Detection of clinically important β-lactamases by using PCR. - Related Documents




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171601.0000Detection 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
171710.9999Integrated 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
222820.9999Accurate 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
168430.9999Plasmid-encoded gene duplications of extended-spectrum β-lactamases in clinical bacterial isolates. INTRODUCTION: The emergence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. METHODS: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). RESULTS: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). CONCLUSION: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.202438469349
86640.9998Opening Pandora's box: High-level resistance to antibiotics of last resort in Gram-negative bacteria from Nigeria. OBJECTIVES: The aim of this study was to determine the percentage of antimicrobial-resistant isolates and the associated resistance mechanisms in Gram-negative bacteria from South Western Nigeria. METHODS: A total of 306 non-duplicate unbiased Gram-negative isolates were recovered from patients admitted to three teaching hospitals in South Western Nigeria in 2011 and 2013. Isolates were from clinical samples as well as from stool samples of inpatients without infection to assess antimicrobial resistance patterns in carriage isolates. Antimicrobial susceptibility testing was performed, and PCR and sequencing were used to identify genes encoding various known β-lactamases. Based on phenotypic and genotypic results, 10 isolates representing the diversity of phenotypes present were selected for whole-genome sequencing (WGS). RESULTS: Antimicrobial susceptibility testing revealed the following resistance rates: fluoroquinolones, 78.1%; third-generation cephalosporins, 92.2%; and carbapenems, 52.6%. More resistant isolates were isolated from stools of uninfected patients compared with clinical infection specimens. Klebsiella (10%) and Escherichia coli (7%) isolates produced a carbapenemase. WGS of selected isolates identified the presence of globally disseminated clones. CONCLUSION: This study illustrates a crisis for the use of first-line antimicrobial therapy in Nigerian patients. It is likely that Nigeria is playing a significant role in the spread of antimicrobial resistance owing to its large population with considerable global mobility.202031654790
171350.9998Conjugative plasmidic AmpC detected in Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae human clinical isolates from Portugal. AmpC is a type of β-lactamase enzyme produced by bacteria; these enzymes are classified in Class C and Group 1, and these confer resistance to cephamycin. Enterobacterales producing AmpC are reported worldwide and have great clinical importance due to therapeutic restriction and epidemiological importance once the easy dissemination by plasmidic genes to other bacteria is a real threat. These genes are naturally found in some enterobacteria as Enterobacter cloacae, Morganella morganii, and Citrobacter freundii, but other species have demonstrated similar resistance phenotype of AmpC production. Genes carried in plasmids have been described in these species conferring resistance to cefoxitin and causing therapeutic failure in some bacterial infections. This work detected and described five clinical strains of Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae that presented plasmid ampC (pAmpC) isolated from the north of Portugal collected in 2009. AmpC production was confirmed by inhibition of the enzyme by cloxacillin and boronic acid in agar diffusion tests. Also, PCR (polymerase chain reaction) was performed for the detection of gene universal to AmpC, bla(ampC), and others to AmpC group: bla(ACC), bla(CIT), bla(CMY), bla(DHA), and bla(EBC). The conjugation in liquid medium for 24 h was realized to determine if gene is localized in chromosome or plasmid. The isolates and their conjugants showed phenotypic characteristics and bla(CMY) and bla(CIT) were detected by PCR corroborating the AmpC characteristics observed in these bacteria. Confirmation of transfer of plasmid containing genes encoding AmpC is of high epidemiological relevance to the hospital studied and demonstrated the importance of AmpC surveillance and studies in hospital and community environments in order to choose the appropriate therapy for bacterial infections.202032740783
