Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. - Related Documents




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222801.0000Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
222710.9999Prophylactic application of antibiotics selects extended-spectrum β-lactamase and carbapenemases producing Gram-negative bacteria in the oral cavity. Prophylactic administration of broad-spectrum antibiotics in surgery can change the oral microbiome and induce colonization of oral cavity with Gram-negative bacteria including multidrug (MDR) or extensively drug resistant (XDR) organisms which can lead to lower respiratory tract infections. The aim of the study was to analyse the Gram-negative isolates obtained from oral cavity of the mechanically ventilated patients in ICUs, after prophylactic application of antibiotics and their resistance mechanisms and to compare them with the isolates obtained from tracheal aspirates from the same patients. The antibiotic susceptibility was determined by broth dilution method. PCR was applied to detect genes encoding β-lactamases. Marked diversity of Gram-negative bacteria and resistance mechanisms was found. High resistance rates and high rate of bla(CTX-M) and carbapenemase encoding genes (bla(VIM-1) , bla(OXA-48) ) were found among Klebsiella pneumoniae. Pseudomonas aeruginosa was found to harbour bla(VIM) and in one strain bla(PER-1) gene, whereas Acinetobacter baumannii produced OXA-23-like and OXA-24/40-like oxacillinases and was XDR in all except one case. All XDR isolates belong to international clonal lineage II (IC II). The main finding of the study is that the prophlylactic application of antibiotics in surgery intensive care units (ICUs) is associated with the colonization of oral cavity and lower respiratory tract with Gram-negative bacteria. The identity of Gram-negative bacteria in oral cavity reflected those found in endotracheal aspirates leading to conclusion that oral swab as non-invasive specimen can predict the colonization of lower respiratory tract with resistant Gram-negative organisms and the risk for development of ventilator-associated pneumonia.202133896011
223020.9999Rapid detection of gram-negative antimicrobial resistance determinants directly from positive blood culture broths using a multiplex PCR system. Currently available rapid blood culture diagnostics detect few gram-negative resistance determinants, limiting their clinical utility. We prospectively evaluated the prototype BIOFIRE FILMARRAY Antimicrobial Resistance (AMR) Panel, a rapid multiplex PCR test that detects 31 AMR genes, on residual positive blood culture broths from patients with gram-negative bacteremia due to five target organisms at a New York City hospital. Predicted antimicrobial resistance based on the AMR Panel was compared to results from broth microdilution testing of bloodstream isolates recovered in culture. A simulated stewardship study assessed opportunities for the optimization of therapy if the AMR Panel results had been available for patient care in real time. We enrolled 148 patients with gram-negative bacteremia (Escherichia coli, n = 75; Klebsiella pneumoniae, n = 44; Pseudomonas aeruginosa, n = 17; Enterobacter cloacae complex, n = 9; and Acinetobacter baumannii, n = 3). The sensitivity of the AMR Panel for predicting antimicrobial resistance was ≥90% for 10/14 antimicrobial agents in E. coli and for 10/16 agents in K. pneumoniae. Specificity was ≥90% for 15/17 agents in E. coli and for all 16 agents in K. pneumoniae. Performance for other organisms was poor. For E. coli or K. pneumoniae bacteremia, use of the AMR Panel could have led to earlier escalation or de-escalation of β-lactam therapy in a majority of patients compared to what actually occurred. This study demonstrates that a rapid multiplex PCR test with a large menu of AMR genes can be applied to positive blood culture broths to rapidly predict resistance to frontline antimicrobial agents in patients with E. coli or K. pneumoniae bacteremia.IMPORTANCEPatients with gram-negative bacteremia require urgent treatment with antimicrobial agents that are effective against their infecting pathogen. However, conventional laboratory work-up of blood cultures takes days to yield results, and during this time, patients may receive ineffective therapies. We evaluated the prototype BIOFIRE FILMARRAY AMR Panel, an assay that detects 31 genes in gram-negative bacteria that confer resistance to β-lactams, fluoroquinolones, and aminoglycosides in approximately 1 hour, directly from positive blood culture broths, and compared these results to antimicrobial susceptibility testing of isolates recovered in culture. We found that the AMR Panel accurately predicted resistance in Escherichia coli and Klebsiella pneumoniae to most antimicrobials. Moreover, if results from this assay had been used for patient care, there would have been opportunities to optimize antimicrobial prescribing more quickly than using conventional methods. These data demonstrate how novel molecular assays could optimize care for patients with E. coli and K. pneumoniae bacteremia.202541117625
