RESUMO
Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 ß-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD600) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 ß-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 ß-lactamase.
Assuntos
Bacteriófagos , Infecções por Klebsiella , Antibacterianos/farmacologia , Humanos , Imipenem/farmacologia , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Mitomicina/farmacologia , beta-Lactamases/genéticaRESUMO
BACKGROUND: Spain is one of the European countries most affected by the COVID-19 pandemic. Serological surveys are a valuable tool to assess the extent of the epidemic, given the existence of asymptomatic cases and little access to diagnostic tests. This nationwide population-based study aims to estimate the seroprevalence of SARS-CoV-2 infection in Spain at national and regional level. METHODS: 35â883 households were selected from municipal rolls using two-stage random sampling stratified by province and municipality size, with all residents invited to participate. From April 27 to May 11, 2020, 61â075 participants (75·1% of all contacted individuals within selected households) answered a questionnaire on history of symptoms compatible with COVID-19 and risk factors, received a point-of-care antibody test, and, if agreed, donated a blood sample for additional testing with a chemiluminescent microparticle immunoassay. Prevalences of IgG antibodies were adjusted using sampling weights and post-stratification to allow for differences in non-response rates based on age group, sex, and census-tract income. Using results for both tests, we calculated a seroprevalence range maximising either specificity (positive for both tests) or sensitivity (positive for either test). FINDINGS: Seroprevalence was 5·0% (95% CI 4·7-5·4) by the point-of-care test and 4·6% (4·3-5·0) by immunoassay, with a specificity-sensitivity range of 3·7% (3·3-4·0; both tests positive) to 6·2% (5·8-6·6; either test positive), with no differences by sex and lower seroprevalence in children younger than 10 years (<3·1% by the point-of-care test). There was substantial geographical variability, with higher prevalence around Madrid (>10%) and lower in coastal areas (<3%). Seroprevalence among 195 participants with positive PCR more than 14 days before the study visit ranged from 87·6% (81·1-92·1; both tests positive) to 91·8% (86·3-95·3; either test positive). In 7273 individuals with anosmia or at least three symptoms, seroprevalence ranged from 15·3% (13·8-16·8) to 19·3% (17·7-21·0). Around a third of seropositive participants were asymptomatic, ranging from 21·9% (19·1-24·9) to 35·8% (33·1-38·5). Only 19·5% (16·3-23·2) of symptomatic participants who were seropositive by both the point-of-care test and immunoassay reported a previous PCR test. INTERPRETATION: The majority of the Spanish population is seronegative to SARS-CoV-2 infection, even in hotspot areas. Most PCR-confirmed cases have detectable antibodies, but a substantial proportion of people with symptoms compatible with COVID-19 did not have a PCR test and at least a third of infections determined by serology were asymptomatic. These results emphasise the need for maintaining public health measures to avoid a new epidemic wave. FUNDING: Spanish Ministry of Health, Institute of Health Carlos III, and Spanish National Health System.
Assuntos
Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , COVID-19 , Criança , Pré-Escolar , Feminino , Humanos , Imunoensaio , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Pandemias , Testes Imediatos , Prevalência , Fatores de Risco , SARS-CoV-2 , Estudos Soroepidemiológicos , Espanha/epidemiologia , Adulto JovemRESUMO
We studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniae isolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group (P < 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Cefotaxima/farmacologia , Farmacorresistência Bacteriana/genética , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , beta-Lactamases/genéticaRESUMO
OBJECTIVES: There is little information about carbapenemase-producing (CP) Citrobacter spp. We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). METHODS: In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. RESULTS: Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM-2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. CONCLUSIONS: We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including co-production of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications.
