Detection of the common resistance genes in Gram-negative bacteria using gene chip technology. - Related Documents




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223101.0000Detection of the common resistance genes in Gram-negative bacteria using gene chip technology. OBJECTIVE: To design a resistance gene detection chip that could, in parallel, detect common clinical drug resistance genes of Gram-negative bacteria. MATERIALS AND METHODS: Seventy clinically significant Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii) were collected. According to the known resistance gene sequences, we designed and synthesized primers and probes, which were used to prepare resistance gene detection chips, and finally we hybridized and scanned the gene detection chips. RESULTS: The results between the gene chip and polymerase chain reaction (PCR) were compared. The rate was consistently 100% in the eight kinds of resistance genes tested (TEM, SHV, CTX-M, DHA, CIT, VIM, KPC, OXA-23). One strain of Pseudomonas aeruginosa had the IMP, but it was not found by gene chip. CONCLUSION: The design of Gram-negative bacteria-resistant gene detection chip had better application value.201323867670
222810.9998Accurate 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
221820.9998Comparison 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
221930.9998Development and validation of a multiplex TaqMan real-time PCR for rapid detection of genes encoding four types of class D carbapenemase in Acinetobacter baumannii. A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D β-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.201222878252
222740.9997Prophylactic 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
222550.9997Evaluation 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
171660.9997Detection 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
222070.9997Rapid detection and molecular survey of blaVIM, blaIMP and blaNDM genes among clinical isolates of Acinetobacter baumannii using new multiplex real-time PCR and melting curve analysis. BACKGROUND: Acinetobacter baumannii is a cosmopolitan bacterium that is frequently reported from hospitalized patients, especially those patients who admitted in the intensive care unit. Recently, multiplex real-time PCR has been introduced for rapid detection of the resistance genes in clinical isolates of bacteria. The current study aimed to develop and evaluate multiplex real-time PCR to detect common resistance genes among clinical isolates of A. baumannii. RESULTS: Multiplex real-time PCR based on melting curve analysis showed different T(m) corresponding to the amplified fragment consisted of 83.5 °C, 93.3 °C and 89.3 °C for blaIMP, blaVIM and blaNDM, respectively. Results of multiplex real-time PCR showed that the prevalence of blaIMP, blaVIM and blaNDM among the clinical isolates of A. baumannii were 5/128(3.9%), 9/128(7.03%) and 0/128(0%), respectively. Multiplex real-time PCR was able to simultaneously identify the resistance genes, while showed 100% concordance with the results of conventional PCR. CONCLUSIONS: The current study showed that blaVIM, was the most prevalent MBL gene among the clinical isolates of A. baumannii while no amplification of blaNDM was seen. Multiplex real-time PCR can be sensitive and reliable technique for rapid detection of resistance genes in clinical isolates.201931182026
169780.9997Detection of betalactamase producing bacterial genes and their clinical features. The present study was aimed to identify the gene of drug resistance betalactamase producing bacteria and clinical features of the infected patients at Hospital University Kebangsaan Malaysia. Blood samples from the patients were collected, processed and betalactamase producing drug resistance bacteria were identified by antibiotic sensitivity testing. Genes of the drug resistance bacteria were detected and characterized by polymerase chain reaction. A total of 34 isolates of drug resistance Betalactamase producing E. coli and Klebsiella spp. were isolated from 2,502 patients. Most common drug resistance gene TEM was found in 50% of the isolates. 11% was found positive for both TEM and SHV. Next 11% of the isolates expressed only SHV genes. Clinical features of the patients were recorded from where the bacteria isolated. Regarding community affiliations 70.5% of the infected patients were Malay 17.6% were Indian and 11.7% were Chinese. Majority of the patients has an underlying pre-morbid condition as reflected by their diagnosis. Better infection control and hygiene in hospitals, plus controlled and prudent use of antibiotics, is required to minimize the impact of drug resistance betalactamase producing bacteria and the spread of infections.201121913496