169260.9998Phenotypic and genotypic detection of antibiotic-resistant bacteria in fresh fruit juices from a public hospital in Rio de Janeiro. Gram-negative bacteria are worrisome because they are becoming resistant to many antibiotic available options, mainly in hospital environment. Several studies have noted the presence of bacteria producing extended-spectrum beta-lactamase, with the presence of antibiotic-resistance genes in fresh vegetables and fruits. This study aimed to detect the presence of phenotypic and genotypic resistance in eight samples of fresh fruit juices served to patients admitted to a hospital in Rio de Janeiro. The growth of microorganisms on MacConkey and XLD agar was carried out to obtain a "pool" of Gram-negative bacteria. The disk diffusion test and the polymerase chain reaction were performed to detect the phenotypic and genotypic resistance of Gram-negative bacteria to the tested antibiotics. The multidrug resistance was detected in all samples and the shv, tem, ctx, tetA, tetB and oxa- 48 genes were found in the samples, including the presence of class 2 and 3 integrons. We can conclude that the selection methodology allows the detection of a greater number of genes and this found warns about the risk of making these foods available to patients in hospitals.202133398401
170070.9998The Prevalence of Multidrug-Resistant Enterobacteriaceae among Neonates in Kuwait. Increasing numbers of neonates with serious bacterial infections, due to resistant bacteria, are associated with considerable morbidity and mortality rates. The aim of this study was to evaluate the prevalence of drug-resistant Enterobacteriaceae in the neonatal population and their mothers in Farwaniya Hospital in Kuwait and to determine the basis of resistance. Rectal screening swabs were taken from 242 mothers and 242 neonates in labor rooms and wards. Identification and sensitivity testing were performed using the VITEK(®) 2 system. Each isolate flagged with any resistance was subjected to the E-test susceptibility method. The detection of resistance genes was performed by PCR, and the Sanger sequencing method was used to identify mutations. Among 168 samples tested by the E-test method, no MDR Enterobacteriaceae were detected among the neonates, while 12 (13.6%) isolates from the mothers' samples were MDR. ESBL, aminoglycosides, fluoroquinolones, and folate pathway inhibitor resistance genes were detected, while beta-lactam-beta-lactamase inhibitor combinations, carbapenems, and tigecycline resistance genes were not. Our results showed that the prevalence of antibiotic resistance in Enterobacteriaceae obtained from neonates in Kuwait is low, and this is encouraging. Furthermore, it is possible to conclude that neonates are acquiring resistance mostly from the environment and after birth but not from their mothers.202337189605
168780.9998Multiple NDM-5-Expressing Escherichia Coli Isolates From an Immunocompromised Pediatric Host. BACKGROUND: Genes conferring carbapenem resistance have disseminated worldwide among Gram-negative bacteria. Here we present longitudinal changes in clinically obtained Escherichia coli isolates from 1 immunocompromised pediatric patient. This report demonstrates potential for antibiotic resistance genes and plasmids to emerge over time in clinical isolates from patients receiving intensive anticancer chemotherapy and broad-spectrum antibiotics. METHODS: Thirty-three isolates obtained over 7 months from 1 patient were included. Clinical data were abstracted from the medical record. For each isolate, studies included phenotypic antibacterial resistance patterns, sequence typing, bacterial isolate sequencing, plasmid identification, and antibiotic resistance gene identification. RESULTS: Sites of isolation included blood, wound culture, and culture for surveillance purposes from the perianal area. Isolates were of 5 sequence types (STs). All were resistant to multiple classes of antibiotics; 23 (69.6%) were phenotypically resistant to all carbapenems. The blaNDM-5 gene was identified in 22 (67%) isolates, all of ST-167 and ST-940, and appeared to coincide with the presence of the IncFII and IncX3 plasmid. CONCLUSIONS: We present unique microbiologic data from 33 multidrug-resistant E. coli isolates obtained over the course of 7 months from an individual patient in the United States. Two E. coli sequence types causing invasive infection in the same patient and harboring the blaNDM-5 gene, encoded on the IncX3 plasmid and the IncFII plasmid, were identified. This study highlights the emergence of multidrug-resistant bacteria on antibiotic therapy and the necessity of adequate neutrophil number and function in the clearance of bacteremia.202032047833