167630.9999Evaluation of carbapenem resistance using phenotypic and genotypic techniques in Enterobacteriaceae isolates. BACKGROUND: Bacterial resistance to antibiotics is increasing worldwide. Antibiotic-resistant strains can lead to serious problems regarding treatment of infection. Carbapenem antibiotics are the final treatment option for infections caused by serious and life-threatening multidrug-resistant gram-negative bacteria. Therefore, an understanding of carbapenem resistance is important for infection control. In the study described herein, the phenotypic and genotypic features of carbapenem-resistant Enterobacteriaceae strains isolated in our hospital were evaluated. METHODS: In total, 43 carbapenem-resistant strains were included in this study. Sensitivity to antibiotics was determined using the VITEK(®)2 system. The modified Hodge test (MHT) and metallo-β-lactamase (MBL) antimicrobial gradient test were performed for phenotypic identification. Resistance genes IMP, VIM, KPC, NDM-1, and OXA-48 were amplified by multiplex PCR. RESULTS: The OXA-48 gene was detected in seven strains, and the NDM-1 gene in one strain. No resistance genes were detected in the remainder of strains. A significant correlation was observed between the MHT test and OXA-48 positivity, and between the MBL antimicrobial gradient test and positivity for resistance genes (p < 0.05). CONCLUSION: The finding of one NDM-1-positive isolate in this study indicates that carbapenem resistance is spreading in Turkey. Carbapenem resistance spreads rapidly and causes challenges in treatment, and results in high mortality/morbidity rates. Therefore, is necessary to determine carbapenem resistance in Enterobacteriaceae isolates and to take essential infection control precautions to avoid spread of this resistance.201526444537
222940.9999A pentaplex real-time PCR assay for rapid identification of major beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 directly from bacteria in blood. Introduction. Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3-5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes (Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia.Aim. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States.Hypothesis/Gap Statement. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40-60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes.Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay.Results. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4-8 c.f.u. ml(-1).Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.202134878374
171650.9999Detection of clinically important β-lactamases by using PCR. Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.202134100944
169960.9999Association between the presence of CRISPR-Cas system genes and antibiotic resistance in Klebsiella pneumoniae isolated from patients admitted in Ahvaz teaching hospitals. BACKGROUND: This study aims to investigate the frequency of cas1 and cas3 and CRISPR1,2,3 genes in Klebsiella pneumoniae isolates, as well as their connection with antibiotic resistance. MATERIALS AND METHODS: 106 K. pneumoniae isolates were identified by biochemical assays and PCR. The susceptibility to antibiotics was determined by Kirby-Bauer disk diffusion method. Screening of ESBLs was undertaken by using double disk diffusion and standard disk diffusion methods. The E-test and mCIM techniques was used to confirm the disc diffusion-based carbapenem resistance profiles. CRISPR-Cas system genes were identified using PCR. RESULTS: ESBL production was found in 19% of isolates. Carbapenemase production was found in 46% of the isolates. Furthermore, the bacteria were classified as multidrug (76%), extensively drug-resistant (4%), or pan-drug-resistant (2%). When CRISPR/Cas systems were present, antibiotic resistance was lower; conversely, when they were absent, resistance was higher. CONCLUSIONS: If the CRISPR/Cas modules aren't present, the bacteria can still acquire foreign DNA, including antibiotic resistance genes. K. pneumoniae isolates with a CRISPR-Cas system were less likely to carry antibiotic-resistance genes than those lacking this defense system.202439375619
222570.9998Evaluation 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
222680.9998Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections. Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients' medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy.202540142509
