RESUMO
OBJECTIVES: To genetically characterize VIM-producing Enterobacter cloacae complex (ECC) isolates recovered in France from 2015 to 2018. METHODS: WGS, species determination, MLST, clonal relationship and genetic characterization were performed on 149 VIM-producing ECC isolates. RESULTS: Among VIM-producing Enterobacterales, the prevalence of ECC increased drastically from 6% in 2012 to 52% in 2018. The most prevalent species were Enterobacter hormaechei subsp. hoffmannii (40.9%), E. hormaechei subsp. steigerwaltii (21.5%), E. hormaechei subsp. xiangfangensis (14.8%) and ECC clade S (17.4%). Major STs were ST-873 (17.5%), ST-66 (12.1%), ST-78 (9.4%), ST-419 (8.1%), ST-145 (4.7%), ST-50 (4.0%), ST-118 (4.0%) and ST-168 (4.0%). Finally, six different integrons were identified, with some being specific to a given blaVIM variant (In916 with blaVIM-1-aacA4'-aphA15-aadA1-catB2 and In416 with blaVIM-4-aacA7-dfrA1b-aadA1b-smr2 genes). CONCLUSIONS: This study demonstrated the genetic diversity among VIM-producing ECC isolates, indicating that their spread is not linked to a single clone.
Assuntos
Enterobacter cloacae , Infecções por Enterobacteriaceae , Antibacterianos/uso terapêutico , Enterobacter/genética , Enterobacter cloacae/genética , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos , beta-Lactamases/genéticaRESUMO
Carbapenemase-producing Enterobacterales (CPE) is a major public-health concern. Here we describe the occurrence of blaVIM-2 in three isolates of Enterobacter hormaechei subsp. steigerwaltii. The blaVIM-2 gene was part of a class II transposon Tn1332 and was embedded in a remnant of a class 1 integron. Tn1332 was carried by a large, conjugative, non-typeable plasmid. The three isolates belonged to sequence type 90 (ST90). Two isolates (90H2 and 90H3) were highly related [<10 single nucleotide polymorphisms (SNPs)], whereas isolate 104D2 exhibited more than 50 SNPs and Tn1332 was inserted in a different place in the plasmid. Another IncHI-type plasmid carrying the extended-spectrum ß-lactamase (ESBL) gene blaCTX-M-15 was identified in 90H2 and 90H3. Among the three isolates, isolate 104D2 was negative for detection of carbapenemase activity using the biochemical Carba NP test, despite the presence of Tn1332 on the same plasmid. Mutants of 104D2 with higher minimum inhibitory concentrations (MICs) for carbapenems were obtained and one mutant (m104D2) was analysed. In contrast to 104D2, mutant m104D2 gave a positive Carba NP test. The mutant possessed two copies of Tn1332 per cell and a nonsense mutation in WecA, an enzyme involved in enterobacterial common antigen and peptidoglycan intermediate biosynthesis. This study describes the first occurrence of Tn1332 in Enterobacterales and the phenotypic diversity of VIM-2-producing E. hormaechei.
Assuntos
Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Enterobacter/efeitos dos fármacos , Enterobacter/genética , Infecções por Enterobacteriaceae/microbiologia , beta-Lactamases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae/epidemiologia , França/epidemiologia , Humanos , Integrons , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Filogenia , Plasmídeos/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The whole-genome sequencing analysis revealed a polyclonal dissemination of NDM-1 and NDM-9 variants in Escherichia coli (n = 20) and Klebsiella pneumoniae (n = 2) in Tahiti since 2015 via interspecies transfer of three different blaNDM-carrying plasmids (IncR, IncHI2, and IncF) and patient-to-patient cross-transmission. It highlights the potential risk of importation of NDM producers in France, where French Polynesia is not considered stricto sensu a foreign country from which repatriated patients have to be screened.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Escherichia coli/genética , França , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Plasmídeos/genética , Polinésia , beta-Lactamases/genéticaRESUMO
