RESUMO
Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.
Assuntos
Antibacterianos , Infecções por Escherichia coli , Antibacterianos/farmacologia , Biologia Computacional/métodos , Farmacorresistência Bacteriana/genética , Escherichia coli , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade MicrobianaRESUMO
This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried blaNDM-1 (n = 11), blaOXA-48 (n = 11), blaNDM-1 + blaOXA-48 (n = 1), blaNDM-1 + blaVIM-1 (n = 1), blaOXA-204 (n = 1), along with blaCTX-M , blaOXA , blaTEM , blaCMY , blaDHA and blaSHV genes variants on conjugative plasmid of IncL/M, IncR, IncFIIK , IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.
Assuntos
Proteínas de Escherichia coli , Infecções por Klebsiella , Pneumonia , Antibacterianos/farmacologia , Proteínas de Bactérias , Carbapenêmicos , Colistina/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/genética , Tunísia , beta-Lactamases/genéticaRESUMO
OBJECTIVE: This 2018 report of Healthcare-Associated Infections Early Warning and Response System (HAI-EWRS) notifications of carbapenemase-producing Enterobacteriaceae (CPE) or glycopeptide-resistant Enterococcus faecium (GRE), and of strains analysed by the National Reference Center for anti-microbial resistance (NRC) aimed to describe the epidemiology of emerging extensively drug-resistant bacteria (eXDR) in France and control measures implemented in hospital settings. PATIENTS AND METHODS: All HAI-EWRS notifications of eXDR received at the national level and all eXDR strains received at the NRC between January 1, 2018 and January 31, 2018 were analysed. Variables analysed were number of cases, number of strains, resistance mechanism, sample type, link with a foreign country, and control measures implemented. RESULTS: In 2018, 1704 CPE notifications and 315 GRE notifications were reported in France, with an increasing trend since 2012 (×6 for CPE, ×3 for GRE), from respectively 364 and 155 hospitals (+66% for CPE, +57% for GRE since 2012). eXDR strains were mainly isolated from rectal screening swabs. Notifications with patients receiving standard precautions were more often associated with outbreaks than notifications with patients receiving contact precautions at admission. NRC received 2674 CPE strains and 775 GRE strains in 2018 (×8.3 and ×2.8 compared with 2012). CONCLUSION: The increasing annual number of eXDR notifications and eXDR strains received by the NRC is multifactorial but reflects a worrying spread of eXDR in France. The number of infections remains low, but this article shows that existing recommendations are not fully implemented.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecção Hospitalar , Infecções por Enterobacteriaceae , Enterococcus faecium , Preparações Farmacêuticas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecções por Enterobacteriaceae/tratamento farmacológico , França/epidemiologia , Humanos , Controle de InfecçõesRESUMO
OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most ß-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.
Assuntos
Shewanella/enzimologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Cromossomos Bacterianos/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Shewanella/efeitos dos fármacos , Shewanella/genética , beta-Lactamases/genética , beta-Lactamas/farmacologiaRESUMO
Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Genoma Bacteriano , Testes de Sensibilidade Microbiana/métodos , Europa (Continente) , InternacionalidadeRESUMO
Acinetobacter baumannii is an emerging threat in healthcare facilities owing to its ability to be multidrug-resistant (MDR) and to be involved in outbreaks. GES-type extended-spectrum ß-lactamases (ESBLs) have been increasingly identified in A. baumannii. In this study, clinical A. baumannii isolates were characterised using standard biochemical methods and antibiotic susceptibility testing. Antibiotic resistance genes were sought by PCR and sequencing. Genetic support was characterised using S1 nuclease pulsed-field gel electrophoresis (PFGE) mapping, conjugation and electroporation assays. The genetic environment was investigated by PCR, and genetic relatedness was investigated by PFGE. Two MDR A. baumannii clinical isolates susceptible only to colistin and rifampicin were isolated from a tracheal aspirate of a 49-year-old woman hospitalised in 2006 at the Military Hospital of Tunis, Tunisia, and from a tracheal aspirate of a 53-year-old man hospitalised in 2010 at the Institut Orthopédique Mohamed El Kassab of Tunis, Tunisia. PCR revealed that the two isolates harboured the acquired carbapenemase blaOXA-23 and ESBL blaGES-11 genes along with chromosomally-encoded blaOXA-51 and blaADC-like genes. PFGE revealed that these A. baumannii isolates were unrelated; nevertheless, plasmid analysis revealed a similar sized plasmid following electrophoresis of the isolates. In addition, A. baumannii CIP70.10 transformants displayed similar resistance patterns. blaGES-11 was integron-borne and the ISAbaI element was identified upstream of blaOXA-23 and blaADC-like. Here we described two unrelated clinical A. baumannii isolates producing GES-11 ESBL and OXA-23 carbapenemase from two Tunisian hospitals. This work further illustrates the emergence of GES-type ß-lactamases in A. baumannii in North Africa as early as 2006.