223290.9997Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.201020356716
2304100.9997Antimicrobial consumption and resistance in five Gram-negative bacterial species in a hospital from 2003 to 2011. BACKGROUND: The misuse of antimicrobial agents increases drug resistance in bacteria. METHODS: The correlation between antimicrobial agent consumption and related resistance in the Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis was analyzed during the period 2003-2011. RESULTS: Among these five bacteria, overall E. coli and K. pneumoniae were more commonly isolated from bloodstream than the other species. Regarding Enterobacteriaceae, E. coli and K. pneumoniae showed annual increases of resistance to the tested antimicrobial agents; conversely, P. mirabilis exhibited reduced resistance to cefuroxime, ceftriaxone and cefepime. In contrast to the relatively low antimicrobial resistance in P. aeruginosa, A. baumannii revealed high resistance, which was over 85% resistant rate to the tested antimicrobial agents and over 80% carbapenem resistance in 2011. E. coli, K. pneumoniae, and P. mirabilis differed in development of antimicrobial resistance after consumption of the antimicrobial agents. K. pneumoniae developed resistance to all antimicrobial groups, whereas resistance in P. mirabilis was not related to any antimicrobial consumption. P. aeruginosa developed resistance to β-lactam antimicrobials and aminoglycosides, whereas A. baumanii developed resistance to carbapenems after their use. CONCLUSION: The development of antimicrobial resistance was related to antimicrobial agents and bacterial species.201524863496
2308110.9997Trends 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
2318120.9997Distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analysis of integron resistance genes in respiratory tract isolates of uninfected patients. BACKGROUND: We studied the distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analyzed the integron resistance genes in respiratory tract isolates of uninfected patients. METHODS: Retrospective analysis was used to select sputum samples from 400 lung cancer patients after chemotherapy admitted in Fuyang People's Hospital from July 2017 to July 2019. Culture, isolation and identification of strains were conducted in accordance with the national clinical examination operating procedures. RESULTS: A total of 134 strains were identified. In 120 patients with pulmonary infection, 114 strains were cultured. Twenty strains of klebsiella pneumoniae were cultured in 280 patients without pulmonary infection. Among the 134 strains, the detection rate of gram-negative bacteria was 79.10%. The first four strains were Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Haemophilus influenzae. The gram-positive bacteria detection rate was 4.47%, mainly Staphylococcus aureus and Streptococcus. The fungus detection rate was 16.42%. The drug sensitivity results showed that the resistance rate of gram-negative bacillus to penicillin and cephalosporin was higher, and were more sensitive to carbapenem, piperacillin tazobactam and cefoperazone sulbactam. Gram-positive cocci were resistant to penicillin, macrolide and clindamycin, and sensitive to linezolid, vancomycin and rifampicin. All strains of fungal culture were candida albicans, which were sensitive to common antifungal drugs. Among the 20 strains of klebsiella pneumoniae cultured in sputum specimens of non-infected patients with lung cancer undergoing chemotherapy, 2 strains were integron-positive strains, and all of them were class I integrons. CONCLUSIONS: Lung cancer patients after chemotherapy have a high resistance to commonly used antimicrobial drugs, so it is necessary to detect the resistance of pathogenic microorganisms in clinical practice. The strains carried by patients with lung cancer without pulmonary infection during chemotherapy can isolate type I integrons, suggesting that the spread of drug resistance at gene level should be closely detected.202032944333
