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222300.9974Evaluation 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
217010.9974Drug 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
143620.9974Characterisation of carbapenem-resistant Gram-negative organisms from clinical specimens in Yola, Nigeria. OBJECTIVES: This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria. METHODS: Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes. RESULTS: Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-β-lactamase (bla(NDM)) gene was detected in 26 isolates and Verona integron-encoded metallo-β-lactamase (bla(VIM)) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates. CONCLUSION: While bla(NDM) and bla(VIM) accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates.202031472281
116130.9973Detection of extended-spectrum β-lactamase-producing Escherichia coli genes isolated from cat rectal swabs at Surabaya Veterinary Hospital, Indonesia. BACKGROUND AND AIM: Escherichia coli causes a bacterial illness that frequently affects cats. Diseases caused by E. coli are treated using antibiotics. Because of their proximity to humans, cats possess an extremely high risk of contracting antibiotic resistance genes when their owners touch cat feces containing E. coli that harbor resistance genes. This study was conducted to identify multidrug-resistant E. coli and extended-spectrum β-lactamase (ESBL)-producing genes from cat rectal swabs collected at Surabaya City Veterinary Hospital to determine antibiotic sensitivity. MATERIALS AND METHODS: Samples of cat rectal swabs were cultured in Brilliant Green Bile Lactose Broth medium and then streaked on eosin methylene blue agar medium for bacterial isolation, whereas Gram-staining and IMViC tests were conducted to confirm the identification results. The Kirby-Bauer diffusion test was used to determine antibiotic sensitivity, and the double-disk synergy test was used to determine ESBL-producing bacteria. Molecular detection of the genes TEM and CTX-M was performed using a polymerase chain reaction. RESULTS: Based on morphological culture, Gram-staining, and biochemical testing, the results of sample inspection showed that of the 100 cat rectal swab samples isolated, 71 (71%) were positive for E. coli. Furthermore, 23 E. coli isolates (32.39%) demonstrated the highest resistance to ampicillin. Four isolates were confirmed to be multidurg-resistant and ESBL-producing strains. Molecular examination revealed that three E. coli isolates harbored TEM and CTX-M. CONCLUSION: In conclusion, pet owners must be educated on the use of antibiotics to improve their knowledge about the risks of antibiotic resistance.202337859949
222240.9973Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis. Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens.201627021662
143750.9973Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany. Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM), bla (OXA-40), bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51), bla (IMI), bla (FIM) and bla (DIM). We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM) and bla (OXA-40), while multiplex-2 included bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51) and bla (IMI).Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.202133448924
91360.9973Intestinal carriage of colistin-resistant Enterobacteriaceae at Saint Georges Hospital in Lebanon. OBJECTIVES: The increase in resistance to antibiotics has led to the revival of colistin as the last option for treatment, which automatically led to an increase of colistin-resistant, Gram-negative bacteria. In this study, we report the presence of clinical colistin-resistant Enterobacteriaceae isolated from a Lebanese hospital. METHODS: From 23 rectal swabs, eight colistin-resistant clinical strains (five Escherichia coli, two Enterobacter cloacae, and one Klebsiella pneumoniae) were isolated. Antibiotic susceptibility testing was performed using the disk diffusion method and Etest. The broth microdilution method was used to determine colistin susceptibility. Reverse transcription polymerase chain reaction (RT-PCR), standard PCR and sequencing were used to investigate genes encoding for extended-spectrum β-lactamases, carbapenemases and colistin resistance. Genotyping of these isolates was conducted by multilocus sequence typing (MLST). RESULTS: Results of