLUSIONS: This study highlights the effectiveness of host-depleted, long-read nanopore metagenomics for direct pathogen identification and accurate genome reconstruction, including antimicrobial resistance gene detection. The approach enables same-day diagnostic reporting within a matter of hours. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-11741-5.202541087996
2237150.9947Evaluation 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
2223160.9946Evaluation of a new real-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM, and NDM carbapenemases using spiked rectal swabs. To prevent the spread of carbapenemase-producing bacteria, a fast and accurate detection of patients carrying these bacteria is extremely important. The Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands) is a new multiplex real-time PCR assay, which has been developed to detect and differentiate between the most prevalent carbapenemase genes encountered in Enterobacteriaceae (blaKPC, blaOXA-48, blaVIM, and blaNDM) directly from rectal swabs. Evaluation of this assay using 83 non-duplicate isolates demonstrated 100% sensitivity and specificity and the correct identification of the carbapenemase gene(s) present in all carbapenemase-producing isolates. Moreover, the limit of detection (LoD) of the real-time PCR assay in spiked rectal swabs was determined and showed comparable LoDs with the ChromID CARBA agar. With an excellent performance on clinical isolates and spiked rectal swabs, this assay appeared to be an accurate and rapid method to detect blaKPC, blaOXA-48, blaVIM, and blaNDM genes directly from a rectal screening swab.201324135412
2218170.9946Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates. BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla (KPC), bla (VIM), bla (NDM) or bla (OXA) were detected. Both RT-PCR assays detected all bla (KPC), bla (VIM) and bla (NDM) in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.201728693493
2249180.9946Tracking Multidrug Resistance in Gram-Negative Bacteria in Alexandria, Egypt (2020-2023): An Integrated Analysis of Patient Data and Diagnostic Tools. BACKGROUND: The rise in carbapenem-resistant Enterobacteriaceae (CRE) in Egypt, particularly in hospital settings, poses a significant public health challenge. This study aims to develop a combined epidemiological surveillance tool utilizing the Microreact online platform (version 269) and molecular microarray technology to track and analyze carbapenem-resistant Escherichia coli strains in Egypt. The objective is to integrate molecular diagnostics and real-time data visualization to better understand the spread and evolution of multidrug-resistant (MDR) bacteria. METHODS: The study analyzed 43 E. coli isolates collected from Egyptian hospitals between 2020 and 2023. Nanopore sequencing and microarray analysis were used to identify carbapenemase genes and other resistance markers, whereas the VITEK2 system was employed for phenotypic antibiotic susceptibility testing. Microreact was used to visualize epidemiological data, mapping the geographic and temporal distribution of resistant strains. RESULTS: We found that 72.09% of the isolates, predominantly from pediatric patients, carried the blaNDM-5 gene, while other carbapenemase genes, including blaOXA-48 and blaVIM, were also detected. The microarray method demonstrated 92.9% diagnostic sensitivity and 87.7% diagnostic specificity compared to whole-genome sequencing. Phenotypic resistance correlated strongly with next-generation sequencing (NGS) genotypic data, achieving 95.6% sensitivity and 95.2% specificity. CONCLUSIONS: This method establishes the utility of combining microarray technology, NGS and real-time data visualization for the surveillance of carbapenem-resistant Enterobacteriaceae, especially E. coli. The high concordance between genotypic and phenotypic data underscores the potential of DNA microarrays as a cost-effective alternative to whole-genome sequencing, especially in resource-limited settings. This integrated approach can enhance public health responses to MDR bacteria in Egypt.202439766575
2170190.9946Drug resistance in bacteria isolated from patients presenting with wounds at a non-profit Surgical Center in Phnom Penh, Cambodia from 2011-2013. BACKGROUND: Emerging antibiotic resistance amongst clinically significant bacteria is a public health issue of increasing significance worldwide, but it is relatively uncharacterized in Cambodia. In this study we performed standard bacterial cultures on samples from wounds at a Non-Governmental-Organization (NGO) Hospital in Phnom Penh, Cambodia. Testing was performed to elucidate pathogenic bacteria causing wound infections and the antibiotic resistance profiles of bacterial isolates. All testing was performed at the Naval Medical Research Unit, No.2 (NAMRU-2) main laboratory in Phnom Penh, Cambodia. METHODS: Between 2011-2013, a total of 251 specimens were collected from patients at the NGO hospital and analyzed for bacterial infection by standard bacterial cultures techniques. Specimens were all from wounds and anonymous. No specific clinical information accompanied the submitted specimens. Antibiotic susceptibility testing, and phenotypic testing for extended-spectrum beta-lactamase (ESBL) were performed and reported based on CLSI guidelines. Further genetic testing for CTX-M, TEM and SHV ESBLs was accomplished using PCR. RESULTS: One-hundred and seventy-six specimens were positive following bacterial culture (70 %). Staphlycoccus aureus was the most frequently isolated bacteria. Antibiotic drug resistance testing revealed that 52.5 % of Staphlycoccus aureus isolates were oxacillin resistant. For Escherichia coli isolates, 63.9 % were ciprofloxacin and levofloxacin resistant and 96 % were ESBL producers. Resistance to meropenem and imipenem was observed in one of three Acinetobacter spp isolates. CONCLUSIONS: This study is the first of its kind detailing the antibiotic resistance profiles of pathogenic bacteria causing wound infections at a single surgical hospital in Cambodia. The reported findings of this study demonstrate significant antibiotic resistance in bacteria from injured patients and should serve to guide treatment modalities in Cambodia.201528883936