936130.9997Occurrence and Diversity of Intra- and Interhospital Drug-Resistant and Biofilm-Forming Acinetobacter baumannii and Pseudomonas aeruginosa. Acinetobacter baumannii and Pseudomonas aeruginosa are the most relevant Gram-negative bacteria associated with hospital and opportunistic infections. This study aimed to evaluate the dynamics of drug-resistant A. baumannii and P. aeruginosa and biofilm formers from two public hospitals in northeastern Brazil. One hundred isolates (35 from A. baumannii and 65 from P. aeruginosa) were identified using the automated Vitek(®)2 Compact method (bioMérieux) and confirmed using the MALDI-TOF (MS) mass spectrometry technique. Molecular experiments were performed by polymerase chain reaction (PCR) to detect the frequency of bla(KPC), bla(IMP), bla(VIM), and bla(SHV) genes. The biofilm formation potential was evaluated using crystal violet in Luria Bertani Miller and trypticase soy broth culture media under the following conditions: at standard concentration, one quarter (25%) of the standard concentration and supplemented with 1% glucose. In addition, the genetic diversity of the isolates was verified by the ERIC-PCR technique. Isolates presented distinct resistance profiles with a high level of beta-lactam resistance. The highest index of genes detected was bla(KPC) (60%), followed by bla(SHV) (39%), bla(VIM) (8%), and bla(IMP) (1%). All the isolates were sensitive to the polymyxins tested and formed biofilms at different intensities. Twelve clones of A. baumannii and eight of P. aeruginosa were identified, of which few were indicative of intra- and interhospital dissemination. This study reveals the dispersion dynamics of these isolates in the hospital environment. The results demonstrate the importance of monitoring programs to combat the spread of these pathogens.202031916896
935140.9997Evaluating the Saliva of Burn ICU Patients for Resistant Infections Harbor Metallo-β-Lactamase Genes. Pseudomonas aeruginosa and Acinetobacter baumannii are the bacteria which increasingly account for nosocomial infections. Due to high virulence, the rate of Multi-Drug Resistance (MDR) and limited availability of new agents, these infections create significant clinical burdens, making it important to identify the possible sources of their occurrence. The aim of this study was to assess non-lactose fermenting bacteria and their metallo-β-lactamase (MBLs) genes expression in the Burn Intensive Care Unit (BICU) patients' saliva samples. This cross-sectional study was conducted from 2017 to 2018 on 124 saliva samples of BICU patients. Identified isolates were evaluated for drug susceptibility by disc diffusion method. MBLs production isolates were detected by Modified Hodge test and Imipenem-EDTA Combined disk. MBLs related genes were evaluated by polymerase chain reaction (PCR). A total of 86 Gram negative non-lactose fermenting bacteria (38; A. baumannii) and (48; P. aeruginosa), were detected. All of the A. baumannii isolates were resistant to Carbapenems, while more than 90% of them were sensitive to Colistin. However, the highest sensitivity in P. aeruginosa isolates was related to Carbapenems and Colistin. More than 95% of A. baumannii and 32% of P. aeruginosa were detected MDR. MBLs production was confirmed in 9 (33.33%) P. aeruginosa and 18 (66.67%) A. baumannii isolates. The blaVIM was the most prevalent gene, while this gene was detected in all of MBLs positive strains. This study confirmed the prevalence of carbapenemase producer Gram-negative bacilli in the saliva of BICU patients. The results of the present study provide a new data set about saliva infection source that could lead to the proper antibiotic regimen and better control of drug resistance.202031930340
2328150.9997Detection of Plasmid-Mediated qnr Genes Among the Clinical Quinolone-Resistant Escherichia coli Strains Isolated in Tehran, Iran. BACKGROUND: Escherichia coli is one of the most important bacterial agents to cause urinary tract infections. Inappropriate and unnecessary administration of antibiotics has led to an increase in the appearance of multidrug-resistant E. coli isolates, limiting treatment options. The increase in a number of resistant strains of bacteria is a major concern of health authorities worldwide. OBJECTIVE: The purpose of this study was to determine the presence of the qnr genes among E. coli isolated from UTIs of patients in Baqiyatallah hospital in Tehran province, Iran. METHOD: Clinical urine samples of patients with suspected urinary tract infection were collected by standard methods in sterile disposable containers. After analysis