antibiotic susceptibility testing revealed that all isolates were resistant to colistin. They had MICs for colistin that ranged from 8 to 32 mg/L. Real-time PCR results showed that five strains harboured bla(TEM-1) and one strain harboured bla(TEM-163). Moreover, four strains were positive for bla(CTX-M-15), bla(CTX-M-103) and bla(CTX-M-189), and K. pneumoniae harboured bla(SHV-1). Observed colistin resistance was linked to amino acid substitutions into protein sequences of pmrA/B, phoP/Q, and mgrB. Interestingly, we report here a mutation in the mgrB regulator and pmrA/B, phoP/Q in colistin-resistant E. cloacae and E. coli clinical isolates for the first time in Lebanon. CONCLUSION: This study highlights the presence of colistin-resistant Gram-negative bacteria in a Lebanese hospital, which is worrisome. An urgent strategy needs to be adopted to avoid the spread of such bacteria.202031838239
145970.9973Molecular characterization of carbapenem-resistance in Gram-negative isolates obtained from clinical samples at Jimma Medical Center, Ethiopia. BACKGROUND: In resource-constrained settings, limited antibiotic options make treating carbapenem-resistant bacterial infections difficult for healthcare providers. This study aimed to assess carbapenemase expression in Gram-negative bacteria isolated from clinical samples in Jimma, Ethiopia. METHODS: A cross-sectional study was conducted to assess carbapenemase expression in Gram-negative bacteria isolated from patients attending Jimma Medical Center. Totally, 846 Gram-negative bacteria were isolated and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Phenotypic antibiotic resistance patterns were determined using the Kirby-Bauer disk diffusion method and Etest strips. Extended-spectrum β-lactamase phenotype was determined using MAST disks, and carbapenemases were characterized using multiplex polymerase chain reactions (PCR). RESULTS: Among the isolates, 19% (157/846) showed phenotypic resistance to carbapenem antibiotics. PCR analysis revealed that at least one carbapenemase gene was detected in 69% (107/155) of these strains. The most frequently detected acquired genes were blaNDM in 35% (37/107), blaVIM in 24% (26/107), and blaKPC42 in 13% (14/107) of the isolates. Coexistence of two or more acquired genes was observed in 31% (33/107) of the isolates. The most common coexisting acquired genes were blaNDM + blaOXA-23, detected in 24% (8/33) of these isolates. No carbapenemase-encoding genes could be detected in 31% (48/155) of carbapenem-resistant isolates, with P. aeruginosa accounting for 85% (41/48) thereof. CONCLUSION: This study revealed high and incremental rates of carbapenem-resistant bacteria in clinical samples with various carbapenemase-encoding genes. This imposes a severe challenge to effective patient care in the context of already limited treatment options against Gram-negative bacterial infections in resource-constrained settings.202438328425
91280.9972Carbapenem and colistin-resistant bacteria in North Lebanon: Coexistence of mcr-1 and NDM-4 genes in Escherichia coli. INTRODUCTION: The increasing incidence of infections caused by multidrug-resistant bacteria is considered a global health problem. This study aimed to investigate this resistance in Gram-negative bacteria isolated from patients hospitalized in North-Lebanon. METHODOLOGY: All isolates were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was achieved using disk diffusion, E-test and Broth microdilution methods. Phenotypic detection of carbapenemase was carried out using the CarbaNP test. RT-PCR, standard-PCR and sequencing were performed to detect resistance genes and oprD gene. Conjugal transfer was carried out between our isolates and Escherichia coli J53 to detect the genetic localization of resistance genes. MLST was conducted to determine the genotype of each isolate. RESULTS: Twenty-three carbapenem-resistant Enterobacterales of which eight colistin-resistant Escherichia coli, and Twenty carbapenem-resistant Pseudomonas aeruginosa were isolated. All isolates showed an imipenem MIC greater than 32 mg/mL with MICs for colistin greater than 2 mg/L for E. coli isolates. All the Enterobacterales isolates had at least one carbapenemase-encoding gene, with E. coli isolates coharboring blaNDM-4 and mcr-1 genes. Moreover, 16/20 Pseudomonas aeruginosa harbored the blaVIM-2 gene and 18/20 had mutations in the oprD gene. MLST revealed that the isolates belonged to several clones. CONCLUSIONS: We report here the first description in the world of clinical E. coli isolates coharboring blaNDM-4 and mcr-1 genes, and K. pneumoniae isolates producing NDM-6 and OXA-48 carbapenemases. Also, we describe the emergence of NDM-1-producing E. cloacae in Lebanon. Screening for these isolates is necessary to limit the spread of resistant microorganisms in hospitals.202134343118