87090.9998Dissemination of multiple carbapenem-resistant clones of Acinetobacter baumannii in the Eastern District of Saudi Arabia. It has previously been shown that carbapenem-resistant Acinetobacter baumannii are frequently detected in Saudi Arabia. The present study aimed to identify the epidemiology and distribution of antibiotic resistance determinants in these bacteria. A total of 83 A. baumannii isolates were typed by pulsed-field gel electrophoresis (PFGE), and screened by PCR for carbapenemase genes and insertion sequences. Antibiotic sensitivity to imipenem, meropenem, tigecycline, and colistin were determined. Eight different PFGE groups were identified, and were spread across multiple hospitals. Many of the PFGE groups contained isolates belonging to World-wide clone 2. Carbapenem resistance or intermediate resistance was detected in 69% of isolates. The bla VIM gene was detected in 94% of isolates, while bla OXA-23-like genes were detected in 58%. The data demonstrate the co-existence and wide distribution of a number of clones of carbapenem-resistant A. baumannii carrying multiple carbapenem-resistance determinants within hospitals in the Eastern Region of Saudi Arabia.201526191044
1710100.9998Carbapenem resistance in bacteria isolated from soil and water environments in Algeria. OBJECTIVES: Recent research has demonstrated that natural populations of bacteria carry large numbers of mobile genetic elements that may harbour antibiotic resistance determinants. This study aimed to investigate carbapenem resistance in Gram-negative bacteria isolated from natural environments in Béjaïa (Algeria) and to determine the horizontal gene transfer potential of a subset of these antibiotic resistance genes (ARGs). METHODS: Antibiotic-resistant bacteria were isolated and the host was identified using MALDI-TOF/MS and 16S rRNA sequencing. ARG carriage was investigated by the double-disk synergy test, metallo-β-lactamase (MBL) production test and PCR screening for carbapenemase genes. Conjugation experiments were performed to determine potential ARG mobility. To identify ARGs, genomic libraries were constructed and functionally screened and inserts were sequenced. RESULTS: A total of 62 antibiotic-resistant strains isolated from soil and water samples were classified as belonging to the Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae and Aeromonadaceae families. Four highly imipenem-resistant (MIC>64μg/mL) and cefotaxime-resistant (MIC>8μg/mL) clinically-relevant strains were selected for further characterisation. All four strains produced extended-spectrum β-lactamases, but MBL production was not confirmed. Imipenem and cefotaxime resistance was transferable to Escherichia coli but was not conferred by bla(AmpC), bla(IMP), bla(NDM), bla(KPC), bla(OXA-48) or bla(GES) genes. Novel putative resistance mechanisms were identified, including a novel DHA β-lactamase conferring clinical resistance to cefotaxime. CONCLUSIONS: The environment is a reservoir of carbapenem-resistant bacteria. Further investigation of the evolution and dissemination of antibiotic resistance in environmental bacteria is required in order to understand and prevent the emergence of resistance in the clinical environment.201830071355
1709110.9998High prevalence of bla(VIM-1) gene in bacteria from Brazilian soil. This study investigated bacteria from soil samples to (i) determine the main bacterial genera and species having resistance to carbapenem and other β-lactams and (ii) establish if the mechanism of resistance was due to the production of metallo-β-lactamases. The isolates were characterized by PCR for metallo-β-lactamases and integrons, by antimicrobial susceptibility testing, and by sequencing. The antimicrobial profile of 40 imipenem-resistant Gram-positive soil isolates from all Brazilian regions demonstrated that 31 (77.5%) of them were multidrug resistant. Among the 40 isolates, 19 presented the bla(VIM) gene and class 1 integrons by PCR. Six of the 19 isolates were identified as Paenibacillus sp., 12 as Bacillus sp., and just 1 was classified as Staphylococcus sp., by sequencing of the 16S rRNA gene. These results suggest that bacteria from soil can act as a source of bla(VIM-1) genes, representing a threat to public health.201627392282
869120.9998The Prevalence of Antibiotic Resistance Phenotypes and Genotypes in Multidrug-Resistant Bacterial Isolates from the Academic Hospital of Jaén, Spain. The heterogenicity of antimicrobial resistance genes described in clinically significant bacterial isolates and their potential role in reducing the efficacy of classically effective antibiotics pose a major challenge for global healthcare, especially in infections caused by Gram-negative bacteria. We analyzed 112 multidrug-resistant (MDR) isolates from clinical samples in order to detect high resistance profiles, both phenotypically and genotypically, among four Gram-negative genera (Acinetobacter, Escherichia, Klebsiella, and Pseudomonas). We found that 9.8% of the total selected isolates were classified as extensively drug-resistant (XDR) (six isolates identified as A. baumannii and five among P. pneumoniae isolates). All other isolates were classified as MDR. Almost 100% of the isolates showed positive results for bla(OXA-23) and bla(NDM-1) genes among the A. baumannii samples, one resistance gene (bla(CTX-M)) among E. coli, and two genetic determinants (bla(CTX-M) and aac(6')-Ib) among Klebsiella. In contrast, P. aeruginosa showed just one high-frequency antibiotic resistance gene (dfrA), which was present in 68.42% of the isolates studied. We also describe positive associations between ampicillin and cefotaxime resistance in A. baumannii and the presence of bla(VEB) and bla(GES) genes, as well as between the aztreonam resistance phenotype and the presence of bla(GES) gene in E. coli. These data may be useful in achieving a better control of infection strategies and antibiotic management in clinical scenarios where these multidrug-resistant Gram-negative pathogens cause higher morbidity and mortality.202438786157