Assuntos
Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Citrobacter/enzimologia , Citrobacter/genética , Infecções por Enterobacteriaceae/microbiologia , Variação Genética , beta-Lactamases/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/classificação , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Citrobacter/classificação , Citrobacter/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Infecções por Enterobacteriaceae/epidemiologia , Genótipo , Humanos , Epidemiologia Molecular , Tipagem Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Espanha/epidemiologiaRESUMO
The presence of colonised patients is one of the main routes for the spread of multiresistant bacteria, and its containment is a clinical and public health priority. Surveillance studies are essential for early detection of colonisation by these bacteria. This article discusses the different microbiological methods, both based on culturing and molecular methods, for detection of carriers of multiresistant bacteria. Those species with a high clinical/epidemiological impact or generating therapeutic difficulties are included: Methicillin-resistant Staphylococcus aureus, Enterococcus spp. resistant to glycopeptides, enterobacteriaceae producing extended spectrum ß-lactamases and plasmid-mediated AmpC, carbapenemases producing enterobacteriaceae, Acinetobacter baumannii and multiresistant Pseudomonas aeruginosa. The information in this document should be considered as a structure matrix to be tailored to the specific needs of each centre.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Técnicas Bacteriológicas , Portador Sadio/microbiologia , Farmacorresistência Bacteriana Múltipla , Bactérias/efeitos dos fármacos , Infecções Bacterianas/transmissão , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Humanos , Técnicas de Diagnóstico Molecular , Vigilância da População , Manejo de EspécimesRESUMO
BACKGROUND: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC ß-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. METHODS: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC ß-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. RESULTS: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. CONCLUSION: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC ß-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/efeitos dos fármacos , Fluoroquinolonas/farmacologia , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Genes Bacterianos , Humanos , Ácido Nalidíxico/farmacologia , Fatores R/genética , Espanha/epidemiologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genéticaRESUMO
We found a low prevalence (0.6%) of carbapenemase-producingEnterobacteriaceae(CPE) in fecal microbiota of companion dogs. A single VIM-1-producingKlebsiella pneumoniaeisolate belonging to sequence type (ST) 2090 was detected.blaVIM-1was carried on a class 1 integron and an untypeable â¼48-kb plasmid. Emergence and spread of CPE in this group of animals may be a threat to public health in human and veterinary medicine. This finding supports the need for active surveillance studies in companion animals that live close to humans, as interspecies transmission may occur within the same household.
Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Plasmídeos/metabolismo , Animais , Cães , Monitoramento Epidemiológico , Fezes/microbiologia , Expressão Gênica , Humanos , Integrons , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/transmissão , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Animais de Estimação , Plasmídeos/química , Prevalência , Espanha/epidemiologia , beta-Lactamases/genéticaRESUMO
A rapid and sensitive (100%) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect OXA-48-type producers, using 161 previously characterized clinical isolates. Ertapenem was monitored to detect carbapenem resistance, and temocillin was included in the assay as a marker for OXA-48-producers. Structural analysis of temocillin is described. Data are obtained within 60 min.
Assuntos
Técnicas de Tipagem Bacteriana , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Ertapenem , Humanos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , beta-Lactamas/farmacologiaRESUMO
OBJECTIVES: The objective of this study was to analyse the microbiological traits and the population structure of carbapenemase-producing (CP) Escherichia coli isolates collected in Spain between 2012 and 2014. METHODS: Two-hundred-and-thirty-nine E. coli isolates non-susceptible to carbapenems were studied. The carbapenemase genes and the phylogenetic groups were characterized using PCR. MLST was carried out using the typing schemes of the University of Warwick and the Institut Pasteur. The diversity of the population structure was estimated by calculating a simple diversity index (SDI). RESULTS: One-hundred-and-twenty-one isolates (50.6%) produced carbapenemases, of which 87 (71.9%) were OXA-48, 27 (22.3%) were VIM-1, 4 (3.3%) were KPC-2, 2 (1.7%) were NDM and 1 (0.8%) was IMP-22; 4 isolates were collected in 2012, 40 in 2013 and 77 in 2014. Ertapenem was more sensitive