211490.9972Clinical, phenotypic, and genotypic characteristics of ESBL-producing Salmonella enterica bloodstream infections from Qatar. BACKGROUND: Resistant Salmonella infections are a major global public health challenge particularly for multidrug-resistant (MDR) isolates manifesting as bloodstream infections (BSIs). OBJECTIVES: To evaluate clinical, phenotypic, and genotypic characteristics of extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica BSIs from Qatar. METHODS: Phenotypic ESBL Salmonella enterica from adult patients presenting with positive BSIs were collected between January 2019 to May 2020. Microbiological identification and characterization were performed using standard methods while genetic characteristics were examined through whole genome sequencing studies. RESULTS: Of 151 episodes of Salmonella enterica BSI, 15 (10%) phenotypic ESBL isolates were collected. Recent travel was recorded in most cases (80%) with recent exposure to antimicrobials (27%). High-level resistance to quinolines, aminoglycosides, and cephalosporins was recorded (80-100%) while meropenem, tigecycline and colistin demonstrated universal susceptibility. Genomic evaluation demonstrated dominance of serotype Salmonella Typhi sequence type 1 (93%) while antimicrobial resistance genes revealed dominance of aminoglycoside resistance (100%), qnrS1 quinolones resistance (80%), bla(CTX-M-15) ESBLs (86.7%), and paucity of AmpC resistance genes (6.7%). CONCLUSIONS: Invasive MDR Salmonella enterica is mainly imported, connected to patients from high prevalent regions with recent travel and antimicrobial use caused by specific resistant clones. In suspected cases of multidrug resistance, carbapenem therapy is recommended.202438742235
1489100.9972Direct detection of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles by multiplex-touchdown PCR assay. Methicillin-resistant staphylococci (MRS) and ESBL(Extended-Spectrum β-Lactamase)-producing bacteria are the most important resistant pathogens in sepsis. In this study, a new multiplex-touchdown PCR method (MT-PCR) was developed to detect rapidly and simultaneously the presence of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles. The technique showed a sensitivity of 10(3 ) CFU ml(-1) for mecA detection and of 10(2)  CFU ml(-1) for other genes, and 100% specificity in the detection of all genes. All genes were detected in the spiked blood culture bottles artificially contaminated with reference strains. Three methicillin-resistant S. aureus (MRSA), two methicillin-resistant S. epidermidis (MRSE) and 32 ESBL-producing bacteria, were isolated from the clinical blood culture specimens in 48 h by standard microbiological procedures. The corresponding genes were detected directly in the three MRSA, two MRSE and 29 ESBL-producing bacteria from the clinical blood culture specimens in 4 h by MT-PCR assay. None of the bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes were detected in three other bottles with ESBL-producing bacteria because of other ESBL genotypes in the pathogens. Likewise, all bottles proven negative by culture remained negative by PCR. The proposed method was rapid, sensitive and specific, and was able to directly detect the genes of MRS and ESBL-producing bacteria from the blood culture bottles. SIGNIFICANCE AND IMPACT OF THE STUDY: Many studies on the development of PCR for the detection of resistance genes have already been published, including multiplex PCR methods. However, cross-amplification reactions can be a major concern in multiplex PCR methods. In this study, we developed a highly sensitive and specific multiplex-touchdown PCR assay for simultaneous detection of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles, cross-amplification was absent and false-positive results were not obtained.201727699804