Assuntos
Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos , Eletroforese em Gel de Campo Pulsado , Feminino , Hospitais , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , TunísiaRESUMO
BACKGROUND: We investigated the molecular mechanism of ß-lactam resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacterial strains isolated in neonatal units of different hospitals in Anatnanarivo, Madagascar. METHODS: Bacteria were identified by standard biochemical methods, disc diffusion antibiograms and Etest. Resistance genes were sought by PCR. Strains were characterized by Rep-PCR (Diversilab), plasmid analysis and rep-typing. RESULTS: From April 2012 to March 2013, 29 ESBL-producing E. cloacae and 15 K. pneumoniae were isolated from blood culture (n = 32) or gastric samples (n = 12) performed at day 0 or 2 from 39/303 newborns suspected of early neonatal infection. These infants were treated with expanded spectrum cephalosporins, due to lack of carbapenems, leading to a high mortality rate (45 %). Isolates recovered were all, but 4, multidrug resistant, particularly to fluoroquinolones (FQ) except for 21 E. cloacae isolates. Isolates produced TEM-1 and CTX-M-15 ß-lactamases and their genes were located on several self-transferable plasmids of variable sizes sizes that could not be linked to a major plasmid incompatibility group. E. cloacae isolates belonged to 6 Rep-types among which two counted for 11 isolates each. The FQ resistant E. cloacae isolates belonged to one clone, whereas the FQ susceptible E. cloacae isolates belonged to four clones. The K. pneumoniae isolates belonged to 9 Rep-types among which one included five isolates. CONCLUSION: This study is the first molecular characterization of ESBL-producing isolates from neonatology units in Madagascar, a country with limited epidemiological data. It revealed an important multi-clonal dissemination of CTX-M-15-producing isolates reflecting both the high community carriage and the very early nosocomial contamination of the neonates.
Assuntos
Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Enterobacter cloacae/genética , Infecções por Enterobacteriaceae/microbiologia , Doenças do Recém-Nascido/microbiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Enterobacter , Enterobacter cloacae/isolamento & purificação , Enterobacter cloacae/metabolismo , Infecções por Enterobacteriaceae/tratamento farmacológico , Feminino , Humanos , Lactente Extremamente Prematuro , Recém-Nascido , Doenças do Recém-Nascido/tratamento farmacológico , Criança Pós-Termo , Recém-Nascido Prematuro , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , Madagáscar , Masculino , Testes de Sensibilidade Microbiana , Plasmídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , beta-Lactamases/metabolismoRESUMO
Escherichia coli is one of the most-studied microorganisms worldwide but its characteristics are continually changing. Extraintestinal E. coli infections, such as urinary tract infections and neonatal sepsis, represent a huge public health problem. They are caused mainly by specialized extraintestinal pathogenic E. coli (ExPEC) strains that can innocuously colonize human hosts but can also cause disease upon entering a normally sterile body site. The virulence capability of such strains is determined by a combination of distinctive accessory traits, called virulence factors, in conjunction with their distinctive phylogenetic background. It is conceivable that by developing interventions against the most successful ExPEC lineages or their key virulence/colonization factors the associated burden of disease and health care costs could foreseeably be reduced in the future. On the other hand, one important problem worldwide is the increase of antimicrobial resistance shown by bacteria. As underscored in the last WHO global report, within a wide range of infectious agents including E. coli, antimicrobial resistance has reached an extremely worrisome situation that 'threatens the achievements of modern medicine'. In the present review, an update of the knowledge about the pathogenicity, antimicrobial resistance and clinical aspects of this 'old friend' was presented.