than imipenem or meropenem for screening for OXA-48-producing E. coli. Using the Warwick typing scheme, 59 different STs were identified, the most prevalent being ST131 (16.5%). The population diversity was higher among VIM-1-producing isolates (SDIâ=â81.5%) than among OXA-48-producing isolates (SDIâ=â44.8%). The Pasteur scheme had a higher discrimination capability (SDIâ=â55.4%) than the Warwick scheme (SDIâ=â48.8%). CONCLUSIONS: A progressive increase in the prevalence of CP E. coli was observed, mainly due to the dissemination of OXA-48 producers. The most sensitive method for detecting decreased susceptibility of CP E. coli to carbapenems was disc diffusion with ertapenem using the EUCAST screening cut-offs. The spread of CP E. coli was due to a polyclonal population. The Pasteur scheme showed the highest discrimination power. Surveillance is crucial for the early detection of CP E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Variação Genética , Filogenia , beta-Lactamases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Ertapenem , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Prevalência , Espanha/epidemiologia , beta-Lactamases/genética , beta-Lactamas/farmacologiaRESUMO
OBJECTIVES: We analysed the microbiological traits and population structure of KPC-producing Enterobacteriaceae isolates collected in Spain between 2012 and 2014. We also performed a comparative WGS analysis of the three major KPC-producing Klebsiella pneumoniae clones detected. METHODS: Carbapenemase and ESBL genes were sequenced. The Institut Pasteur MLST scheme was used. WGS data were used to construct phylogenetic trees, to identify the determinants of resistance and to de novo assemble the genome of one representative isolate of each of the three major K. pneumoniae clones. RESULTS: Of the 2443 carbapenemase-producing Enterobacteriaceae isolates identified during the study period, 111 (4.5%) produced KPC. Of these, 81 (73.0%) were K. pneumoniae and 13 (11.7%) were Enterobacter cloacae. Three major epidemic clones of K. pneumoniae were identified: ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3. ST11/KPC-2 differed from ST101/KPC-2 and ST512/KPC-3 by 27â819 and 6924 SNPs, respectively. ST101/KPC-2 differed from ST512/KPC-3 by 28â345 SNPs. Nine acquired resistance genes were found in ST11/KPC-2, 11 in ST512/KPC-3 and 13 in ST101/KPC-2. ST101/KPC-2 had the highest number of virulence genes (20). An 11 bp deletion at the end of the mgrB sequence was the cause of colistin resistance in ST512/KPC-3. CONCLUSIONS: KPC-producing Enterobacteriaceae are increasing in Spain. Most KPC-producing K. pneumoniae isolates belonged to only five clones: ST11 and ST512 caused interregional spread, ST101 caused regional spread and ST1961 and ST678 produced independent hospital outbreaks. ST101/KPC-2 had the highest number of resistance and virulence genes. ST101/KPC-2 and ST512/KPC-3 were recently implicated in the spread of KPC in Italy.
Assuntos
Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Genoma Bacteriano , Genótipo , Análise de Sequência de DNA , beta-Lactamases/genética , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Humanos , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Prevalência , Espanha/epidemiologiaRESUMO
OBJECTIVES: The global emergence of OXA-48-producing Klebsiella pneumoniae clones is a significant threat to public health. We used WGS and phylogenetic analysis of Spanish isolates to investigate the population structure of blaOXA-48-like-expressing K. pneumoniae ST11 and ST405 and to determine the distribution of resistance genes and plasmids encoding blaOXA-48-like carbapenemases. METHODS: SNPs identified in whole-genome sequences were used to reconstruct phylogenetic trees, identify resistance determinants and de novo assemble the genomes of 105 blaOXA-48-like-expressing K. pneumoniae isolates. RESULTS: Genome variation was generally lower in outbreak-associated isolates compared with those associated with sporadic infections. The relatively limited variation observed within the outbreak-associated isolates was on average 7-10 SNPs per outbreak. Of 24 isolates from suspected sporadic infections, 7 were very closely related to isolates causing hospital outbreaks and 17 were more diverse and therefore probably true sporadic cases. On average, 14 resistance genes were identified per isolate. The 17 ST405 isolates from sporadic cases of infection had four distinct resistance gene profiles, while the resistance gene profile differed in all ST11 isolates from sporadic cases. Sequence analysis of 94 IncL/M plasmids carrying blaOXA-48-like genes revealed an average of two SNP differences, indicating a conserved plasmid clade. CONCLUSIONS: Whole-genome sequence analysis enabled the discrimination of outbreak and sporadic isolates. Significant inter-regional spread within Spain of highly related isolates was evident for both ST11 and ST405 K. pneumoniae. IncL/M plasmids carrying blaOXA-48-like carbapenemase genes were highly conserved geographically and across the outbreaks, sporadic cases and clones.