221890.9998Comparison 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
5040100.9998Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis. BACKGROUND: The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. AIM: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. METHODS: All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. FINDINGS: In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. CONCLUSIONS: The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.202133485969
928110.9998Phenotypic and genotypic characterization of carbapenem encoding genes among carbapenem-resistant Gram-negative bacteria isolated from North Casablanca, Morocco. Carbapenem resistance genes in Gram-negative bacteria (CR-GNB) are a major cause of critical infections and are considered an urgent public health concern. The present study aimed to describe the prevalence of CR-GNB and the dissemination of extended-spectrum beta-lactamase (ESBL) and carbapenemase genes in clinical isolates from Casablanca, Morocco. Firstly, the strains were collected and identified using phenotypic and biochemical methods, then the antibiotic susceptibility was evaluated by the disc diffusion assay to screen isolates resistant to carbapenems. Secondly, three traditional methods, the carbapenem inactivation method, the modified Hodge, and the in-house carba-NP, were performed to predict the carbapenemase production by the included strains. Finally, conventional PCR was utilized to validate and detect the carbapenemase- and ESBL-related genes. Concerning the results, out of the identified 122 strains, 48 were CR isolates, including 30 Klebsiella pneumoniae, 13 Escherichia coli, and 5 Pseudomonas aeruginosa. Furthermore, these strains presented a high level of resistance. Moreover, the prediction of carbapenemase production by the phenotypic methods showed variable results. Also, the PCR analysis revealed a high occurrence of β-lactamase (ESBL and carbapenemase) genes in the included clinical strains, and most strains harbored multiple resistance genes. Our findings suggest that the three existing methods have some limitations, and a validation study is still necessary for the carbapenemase diagnostics.202540857960
2308120.9998Trends of Antibiotic Resistance in Multidrug-Resistant Pathogens Isolated from Blood Cultures in a Four-Year Period. BACKGROUND: Multidrug-resistant organisms cause serious infections with significant morbidity and mortality in the worldwide. These organisms have been identified as urgent and serious threats by CDC. The aim of this study was to determine the prevalence and changes of antibiotic resistance of multidrug-resistant pathogens isolated from blood cultures over a four-year period in a tertiary-care hospital. METHODS: Blood cultures were incubated in a blood culture system. Positive signalling blood cultures were subcultured on 5% sheep-blood agar. Identification of isolated bacteria was performed using conventional or automated identification systems. Antibiotic susceptibility tests were performed by disc diffusion and/or gradient test methods, if necessary, by automated systems. The CLSI guidelines were used for interpretation of antibiotic susceptibility testing of bacteria. RESULTS: The most frequently isolated Gram-negative bacteria was Escherichia coli (33.4%) followed by Klebsiella pneumoniae (21.5%). ESBL positivity was 47% for E. coli, 66% for K. pneumoniae. Among E. coli, K. pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii isolates, carbapenem resistance was 4%, 41%, 37%, and 62%, respectively. Carbapenem resistance of K. pneumoniae isolates has increased from 25% to 57% over the years, and the highest rate (57%) occured during the pandemic period. It is noteworthy that the aminoglycoside resistance in E. coli isolates gradually increased from 2017 to 2021. The rate of methicillin-resistant S. aureus (MRSA) was found to be 35.5%. CONCLUSIONS: Increased carbapenem resistance in K. pneumoniae and A. baumannii isolates is noteworthy, but carbapenem resistance in P. aeruginosa decreased. It is of great importance for each hospital to monitor the increase in resistance in clinically important bacteria, especially isolated from invasive samples, in order to take the necessary precautions in a timely manner. Future studies involving clinical data of patients and bacterial resistance genes are warranted.202337307126
1717130.9998Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).201020007393
2253140.9998Biofilm Formation and Antibiotic Resistance Profiles in Carbapenemase-Producing Gram-Negative Rods-A Comparative Analysis between Screening and Pathological Isolates. (1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes bla(KPC), bla(OXA-48-like), and bla(NDM) were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.202439199988