889110.9972First Known Report of mcr-Harboring Enterobacteriaceae in the Dominican Republic. Colistin is a last-resort antibiotic used to treat infections caused by multidrug-resistant Gram-negative bacteria. People with a history of travel to the Dominican Republic have become sick with pathogenic bacteria carrying the mobile colistin resistance gene, mcr-1, during and after traveling. This investigation aimed to identify mcr genes in Enterobacteriaceae isolated from food animal sources in the Dominican Republic. Three hundred and eleven samples were tested, from which 1354 bacterial isolates were obtained. Real-time PCR tests showed that 70.7% (220 out of 311) of the samples and 3.2% (44 out of 1354) of the isolates tested positive for the mcr gene. All RT-PCR presumptive mcr-positive isolates (n = 44) and a subset (n = 133) of RT-PCR presumptive mcr-negative isolates were subjected to whole-genome sequencing. WGS analysis showed that 39 isolates carried the mcr gene, with 37 confirmed as positive through RT-PCR and two as negative. Further, all of the mcr-positive genomes were identified as Escherichia coli and all contained a IncX4 plasmid replicon. Resistant determinants for other antibiotics important for human health were found in almost all isolates carrying mcr genes.202336982034
1500120.9972Microbiological surveillance of plasmid mediated colistin resistance in human Enterobacteriaceae isolates in Romagna (Northern Italy): August 2016-July 2017. OBJECTIVES: To start a surveillance program to investigate the possible diffusion of mobilized colistin resistance genes in Enterobacteriaceae strains isolated in the Unit of Microbiology of the Great Romagna Hub Laboratory. METHODS: All the colistin resistant Enterobacteriaceae, isolated from August 1st 2016 to July 31st 2017, were prospectively evaluated for mcr-1 and mcr-2. Backdated survey of mcr-3, mcr-4 and mcr-5 was performed on the same group of isolates. Species identification was achieved by Vitek MS and the antibiotic susceptibility testing was performed both with Vitek-2 and Sensititre systems. Colistin resistant isolates were screened by PCR for the presence of the plasmid-mediated colistin resistance genes and amplicons were verified by sequencing. All mcr-1 positive isolates were subjected to MLST analysis. RESULTS: Over the total of 19053 isolates belonging to Enterobacteriaceae, 90 were colistin resistant. The presence of mcr-1 was detected in 26 Escherichia coli. The overall prevalence of mcr-1 was 0.14%. The mcr-1 positive E. coli strains were assigned to 13 distinct sequence types (STs) according to MLST. CONCLUSIONS: The prospective epidemiological survey carried out in our study gave a glimpse of the plasmid-mediated colistin resistance dissemination in Romagna. Since the prevalence rate of carbapenem resistant Enterobacteriaceae (CRE) in some hospital wards in our area is alarming, we underline the importance of a Surveillance Program to monitor the spread of the plasmid-mediated colistin resistance genes into MDR Gram-negative bacteria.201829447913
928130.9972Phenotypic 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
2218140.9971Comparison 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
2217150.9971MALDI-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
915160.9971Detection of Plasmid-Mediated Mobile Colistin Resistance Gene (mcr-1) in Enterobacterales Isolates from a University Hospital. PURPOSE: Colistin represents one of the last treatment options for infections caused by multi-drug resistant (MDR) Enterobacterales. The emergence of a plasmid-mediated mobile colistin resistance-1 (mcr-1) gene has raised serious concerns about its potential dissemination among bacteria. METHODS: In this study, we evaluated the chromogenic medium, CHROMID(®) Colistin Resistance (COLR) agar, for the rapid detection of colistin-resistant Enterobacterales using broth microdilution (BMD) as a reference method. We also attempted to detect mcr-1, -2, -3, -4, and -5 genes, as well as the insertion sequence ISApl1 via polymerase chain reaction (PCR), followed by sequencing of mcr gene(s). RESULTS: Among the 100 studied Enterobacterales isolates, 53% of them were colistin-resistant, with higher rate among Klebsiella pneumoniae (75%) as compared to Escherichia coli (44.4%). The COLR agar showed 83.2% sensitivity and 97.9% specificity for the detection of colistin resistance. Among colistin-resistant isolates, mcr-1 gene was only detected in four (7.5%) E. coli isolates. The ISApl1 was not found among mcr-1 positive isolates. Sequencing of mcr-1 gene revealed nucleotide sequence homogeneity with the wild-type mcr-1 gene in BLAST. CONCLUSION: The COLR agar is a promising phenotypic method for the detection of colistin-resistant Enterobacterales. Multiplex PCR followed by sequencing can be used for mcr genes' detection and characterization.202134408450