Assuntos
Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Filogenia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Infecção Hospitalar , DNA Bacteriano/genética , Surtos de Doenças , Variação Genética , Humanos , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , Epidemiologia Molecular , Plasmídeos/genética , Espanha/epidemiologia , beta-Lactamases/biossíntese , beta-Lactamases/genéticaRESUMO
The aim of this study was to determine the impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceae using the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined for Klebsiella pneumoniae and Escherichia coli. A total of 702 Enterobacteriaceae isolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae (74.4%), Enterobacter cloacae (10.3%), and E. coli (8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 for K. pneumoniae and ST131 for E. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. For K. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producing K. pneumoniae (e.g., ST11 and ST405). The dissemination of OXA-48-producing E. coli is a new finding of public health concern. According to the susceptibilities determined in vitro, most of the CPE (94.5%) had three or more options for antibiotic treatment.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Tienamicinas/farmacologia , beta-Lactamases/metabolismo , Idoso , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Meropeném , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Estudos Prospectivos , EspanhaRESUMO
OBJECTIVES: There are different methodological recommendations for in vitro testing of the co-amoxiclav combination. Performance of co-amoxiclav MIC testing for Escherichia coli by the standard ISO microdilution method (ISO 20776-1) was compared using EUCAST (fixed 2 mg/L clavulanate concentration) and CLSI (2â:â1 ratio) interpretive criteria. METHODS: MICs were determined by broth microdilution using a 2â:â1 ratio and fixed clavulanate concentrations (2 and 4 mg/L) for 160 clinical E. coli isolates with characterized resistance mechanisms. Essential agreements, categorical agreements and relative errors were determined. RESULTS: For all isolates, essential agreement between microdilution using 2 mg/L clavulanate and a 2â:â1 ratio was 25.6%. For ESBL-producing isolates, considering EUCAST breakpoints, 55% of isolates tested with 2 mg/L clavulanate were classified as resistant; conversely, 95% of isolates tested with 4 mg/L clavulanate were susceptible. When using CLSI breakpoints and a 2â:â1 ratio, 90% of isolates were susceptible and 10% were intermediate. CONCLUSIONS: Variation in the clavulanate concentration gave different susceptibility testing results, particularly among ESBL-producing E. coli isolates. The in vitro concentration of clavulanate that better correlates with clinical outcome is still under debate and should be established.
Assuntos
Combinação Amoxicilina e Clavulanato de Potássio/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , FenótipoRESUMO
Multi-drug resistance in bacterial pathogens increases morbidity and mortality in infected patients and it is a threat to public health concern by their high capacity to spread. For both reasons, the rapid detection of multi-drug resistant bacteria is critical. Standard microbiological procedures require 48-72 h to provide the antimicrobial susceptibility results, thus there is emerging interest in the development of rapid detection techniques. In recent years, the use of selective and differential culture-based methods has widely spread. However, the capacity for detecting antibiotic resistance genes and their low turnaround times has made molecular methods a reference for diagnosis of multidrug resistance. This review focusses on the molecular methods for detecting some mechanisms of antibiotic resistance with a high clinical and epidemiological impact: a) Enzymatic resistance to broad spectrum ß-lactam antibiotics in Enterobacteriaceae, mainly extended spectrum ß-lactamases (ESBL) and carbapenemases; and b) methicillin resistance in Staphylococcus aureus.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/genética , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Enterobacteriaceae/efeitos dos fármacos , Humanos , Resistência a Meticilina/genética , Reação em Cadeia da Polimerase/métodos , Especificidade por Substrato , Resistência beta-Lactâmica/genética , beta-Lactamases/genéticaRESUMO
In a previous prospective multicenter study in Spain, we found that OXA-1 and inhibitor-resistant TEM (IRT) ß-lactamases constitute the most common plasmid-borne mechanisms of genuine amoxicillin-clavulanate (AMC) resistance in Escherichia coli. In the present study, we investigated the population structure and virulence traits of clinical AMC-resistant E. coli strains expressing OXA-1 or IRT and compared these traits to those in a control group of clinical AMC-susceptible E. coli isolates. All OXA-1-producing (n = 67) and IRT-producing (n = 45) isolates were matched by geographical and temporal origin to the AMC-susceptible control set (n = 56). We performed multilocus sequence typing and phylogenetic group characterization for each isolate and then studied the isolates for the presence of 49 virulence factors (VFs) by PCR and sequencing. The most prevalent clone detected was distinct for each group: group C isolates of sequence type (ST) 88 (C/ST88) were the most common in OXA-1 producers, B2/ST131 isolates were the most common in IRT producers, and B2/ST73 isolates were the most common in AMC-susceptible isolates. The median numbers of isolates per ST were 3.72 in OXA-1 producers, 2.04 in IRT producers, and 1.69 in AMC-susceptible isolates; the proportions of STs represented by one unique isolate in each group were 19.4%, 31.1%, and 48.2%, respectively. The sum of all VFs detected, calculated as a virulence score, was significantly higher in AMC-susceptible isolates than OXA-1 and IRT producers (means, 12.5 versus 8.3 and 8.2, respectively). Our findings suggest that IRT- and OXA-1-producing E. coli isolates resistant to AMC have a different and less diverse population structure than AMC-susceptible clinical E. coli isolates. The AMC-susceptible population also contains more VFs than AMC-resistant isolates.