of urine, microscopic observations and culture analysis, the bacterial genome was extracted by boiling method. PCR for detection of qnr genes including qnrA, qnrB and qnrS was done by specific primers, then PCR products were run using gel electrophoresis and visualized by gel documentation system. RESULTS: In the present study among the 95 isolates, 60 strains were resistant to nalidixic acid. PCR showed that 92 strains were positive for qnrS. The qnrA and qnrB genes were not found among the clinical isolates. CONCLUSION: Our finding indicates a high level of resistance against nalidixic acid among E. coli isolates recovered from the patients with UTI. Also, the high frequency of qnrS imposes the importance of survey of molecular and genetic analysis of mechanisms of quinolone resistance in E. coli strains.201830197698
1676160.9997Evaluation 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
1717170.9997Integrated 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
2217180.9997MALDI-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
2303190.9997Patterns of Drug-Resistant Bacteria in a General Hospital, China, 2011-2016. Drug-resistant bacteria has been a threat to public life and property. We described the trends and changes in antibiotic resistance of important pathogens in a general hospital in Zhengzhou, China from 2011 to 2016, to control antimicrobial-resistant bacteria in hospital and provide support to clinicians and decision-making departments. Five dominant bacteria were enrolled based on the data from the general hospital during 6 years. The results of antimicrobial susceptibility testing were interpreted according to Clinical and Laboratory Standards Institute (CLSI). From 2011 to 2016, a total of 19,260 strains of bacteria were isolated, of which Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii accounted for 51.98%. The resistance rate of K. pneumoniae and E. coli to carbapenem was less than 15%, but resistance of K. pneumoniae to carbapenems increased with time and resistance of E. coli to meropenem increased. The rate of extended-spectrum beta-lactamase (ESBL) production among K. pneumoniae and E. coli was decreasing. For most antibiotics, the resistance rate of ESBL-positive isolates was higher than that of ESBL-negative isolates, excluding carbapenems and cefoxitin. For S. aureus, the rate of methicillin-resistant S. aureus (MRSA) was stable. Resistance of S. aureus to mostly antibiotics decreased with time. Besides polymyxin B, P. aeruginosa and A. baumannii showed high resistance to other antibiotics. For A. baumannii, the resistance rate to mostly antibiotics was increasing. The bacteria showed high levels of resistance and multiple drug resistance. Continuous surveillance and optimizing the use of antibiotics are essential. Drug-resistant bacteria has been a threat to public life and property. We described the trends and changes in antibiotic resistance of important pathogens in a general hospital in Zhengzhou, China from 2011 to 2016, to control antimicrobial-resistant bacteria in hospital and provide support to clinicians and decision-making departments. Five dominant bacteria were enrolled based on the data from the general hospital during 6 years. The results of antimicrobial susceptibility testing were interpreted according to Clinical and Laboratory Standards Institute (CLSI). From 2011 to 2016, a total of 19,260 strains of bacteria were isolated, of which Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii accounted for 51.98%. The resistance rate of K. pneumoniae and E. coli to carbapenem was less than 15%, but resistance of K. pneumoniae to carbapenems increased with time and resistance of E. coli to meropenem increased. The rate of extended-spectrum beta-lactamase (ESBL) production among K. pneumoniae and E. coli was decreasing. For most antibiotics, the resistance rate of ESBL-positive isolates was higher than that of ESBL-negative isolates, excluding carbapenems and cefoxitin. For S. aureus, the rate of methicillin-resistant S. aureus (MRSA) was stable. Resistance of S. aureus to mostly antibiotics decreased with time. Besides polymyxin B, P. aeruginosa and A. baumannii showed high resistance to other antibiotics. For A. baumannii, the resistance rate to mostly antibiotics was increasing. The bacteria showed high levels of resistance and multiple drug resistance. Continuous surveillance and optimizing the use of antibiotics are essential.201931250593