Assuntos
Infecções por Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Humanos , Sepse , Infecções Urinárias , Fatores de VirulênciaRESUMO
BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are becoming of immediate concern for infection control policies. Prompt detection of CPE on health care setting admission is crucial to halt the spread of an outbreak. We report a cluster of 13 Klebsiella pneumoniae carbapenemase (KPC)-2-producing K pneumoniae cases in a tertiary care hospital.The objective of this study was to identify contributing factors originating the outbreak. METHODS: An outbreak investigation was conducted using descriptive epidemiology, observation of health care practices, and interviews of management staff. A root cause analysis was performed to identify patent and latent failures of infection control measures using the association of litigation and risk management method. RESULTS: The main patent failure was the delay in identifying KPC-2-producing K pneumoniae carriers. Contributing factors were work and environmental factors: understaffing, lack of predefined protocols, staff members' characteristics, and underlying patients' characteristics. Latent failures were as follows: no promotion of the national guidelines for prevention of CPE transmission, no clear procedure for the management of patients hospitalized abroad, no clear initiative for promoting a culture of quality in the hospital, biologic activity recently outsourced to a private laboratory, and poor communication among hospital members. CONCLUSION: Clinical management should be better promoted to control hospital outbreaks and should include team work and safety culture.
Assuntos
Infecção Hospitalar/epidemiologia , Surtos de Doenças , Transmissão de Doença Infecciosa/prevenção & controle , Controle de Infecções/métodos , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/enzimologia , beta-Lactamases/metabolismo , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/transmissão , Humanos , Controle de Infecções/organização & administração , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/prevenção & controle , Infecções por Klebsiella/transmissão , Klebsiella pneumoniae/isolamento & purificação , Fatores de Risco , Centros de Atenção Terciária , Fatores de TempoRESUMO
OBJECTIVES: Klebsiella oxytoca is a member of the family of Enterobacteriaceae and often contains the ß-lactamase blaOXY gene. Although this ß-lactamase does not naturally hydrolyse ceftazidime, this study describes possible in vivo selection of a clinical K. oxytoca isolate showing increased MICs of ceftazidime. METHODS: To reveal the molecular mechanism underlying this unusual resistance phenotype, WGS, cloning, overexpression, MIC and steady-state kinetic studies were performed. RESULTS: A patient was treated for a septic episode with ceftazidime (4 g/day). This therapy was based on earlier culture results in which, amongst others, a K. oxytoca (Velp-1) isolate was identified. After 11 days of treatment, K. oxytoca Velp-2 was isolated from a pus sample drained from the wound. The isolate showed increased resistance to ceftazidime (MIC ≥64 mg/L) compared with the original K. oxytoca isolate (Velp-1). WGS revealed the presence of a novel blaOXY-2 allele, designated blaOXY-2-15, with a two amino acid deletion at Ambler positions 168 and 169 compared with OXY-2-2. Cloning blaOXY-2-15 into Escherichia coli TOP10 resulted in increased MICs of ceftazidime, but reduced MICs of most other ß-lactams compared with OXY-2-2. Steady-state kinetics confirmed the results of the MIC data, showing clearly significant ceftazidime hydrolysis. CONCLUSIONS: This report shows the risk of in vivo selection of ceftazidime-resistant K. oxytoca isolates after prolonged ceftazidime treatment. Furthermore, it is the first known report of a K. oxytoca isolate conferring resistance to ceftazidime by a two amino acid deletion in the omega loop of OXY-2-2.