BACKGROUND: Enterobacter cloacae complex contains nosocomial pathogens responsible for infection outbreaks. Identification at the species level within the E cloacae complex remains difficult. Using whole genome sequencing, our aim was to decipher the transmission routes that led to the death of six of seven neonates who had bacteraemia caused by E cloacae complex isolates in a neonatal intensive care unit (NICU) over a 13 month period. METHODS: In this cohort study, E cloacae complex isolates were taken from 186 newborns in an NICU: 14 were clinical samples (eg, blood cultures), 728 rectal swab samples, and 38 environmental samples (20 from siphons, 18 from incubators, and one from a mattress). The samples were analysed to decipher the possible role of manual cross transmission or environmental source in an outbreak of fatal septic shocks related to E cloacae complex bacteraemia. After the replacement of the incubators suspected to be the reservoir of some outbreak-related isolates on Feb 1, 2018, E cloacae complex strains were screened again for 10 months (503 rectal swab samples from 163 newborns). The 71 E cloacae complex isolates recovered from screening, clinical, and environmental samples across both study periods were compared by whole genome sequencing. The pathogenicity of E cloacae complex isolates responsible for fatal septic shocks was assessed using a Galleria mellonella in-vivo model. FINDINGS: From Dec 9, 2016, to Jan 31, 2018, 249 (34%) of 728 rectal swab samples were positive for E cloacae complex, with 66 (35%) of 186 newborns colonised. E cloacae complex were also recovered from four (20%) of 20 siphons and 11 (61%) of 18 incubators. During this period, whole genome sequencing identified that the isolates responsible for the six fatal septic shocks were all Enterobacter bugandensis. A G mellonella infection model showed a higher virulence of E bugandensis. Genomic analysis highlighted the role of incubators as a long-term reservoir and source of cross contamination, leading to their replacement on Feb 1, 2018. Following incubator replacement, a 10-month follow-up investigation identified a physiological gut colonisation with polyclonal E cloacae complex in 52 (34%) of 163 neonates within a median of 9 days (5-14), but no E cloacae complex-related septic shocks. INTERPRETATION: Despite around 30% of neonates being physiologically colonised with E cloacae complex, fatal sepsis was systematically linked with bacteraemia caused by E bugandensis. Our findings highlight the need for accurate identification methods to detect the hypervirulent species within the E cloacae complex recovered in neonates. FUNDING: Assistance Publique-Hôpitaux de Paris.
Assuntos
Bacteriemia , Infecções por Enterobacteriaceae , Choque Séptico , Bacteriemia/complicações , Estudos de Coortes , Enterobacter cloacae/genética , Infecções por Enterobacteriaceae/epidemiologia , Humanos , Recém-Nascido , Choque Séptico/complicaçõesRESUMO
In Enterobacterales, the most common carbapenemases are Ambler's class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.
Assuntos
Proteínas de Bactérias/biossíntese , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , beta-Lactamases/biossíntese , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Bélgica , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , França , Genes Bacterianos/genética , Humanos , Plasmídeos/genética , Proteus mirabilis/isolamento & purificação , beta-Lactamases/classificação , beta-Lactamases/genéticaRESUMO
BACKGROUND: Carbapenem-resistant Enterobacteriaceae are considered by WHO as "critical" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes. METHODS: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing. RESULTS: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse ß-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination. CONCLUSIONS: Lineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by ß-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli Enteropatogênica/genética , Evolução Molecular , Transferência Genética Horizontal , Resistência beta-Lactâmica , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Mutação , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Filogenia , Porinas/genética , beta-Lactamases/metabolismoRESUMO
A carbapenem-resistant Citrobacter sp. was recovered from routine screening of multidrug-resistant bacteria. This isolate coproduced OXA-48 and OXA-198. OXA-48 was carried by the prototypical IncL plasmid, whereas OXA-198 was carried by a peculiar IncHI-type plasmid. This carbapenemase gene was inserted within a class 1 integron located on a conjugative plasmid. This report describes the first occurrence of OXA-198 in Enterobacterales.
Assuntos
Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Citrobacter/efeitos dos fármacos , Citrobacter/genética , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Citrobacter/isolamento & purificação , Humanos , Masculino , Testes de Sensibilidade Microbiana , Penicilinas/farmacologia , Plasmídeos/genéticaRESUMO
In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The blaACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the blaACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the blaACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests.