929130.9998Prevalence, antibiotic susceptibility and characterization of antibiotic resistant genes among carbapenem-resistant Gram-negative bacilli and yeast in intestinal flora of cancer patients in North Lebanon. The emergence and spread of carbapenem-resistant bacteria are a significant clinical and public health concern. The aim of the study is to determine the prevalence of intestinal carriage of carbapenem-resistant bacteria and yeasts in cancer patients under chemotherapy. 41 stool samples collected from cancer patients in Nini hospital in Tripoli, North Lebanon have been analyzed. After isolating yeasts and carbapenem-resistant bacteria, a biochemical identification and antimicrobial susceptibility profile were determined. The mechanism of enzymatic carbapenem-resistance was detected by searching for carbapenemases by both Hodge test and PCR assays. The association of several mechanisms of resistance was also searched. 46.3% (19/41) of patients were colonized by yeast. Candida glabrata (6/19) was the major species. The prevalence of carbapenem-resistant bacteria was 24.4% (10/41) including Escherichia coli (5/10), Enterobacter cloacae (1/10), Enterobacter aerogenes (1/10) Edwardsiella hoshinae (1/10) Pantoea agglomerans (1/10) and Pseudomonas stutzeri (1/10). PCR and sequencing of the amplified fragments revealed that Pseudomonas stutzeri (1/1) carried VIM gene and Enterobacter aerogenes (1/1) and E. coli (1/5) carried OXA-48 gene. The other Enterobacteriaceae were resistant to carbapenems by mechanisms other than a carbapenemase including hyperproduction of cephalosporinase (4/10), extended spectrum beta-lactamases (1/10) and both cephalosporinase and extended spectrum beta-lactamases (2/10). High prevalence of intestinal carriage of carbapenem-resistant bacteria and yeasts were detected in cancer patients under chemotherapy. In order to prevent the development of endogenous infection and the dissemination of antimicrobial resistance, an implementation of antibiotic stewardship programs and infection control measures is required in hospitals particularly in the department of chemotherapy.201728216021
1647140.9998Genomic and antimicrobial resistance genes diversity in multidrug-resistant CTX-M-positive isolates of Escherichia coli at a health care facility in Jeddah. BACKGROUND: Whole genome sequencing has revolutionized epidemiological investigations of multidrug-resistant pathogenic bacteria worldwide. Aim of this study was to perform comprehensive characterization of ESBL-positive isolates of Escherichia coli obtained from clinical samples at the King Abdulaziz University Hospital utilizing whole genome sequencing. METHODS: Isolates were identified by MALDI-TOF mass spectrometry. Genome sequencing was performed using a paired-end strategy on the MiSeq platform. RESULTS: Nineteen isolates were clustered into different clades in a phylogenetic tree based on single nucleotide polymorphisms in core genomes. Seventeen sequence types were identified in the extended-spectrum β-lactamase (ESBL)-positive isolates, and 11 subtypes were identified based on distinct types of fimH alleles. Forty-one acquired resistance genes were found in the 19 genomes. The bla(CTX-M-15) gene, which encodes ESBL, was found in 15 isolates and was the most predominant resistance gene. Other antimicrobial resistance genes (ARGs) found in the isolates were associated with resistance to tetracycline (tetA), aminoglycoside [aph(3″)-Ib, and aph(6)-Id], and sulfonamide (sul1, and sul2). Nonsynonymous chromosomal mutations in the housekeeping genes parC and gyrA were commonly found in several genomes. CONCLUSION: Several other ARGs were found in CTX-M-positive E. coli isolates confer resistance to clinically important antibiotics used to treat infections caused by Gram-negative bacteria.202031279801