Assuntos
Antibacterianos/metabolismo , Ceftazidima/metabolismo , Klebsiella oxytoca/efeitos dos fármacos , Klebsiella oxytoca/enzimologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Ceftazidima/farmacologia , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Hidrólise , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella oxytoca/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Seleção Genética , Sepse/tratamento farmacológico , Sepse/microbiologia , Análise de Sequência de DNARESUMO
The Check-MDR Carba test (Check-Points, Wageningen, Netherlands), which is based on specific molecular recognition of blaNDM, blaKPC, blaOXA-48, blaVIM, and blaIMP genes by DNA probe ligation and real-time PCR detection, was evaluated on 183 well-characterized Gram-negative rods. Representatives of the 5 gene families were accurately identified (specificities and sensitivities of 100%) within 4.5 hours. This test may be helpful to differentiate carbapenem resistance mediated by carbapenemases from those involving other mechanisms.
Assuntos
Proteínas de Bactérias/genética , Bioensaio/normas , Bactérias Gram-Negativas/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Primers do DNA/genética , Sondas de DNA/genética , Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , beta-Lactamases/classificação , beta-Lactamases/metabolismoRESUMO
Multidrug-resistant and New Delhi metallo-ß-lactamase 1 (NDM-1) -producing Acinetobacter baumannii are increasingly reported. A collection of five NDM-1-positive A. baumannii isolates recovered in four European countries were analysed. Genotyping was performed by pulsed-field gel electrophoresis, multiplex PCR sequence typing, Diversilab and multilocus sequence typing. Three distinct sequence types were identified. All isolates harboured a chromosomally located bla(NDM-1) gene within a Tn125-like transposon. One isolate co-expressed another unrelated carbapenemase OXA-23. This report constitutes the first epidemiological study of NDM-1-producing A. baumannii from four countries.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/enzimologia , beta-Lactamases/biossíntese , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Europa (Continente) , Humanos , Testes de Sensibilidade Microbiana , Resistência beta-Lactâmica , beta-Lactamases/genéticaRESUMO
Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most ß-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum ß-lactamase (mainly CTX-M) producers in all European countries.
Assuntos
Proteínas de Bactérias/genética , Infecção Hospitalar/transmissão , Infecções por Enterobacteriaceae/transmissão , Enterobacteriaceae/enzimologia , Evolução Molecular , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Carbapenêmicos/farmacologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Prevalência , Fatores de Tempo , beta-Lactamases/biossínteseRESUMO
Extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ss-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ss-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ss-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.
Assuntos
Perfilação da Expressão Gênica , Bactérias Gram-Negativas/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , beta-Lactamases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , beta-Lactamas/farmacologiaRESUMO
OBJECTIVES: International adoption from developing countries has become an increasing phenomenon in recent years. Given the high prevalence of multidrug-resistant (MDR) bacteria in these countries, the adopted children represent a group at risk for both carriage and infection with MDR bacteria. The dynamics of intrafamilial transmission of MDR bacteria after adoption was studied in a prospective study from January 2002 to January 2005. METHODS: Stool samples, taken at the first visit to the outpatient adoption practice and subsequently every month, from the adopted children of an orphanage of Bamako (Mali) and from all the members of their adoptive families were screened for MDR bacteria and bacterial pathogens. Bacteria were characterized by standard biochemical methods, disc diffusion antibiograms, PFGE and plasmid analysis. beta-Lactamase genes were sought by PCR. RESULTS: Over the study period, 52 ESBL-producing Enterobacteriaceae (E-ESBL), with Escherichia coli (56%) being the most prevalent, were isolated from 24/25 adoptees at arrival in France. During follow-up, the transmission of ESBL-producing E. coli and Salmonella enterica Babelsberg between the adoptees and their adoptive family members has clearly been demonstrated for 5/22 families (23%). The mean duration of the carriage for the adopted children was 9 months (1-15 months). CTX-M-15 was the most prevalent resistance gene among the E-ESBLs (93%), while SHV-12 was found among the S. enterica Babelsberg studied. CONCLUSIONS: International travellers, transfer of patients and now adoption may contribute to the global emergence of MDR bacteria. Thus, in addition to the usual screening of adopted children for infectious diseases, additional screening for MDR bacteria should be recommended, at least for children coming from countries with a high prevalence of MDR bacteria.