Assuntos
Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Carbapenêmicos/farmacologia , Enterobacter/efeitos dos fármacos , beta-Lactamases/metabolismo , Cefalosporinase/genética , Cefalosporinase/metabolismo , Enterobacter/enzimologia , Hidrólise , Cinética , Testes de Sensibilidade MicrobianaRESUMO
Objectives: Available commercial tools (molecular methods or immunochromatographic assays) usually allow the detection of the five most prevalent carbapenemases (KPC, NDM, VIM, IMP and OXA-48-like), but miss minor carbapenemases. Here, we characterize two enterobacterial isolates with reduced susceptibility to carbapenems and negative for the most commonly encountered carbapenemase genes. Methods: Enterobacter hormaechei and Citrobacter freundii isolates were recovered from a bile sample and rectal screening, respectively. Both isolates were investigated by WGS. Resistance genes were detected using ResFinder. The blaTMB-1-harbouring plasmid was reconstructed using CLC genomic workbench 10.0 and was annotated using the RAST tool. Transfer frequency was determined by conjugation experiments using the laboratory strain Escherichia coli J53. Results: The two isolates were resistant to broad-spectrum cephalosporins and carbapenems. WGS revealed the presence of blaTMB-1, which has previously only been described in non-fermenters. blaTMB-1 was located within an ISKpn19-based composite class 1 transposon. Comparative genomics revealed that this structure was carried on a conjugative IncN-type plasmid within an integration hotspot. Conjugation experiments revealed high transfer frequencies of â¼1â×â10-3. Conclusions: To the best of our knowledge, this study corresponds to the first report of Tripoli MBL 1-producing Enterobacteriaceae. Despite always being described as likely to be chromosomally located in non-fermenters, the blaTMB-1 gene is now found to be carried by a conjugative plasmid among Enterobacteriaceae, raising concern about the possible dissemination of this carbapenemase. The blaTMB-1 gene should now be suspected when PCRs targeting the main carbapenemases remain negative.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Citrobacter freundii/isolamento & purificação , Enterobacter/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , beta-Lactamases/genética , Bile/microbiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Citrobacter freundii/enzimologia , Citrobacter freundii/genética , Conjugação Genética , Enterobacter/enzimologia , Enterobacter/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Transferência Genética Horizontal , Genes Bacterianos , Humanos , Plasmídeos/análise , Reto/microbiologia , Sequenciamento Completo do Genoma , Resistência beta-LactâmicaRESUMO
Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.
Assuntos
Cromossomos Bacterianos/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Plasmídeos/genética , Polimixinas/farmacologia , Proteínas de Escherichia coli/genética , Lipídeo A/genética , Testes de Sensibilidade Microbiana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.
Assuntos
Antibacterianos/farmacologia , Infecção Hospitalar , Surtos de Doenças , Enterobacter cloacae/enzimologia , Infecções por Enterobacteriaceae/microbiologia , beta-Lactamases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Farmacorresistência Bacteriana Múltipla , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , beta-Lactamases/genéticaRESUMO
With the dissemination of carbapenemase-producing Enterobacteriaceae (CPE) strains worldwide, carbapenem-hydrolyzing enzymes are increasingly reported among isolates of Escherichia coli, the first hospital and community-acquired opportunistic pathogen. Here, we have performed an epidemiological survey of carbapenemase-producing E. coli (CP-Ec) isolates received at the French National Reference Centre (F-NRC) in 2012 and 2013. Antimicrobial susceptibilities for last-resort antibiotics and antimicrobial compounds commonly used to treat urinary tract infections were determined by broth microdilution. Clonal relationship was assessed using repetitive sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). From this collection of 140 carbapenemase-producing E. coli isolates, 74% produced an OXA-48-like carbapenemase and 21% produced an NDM carbapenemase. A link with a foreign country was suspected for 37% of infected/colonized patients. Most of the isolates were from screening (56%) and from urine samples (26%). Colistin, fosfomycin, and nitrofurantoin possessed the most consistent activity, with 100%, 95%, and 96% isolates susceptible, respectively. A wide diversity of carbapenemase-producing E. coli isolates has been found (50 different sequence types [STs]). The most prevalent clones were (i) E. coli sequence type 38 (ST38) producing OXA-48 (n = 21), a clone linked to Turkey and North African countries, (ii) E. coli ST-90 producing OXA-204 (n = 9), which was responsible for an outbreak related to a contaminated duodenoscope, and (iii) E. coli ST-410 producing OXA-181 (n = 5), which was recovered from patients of different geographical origins. These specific clones might be considered high-risk clones for the dissemination of carbapenemases in E. coli The wide diversity of STs, combined with the increasing number of CP-Ec isolates received by the F-NRC, suggests a likely dissemination of CP-Ec isolates in the community.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Infecções por Escherichia coli/epidemiologia , Escherichia coli/genética , Variação Genética , Infecções Urinárias/epidemiologia , beta-Lactamases/genética , África do Norte/epidemiologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Colistina/farmacologia , Monitoramento Epidemiológico , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Fosfomicina/farmacologia , França/epidemiologia , Expressão Gênica , Humanos , Incidência , Tipagem de Sequências Multilocus , Nitrofurantoína/farmacologia , Filogenia , Turquia/epidemiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , beta-Lactamases/metabolismoRESUMO