2125170.9971Emergence of Carbapenem-Resistant Gram-Negative Isolates in Hospital Settings in Djibouti. Introduction: The antimicrobial resistance (AMR) of bacteria is increasing rapidly against all classes of antibiotics, with the increasing detection of carbapenem-resistant isolates. However, while growing prevalence has been reported around the world, data on the prevalence of carbapenem resistance in developing countries are fairly limited. In this study, we investigated and determined the resistance rate to carbapenems among multidrug-resistant Gram-negative bacteria (MDR-GNB) isolated in Djibouti and characterized their resistance mechanisms. Results: Of the 256 isolates, 235 (91.8%) were identified as Gram-negative bacteria (GNB). Of these GNBs, 225 (95.7%) isolates exhibited a multidrug resistance phenotype, and 20 (8.5%) isolates were resistant to carbapenems, including 13 Escherichia coli, 4 Acinetobacter baumannii, 2 Klebsiella pneumoniae and 1 Proteus mirabilis. The most predominant GNB in this hospital setting were E. coli and K. pneumoniae species. Carbapenemase genes such as bla(OXA-48) and bla(NDM-5) were identified, respectively, in six and four E. coli isolates, whereas the carbapenemase bla(NDM-1) was identified in three E. coli, two K. pneumoniae, one P. mirabilis and one A. baumannii. Moreover, three A. baumannii isolates co-hosted bla(OXA-23) and bla(NDM-1). Materials and Methods: A total of 256 clinical strains collected between 2019 and 2020 were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). Antibiotic susceptibility testing was performed using disk diffusion and E-test methods. Real-time polymerase chain reaction (RT-PCR), standard PCR and sequencing were used to investigate genes encoding for extended-spectrum-β-lactamases, carbapenemases and colistin resistance genes. Conclusions: We report, for the first time, the presence of MDR-GNB clinical isolates and the emergence of carbapenem-resistant isolates in Djibouti. In addition to performing antimicrobial susceptibility testing, we recommend phenotypic and molecular screening to track the spread of carbapenemase genes among clinical GNB isolates.202337508230
2216180.9971Ultrafast 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
1502190.9971Tunisian Multicenter Study on the Prevalence of Colistin Resistance in Clinical Isolates of Gram Negative Bacilli: Emergence of Escherichia coli Harbouring the mcr-1 Gene. BACKGROUND: Actually, no data on the prevalence of plasmid colistin resistance in Tunisia are available among clinical bacteria. OBJECTIVES: This study aimed to investigate the current epidemiology of colistin resistance and the spread of the mcr gene in clinical Gram-negative bacteria (GNB) isolated from six Tunisian university hospitals. METHODS: A total of 836 GNB strains were inoculated on COL-R agar plates with selective screening agar for the isolation of GNB resistant to colistin. For the selected isolates, mcr genes, beta-lactamases associated-resistance genes and molecular characterisation were screened by PCRs and sequencing. RESULTS: Colistin-resistance was detected in 5.02% (42/836) of the isolates and colistin-resistant isolates harboured an ESBL (bla(CTX-M-15)) and/or a carbapenemase (bla(OXA-48), bla(VIM)) encoding gene in 45.2% of the cases. The mcr-1 gene was detected in four E. coli isolates (0.59%) causing urinary tract infections and all these isolates also contained the bla(TEM-1) gene. The bla(CTX-M-15) gene was detected in three isolates that also carried the IncY and IncFIB replicons. The genetic environment surrounding the mcr-carrying plasmid indicated the presence of pap-2 gene upstream mcr-1 resistance marker with unusual missing of ISApl1 insertion sequence. THE CONCLUSIONS: This study reports the first description of the mcr-1 gene among clinical E. coli isolates in Tunisia and provides an incentive to conduct routine colistin susceptibility testing in GNB clinical isolates.202236290048