Assuntos
Adoção , Infecções por Escherichia coli/transmissão , Escherichia coli/enzimologia , Saúde da Família , Infecções por Salmonella/transmissão , Salmonella enterica/enzimologia , beta-Lactamases/biossíntese , Técnicas de Tipagem Bacteriana , Portador Sadio/microbiologia , Portador Sadio/transmissão , Criança , Pré-Escolar , Análise por Conglomerados , Impressões Digitais de DNA , Farmacorresistência Bacteriana Múltipla , Eletroforese em Gel de Campo Pulsado , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Feminino , França , Genótipo , Humanos , Masculino , Mali , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Plasmídeos/análise , Infecções por Salmonella/microbiologia , Salmonella enterica/classificação , Salmonella enterica/genética , Salmonella enterica/isolamento & purificaçãoRESUMO
Emergence and dissemination of carbapenem resistance in the world represent a significant threat for management of hospital-acquired infections. From the early 2000s, Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases (KPC) have initially been reported from the USA and now worldwide, becoming the most important carbapenemase. These KPC producing-bacteria are mostly involved in nosocomial and systemic infections. They are mostly Enterobacteriaceae and more rarely Pseudomonas aeruginosa. KPC beta-lactamases confer decreased susceptibility or resistance to virtually all beta-lactams. Therefore, carbapenems (imipenem, meropenem, ertapenem) may become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are in addition resistant to many other non beta-lactam molecules, leaving only few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. Several phenotypic tests have been proposed, but until now, only molecular methods are reliable techniques for their identification. It is therefore critical to implement efficient infection control measures to detect patients who are colonized or infected with these pathogens in order to limit their spread.
Assuntos
Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Klebsiella pneumoniae/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/epidemiologia , França/epidemiologia , Humanos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana/métodos , Fenótipo , Fatores R/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Especificidade da Espécie , Especificidade por Substrato , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamases/isolamento & purificaçãoRESUMO
Carbapenem-resistant Acinetobacter baumannii (CR-Ab) ranked third, with a frequency of 24.8%, among 202 strains of multidrug-resistant bacteria isolated from clinical samples in the main hospital of New Caledonia in 2004. All CR-Ab isolates were analysed by isoelectric focusing, conjugation, pulsed-field gel electrophoresis and PCR for the presence of carbapenemase genes. Fifty CR-Ab isolates produced carbapenemase OXA-23. The isolates belonged to a single clone presenting several subtypes, suggesting an endemic situation. This study further illustrates the widespread prevalence of carbapenemase OXA-23-producing CR-Ab isolates in the South Pacific.
Assuntos
Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana , Epidemiologia Molecular , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Análise por Conglomerados , Conjugação Genética , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Focalização Isoelétrica , Nova Caledônia/epidemiologia , Reação em Cadeia da Polimerase , beta-Lactamases/genéticaAssuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Resistência beta-Lactâmica , Criança , Feminino , Humanos , Klebsiella pneumoniae/isolamento & purificação , Líbano , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. METHODS AND RESULTS: One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5'ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5' splice site by site-directed mutagenesis. CONCLUSION: These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.