Objectives: Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods: The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion: The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions: The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Carbapenêmicos/metabolismo , Programas de Rastreamento/métodos , beta-Lactamases/análise , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacter cloacae/crescimento & desenvolvimento , Infecções por Enterobacteriaceae/microbiologia , Nefelometria e Turbidimetria , Estudos Prospectivos , Estudos Retrospectivos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: There is an urgent need for accurate and rapid diagnostic tests to identify carbapenemase producing enterobacteria (CPE). Here, we have evaluated the Carbapenem Inactivation Method (CIM) test to detect CPEs from cultured colonies. METHODS: A total of 256 enterobacterial isolates were used to evaluate the performance of the CIM in comparison to Carba NP test and molecular detection used a reference method. Ninety three well-characterized isolates (including 29 non-CPE and 63 CPEs of worldwide origin) with decreased susceptibility to at least one carbapenem were used to (i) evaluate the efficacy of CIM test and (ii) to compare it to the Carba NP test. We also tested different carbapenems to determine the best substrate for this test. Finally, the CIM test was then evaluated prospectively against 164 isolates referred to the French National Reference Center (NRC) for Antimicrobial Resistance from may 2016 to july 2016. RESULTS: Based on the results of this retrospective study, sensitivity and specificity of the CIM and the Carba NP test were 92.1% and 100%, respectively. We demonstrated that the meropenem was the best substrate to perform the CIM test since sensitivity and specificity were 81.1% and 100% using ertapenem disk, and 100% and 65,6% using imipenem disk, and respectively. Taking in account the results of retrospective and prospective studies, CIM and Carba NP tests have similar sensitivity, specificity, positive predictive value and negative predictive values being 96.3%, 98.9%, 99.0% and 98.4% for the CIM test versus 96.9%, 100%, 100% and 100% for the Carba NP test. CONCLUSIONS: Our results confirm that the CIM test may be a useful tool for the reliable confirmation of carbapenemase-activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources, no trained personnel, and no specialized equipment.
Assuntos
Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , beta-Lactamases/metabolismo , Testes de Sensibilidade Microbiana , Estudos Prospectivos , Estudos RetrospectivosAssuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Klebsiella pneumoniae/genética , Plasmídeos/química , beta-Lactamases/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Carbapenêmicos/farmacologia , Mapeamento Cromossômico , França , Expressão Gênica , Humanos , Integrons , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Plasmídeos/metabolismo , Tailândia , beta-Lactamases/metabolismoRESUMO
BACKGROUND: Neutrophils are key effectors against the widely distributed mold Aspergillus fumigatus, which is a major threat for immunocompromised patients, including allogeneic hematopoietic stem cell transplant (HSCT) recipients. Yet little is known about neutrophil activity over time after cell transplantation, especially regarding A fumigatus. OBJECTIVE: We aimed at assessing the activity of neutrophils on A fumigatus in allogeneic HSCT recipients at different posttransplantation time points. METHODS: We performed a longitudinal study involving 37 patients undergoing HSCT, drawing blood samples at engraftment and at 2, 6, and 10 months after the HSCT. Posttransplantation neutrophil activity in the recipients was compared with that of the respective donors. Neutrophil/A fumigatus coculture, flow cytometry, and video microscopy were used to assess neutrophil inhibition of fungal growth, cell/fungus interactions, reactive oxygen species production, major surface molecule expression, and neutrophil extracellular trap (NET) formation. RESULTS: The ability of neutrophils to interfere with Aspergillus species hyphal growth was impaired after HSCT. The administration of calcineurin inhibitors appeared to play an important role in this impairment. We also observed that post-HSCT neutrophils produced less NETs, which was correlated with increased fungal growth. Tapering immunosuppression led to the recuperation of inhibition capacity 10 months after HSCT. CONCLUSION: In HSCT recipients neutrophil-driven innate immunity to fungi is altered in the early posttransplantation period (between recovery from neutropenia and up to 6 months). This alteration is at least partly related to administration of calcineurin inhibitors and diminution of NETs production.