Assuntos
Combinação Amoxicilina e Clavulanato de Potássio/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/biossíntese , beta-Lactamases/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/genética , beta-Lactamases/genéticaRESUMO
OBJECTIVES: The epidemiology of invasive Haemophilus influenzae has changed in recent years. ß-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to ß-lactam antibiotics indicated in the treatment of invasive infections. METHODS: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven ß-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins. RESULTS: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to ß-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to ß-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively. CONCLUSIONS: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of ß-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.
Assuntos
Resistência a Ampicilina , Antibacterianos/farmacologia , Cefotaxima/farmacologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/efeitos dos fármacos , Imipenem/farmacologia , Adulto , Criança , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Infecções por Haemophilus/epidemiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Mutação de Sentido Incorreto , Proteínas de Ligação às Penicilinas/genética , Espanha/epidemiologia , beta-Lactamases/genéticaRESUMO
OBJECTIVES: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to ß-lactams. METHODS: A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. RESULTS: In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to ß-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by ß-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). CONCLUSIONS: These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of ß-lactam resistance mechanisms, particularly PBP3 mutations.
Assuntos
Biofilmes/crescimento & desenvolvimento , Genes Bacterianos , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/fisiologia , Otite Média/microbiologia , Resistência beta-Lactâmica , Pré-Escolar , Infecções por Haemophilus/epidemiologia , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Otite Média/epidemiologia , Recidiva , Falha de TratamentoRESUMO
In recent years, Enterobacteriaceae isolates have increased their potential to become highly drug resistant by acquiring resistance to carbapenems, primarily due to the production of acquired carbapenemases. The carbapenemases detected in Enterobacteriaceae are largely of the KPC, VIM, NDM, IMP and OXA-48 types. Although the epidemiological origin and geographic distribution of carbapenemases are clearly different, they all first appeared in the late 20th Century. Only a decade later, these enzymes have already become established and have expanded globally. An important epidemiological change has occurred in Spain in recent years, characterized by a rapid increase in the number of cases of carbapenemase-producing Enterobacteriaceae (CPE), causing both nosocomial outbreaks and single infections. The impact of CPE in Spain is primarily due to OXA-48-producing and VIM-1-producing Klebsiella pneumoniae isolates, although other species such as Escherichia coli and Enterobacter cloacae are also increasing. The emergence of CPE as a cause of community-onset infections is a matter of great concern. Taking into account recent experience, and considering the fact that increasing numbers of patients are becoming infected by CPE and reservoirs of carbapenemases are growing globally, the trend of the CPE epidemic points toward a rise in its incidence. To prevent a massive CPE pandemic, a well-coordinated response from all health professionals and national and supranational authorities is clearly needed.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/metabolismo , Antibacterianos/uso terapêutico , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Carbapenêmicos/uso terapêutico , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Surtos de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/epidemiologia , Evolução Molecular , Previsões , Saúde Global , Humanos , Controle de Infecções , Cooperação Internacional , Espanha , Resistência beta-Lactâmica/genética , beta-Lactamases/classificação , beta-Lactamases/genéticaRESUMO
The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE), as the current paradigm of extensive drug-resistance and multi-drug resistance to antibiotics, is a serious threat to patient health and public health. The increase in OXA-48- and VIM-1-producing Klebsiella pneumoniae isolates represents the greatest impact of CPE in Spain. This evidence has lead the members of a representative panel of the Spanish Study Groups of Nosocomial Infections and Mechanisms of Action and Resistance to Antimicrobials of the Spanish Society of Clinical Microbiology and Infectious Diseases (GEIH/GEMARA-SEIMC) to make a position statement expressing the need for: (i) definitive and coordinated action by all health professionals and authorities involved, and (ii) an adaptation of health systems to facilitate their early control and minimize their impact.