1822150.9998Carriage of two carbapenem-resistance genes in Pseudomonas aeruginosa isolated from hospital-acquired infections in children from Costa Rica: the importance of local epidemiology. BACKGROUND: The assessment of Hospital-acquired infections due to multidrug-resistant bacteria involves the use of a variety of commercial and laboratory-developed tests to detect antimicrobial resistance genes in bacterial pathogens; however, few are evaluated for use in low- and middle-income countries. METHODS: We used whole-genome sequencing, rapid commercial molecular tests, laboratory-developed tests and routine culture testing. RESULTS: We identified the carriage of the metallo-β-lactamase bla(VIM-2) and bla(IMP-18) alleles in Carbapenem-Resistant Pseudomonas aeruginosa infections among children in Costa Rica. CONCLUSIONS: The bla(IMP-18) allele is not present in the most frequently used commercial tests; thus, it is possible that the circulation of this resistance gene may be underdiagnosed in Costa Rica.202133910633
2217160.9998MALDI-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
1703170.9998Acinetobacter baumannii clinical isolates from outbreaks in Erbil hospitals after the COVID-19 pandemic. INTRODUCTION: Acinetobacter baumannii is endemic in hospital environments, and since the coronavirus disease 2019 (COVID-19) pandemic, multidrug-resistant A. baumannii has become more potent. This potential evolution is driven by the undetectable numbers of gene resistances it has acquired. We evaluated the antibiotic-resistance genes in isolates from patients in Erbil hospitals. METHODOLOGY: This is the first study to demonstrate the antimicrobial resistance epidemic in Erbil, Iraq. A total of 570 patients, including 100 COVID-19 patients were tested. Isolate identification, characterization, antibiotics susceptibility test, polymerase chain reaction (PCR) amplification of the antibiotic resistance genes in both bacterial chromosome and plasmid, 16S-23S rRNA gene intergenic spacer (ITS) sequencing using the Sanger DNA sequencing, and phylogenetic analysis were used in this study. RESULTS: Only 13% of A. baumannii isolates were from COVID-19 patients. All isolates were multi-drug resistant due because of 24 resistance genes located in both the bacterial chromosome or the plasmid. blaTEM gene was detected in the isolates; however, aadB was not detected in the isolated bacteria. New carbapenemase genes were identified by Sanger sequencing and resistance genes were acquired by plasmids. CONCLUSIONS: The study identified metabolic differences in the isolates; although all the strains used the coumarate pathway to survive. Several resistance genes were present in the isolates' plasmids and chromosome. There were no strong biofilm producers. The role of the plasmid in A. baumannii resistance development was described based on the results.202439499748
2249180.9998Tracking 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
1674190.9998Bloodstream infections caused by multidrug-resistant gram-negative bacteria: epidemiological, clinical and microbiological features. BACKGROUND: Bloodstream infections (BSI) are associated with high morbidity and mortality. This scenario worsens with the emergence of drug-resistant pathogens, resulting in infections which are difficult to treat or even untreatable with conventional antimicrobials. The aim of this study is to describe the epidemiological aspects of BSI caused by multiresistant gram-negative bacilli (MDR-GNB). METHODS: We conducted a laboratory-based surveillance for gram-negative bacteremia over a 1-year period. The bacterial isolates were identified by MALDI-TOF/MS and the antimicrobial susceptibility testing was performed by VITEK®2. Resistance genes were identified through PCR assays. RESULTS: Of the 143 patients, 28.7% had infections caused by MDR-GNB. The risk factors for MDR bacteremia were male sex, age ≥ 60, previous antimicrobial use, liver disease and bacteremia caused by K. pneumoniae. K. pneumoniae was the most frequently observed causative agent and had the highest resistance level. Regarding the resistance determinants, SHV, TEM, OXA-1-like and CTX-M-gp1 were predominant enzymatic variants, whereas CTX-M-gp9, CTX-M-gp2, KPC, VIM, GES, OXA-48-like, NDM and OXA-23-like were considered emerging enzymes. CONCLUSIONS: Here we demonstrate that clinically relevant antibiotic resistance genes are prevalent in this setting. We hope our findings support the development of intervention measures by policy makers and healthcare professionals to face antibiotic resistance.201931296179