888150.9998Identification of New Delhi metallo-β-lactamase 1 in Acinetobacter lwoffii of food animal origin. BACKGROUND: To investigate the presence of metallo-β-lactamase (MBL) genes and the genetic environment of the New Delhi metallo-β-lactamase gene bla(NDM-1) in bacteria of food animal origin. METHODOLOGY/PRINCIPAL FINDINGS: Gram-negative bacteria with low susceptibility to imipenem (MIC>8 µg/mL) were isolated from swab samples collected from 15 animal farms and one slaughterhouse in eastern China. These bacteria were selected for phenotypic and molecular detection of known MBL genes and antimicrobial susceptibility testing. For the bla(NDM-1) positive isolate, conjugation and transformation experiments were carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla(NDM-1) genes, and DNA sequencing was performed to determine the sequences of bla(NDM-1) and the flanking genes. In total, nine gram-negative bacteria of four different species presented a MBL phenotype. bla(NDM-1) was identified on a mobile plasmid named pAL-01 in an Acinetobacter lwoffii isolate of chicken origin. Transfer of pAL-01 from this isolate to E. coli J53 and JM109 resulted in resistance to multiple β-lactams. Sequence analysis revealed that the bla(NDM-1) gene is attached to an intact insertion element ISAba125, whose right inverted repeat (IR-R) overlaps with the promoter sequence of bla(NDM-1). Thus, insertion of ISAba125 likely enhances the expression of bla(NDM-1). CONCLUSION: The identification of a bla(NDM-1)- carrying strain of A. lwoffii in chickens suggests the potential for zoonotic transmission of bla(NDM-1) and has important implications for food safety.201222629360
1648160.9998Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance. The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for bla(KPC), bla(OXA), bla(VIM), bla(IMP), bla(MUS), bla(TUS), bla(NDM), and bla(B) genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the bla(MUS-1) gene. WGS identified bla(MUS-1), erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.202438127105
1708170.9998High-level of resistance to β-lactam and presence of β-lactamases encoding genes in Ochrobactrum sp. and Achromobacter sp. isolated from soil. OBJECTIVES: Bacteria belonging to the genera Ochrobactrum and Achromobacter are bacteria considered opportunistic, causing infections mainly in immunocompromised patients. β-lactamases are the main cause of resistance to β-lactam antibiotics. This study aimed to investigate the antimicrobial resistance profile and the presence of β-lactamases encoding genes in Ochrobactrum sp. and Achromobacter sp. isolated from Brazilian soils. METHODS: Soil samples from the five regions of Brazil were collected for the isolation of bacteria, which were identified molecularly and then, the minimum inhibitory concentration and detection of β-lactamases encoding genes were performed. RESULTS: High-level of resistance to β-lactam antibiotics and different β-lactamases encoding genes were found (bla(CTX-M-Gp1), bla(SHV), bla(OXA-1-like) and bla(KPC)), including the first report of the presence of bla(KPC) in bacteria belonging to the genera Ochrobactrum and Achromobacter. CONCLUSION: The results showed that the bacteria from this study, belonging to genera Ochrobactrum and Achromobacter isolated from soil, harbor different β-lactamases encoding genes and can act as a reservoir of these genes.201729111479
1714180.9998Carbapenemase-producing enterobacteriaceae recovered from a Spanish river ecosystem. The increasing resistance to carbapenems is an alarming threat in the fight against multiresistant bacteria. The dissemination properties of antimicrobial resistance genes are supported by their detection in a diverse population of bacteria, including strains isolated from the environment. The objective of this study was to investigate the presence of carbapenemase-producing Enterobacteriaceae (CPE) collected from a river ecosystem in the Barcelona metropolitan area (Spain). Identification of β-lactamases and other resistance determinants was determined as was the antimicrobial susceptibility profile. Moreover, screening of virulence factors, plasmid addiction systems, plasmid partition systems and replicon typing was performed. The results identified 8 isolates belonging to different species (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica). The most prevalent enzyme was KPC-2 (n = 6), followed by VIM-1 (n = 2) and IMI-2 (n = 1), whereas no OXA-48-type was detected. In addition, one strain was positive for both KPC-2 and VIM-1 enzymes. All the carbapenemase-encoding plasmids carried at least one plasmid addiction or partition system, being vagCD and parAB the most frequently detected, respectively. E. coli and K. pneumoniae isolates carried a low number of virulence-associated factors and none of the detected clones has previously been identified in the clinical setting. These findings support the high dissemination potential of the carbapanemase-encoding genes and reinforce the idea that the environment is another reservoir that may play an important role in the capture, selection and dissemination of carbapenem resistance genes.201728380016
2219190.9998Development 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