RESUMEN
AIM: To describe an outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit. METHODS: Isolates of linezolid-resistant E. faecium were typed by pulsed-field gel electrophoresis (PFGE), and examined by polymerase chain reaction (PCR) and sequencing for the transmissible cfr gene that confers resistance to linezolid. Enhanced environmental cleaning, initial and weekly screening of all patients, and monitoring of adherence to standard infection control precautions were implemented. FINDINGS: Five patients with pre-existing renal disease were found to have rectal colonization with linezolid-resistant E. faecium over a two-week period. The index case was a 57-year-old male from India who had travelled to the UK. One patient also had a linezolid-resistant E. faecium of a different PFGE profile isolated from a heel wound. All isolates were confirmed to harbour the cfr gene by PCR and Sanger sequencing, and all were resistant to glycopeptides (VanA phenotype). CONCLUSIONS: This article describes the first UK outbreak with a single strain of linezolid- and glycopeptide-resistant E. faecium harbouring the cfr gene, affecting five patients in a nephrology unit. Following the implementation of aggressive infection control measures, no further cases were detected beyond a two-week period.
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Antibacterianos/farmacología , Portador Sano/epidemiología , Farmacorresistencia Bacteriana , Enterococcus faecium/efectos de los fármacos , Glicopéptidos/farmacología , Infecciones por Bacterias Grampositivas/epidemiología , Linezolid/farmacología , Portador Sano/microbiología , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Electroforesis en Gel de Campo Pulsado , Enterococcus faecium/clasificación , Enterococcus faecium/genética , Enterococcus faecium/aislamiento & purificación , Genes Bacterianos , Genotipo , Infecciones por Bacterias Grampositivas/microbiología , Departamentos de Hospitales , Humanos , Control de Infecciones/métodos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Reino UnidoRESUMEN
Objectives: Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were first seen in the UK in 2003 and have been increasingly reported since 2010, largely owing to an ongoing outbreak in North-West England. We examined the role of clonal spread and plasmid transmission in their emergence. Methods: Isolates comprised KPC-positive K. pneumoniae ( n = 33), Escherichia coli ( n = 7) and Enterobacter spp. ( n = 4) referred to the national reference laboratory between 2008 and 2010 from 17 UK centres, including three in North-West England. Isolates were typed by MLST. Plasmids were transferred by electroporation and characterized by PCR or sequencing. PCR screening assays were developed to distinguish plasmid pKpQIL variants. Results: The K. pneumoniae isolates included 10 STs, of which three belonged to clonal group (CG) 258. CG258 ( n = 19) isolates were detected in 13 centres but accounted for only 7/19 (36.8%) of those from North-West England. Most KPC-producers (37/44, 84.1%), including 16/19 CG258 K. pneumoniae , carried bla KPC on IncFII K2 plasmids. Sequencing of a subset of these plasmids ( n = 11) revealed similarities with published pKpQIL. One variant, pKpQIL-UK [identified in K. pneumoniae CG258 ( n = 5) and ST468 ( n = 1) isolates from distinct centres] had only a few nucleotide changes from classical pKpQIL, whereas pKpQIL-D1 ( n = 1) and pKpQIL-D2 ( n = 4), from isolates of various species in the North-West, harboured large variations, reflecting replacement of the partitioning and replication functions and potentially thereby facilitating spread. PCR revealed that 36/37 (97.3%) IncFII K2 -type plasmids in KPC-positive isolates had pKpQIL markers. Conclusions: pKpQIL-like plasmids played a major role in the early dissemination of KPC enzymes in the UK.
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Proteínas Bacterianas/genética , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/enzimología , Plásmidos , beta-Lactamasas/genética , Transmisión de Enfermedad Infecciosa , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Transferencia de Gen Horizontal , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Reino Unido/epidemiologíaRESUMEN
OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (nâ=â10) and from healthy urines 'spiked' with multiresistant Escherichia coli (nâ=â5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.
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Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Metagenómica/métodos , Pruebas de Sensibilidad Microbiana/métodos , Nanoporos , Infecciones Urinarias/diagnóstico , Orina/microbiología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/microbiología , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Factores de Tiempo , Infecciones Urinarias/microbiologíaRESUMEN
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.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana/métodos , Europa (Continente) , InternacionalidadRESUMEN
Limited data are available on the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in the UK. We tested 124 raw meat samples for MRSA including pork (n = 63), chicken (n = 50) and turkey (n = 11) collected from retail outlets in North West England between March and July 2015. MRSA was recovered from nine (7·3%) samples (four chicken, three pork and two turkey) from different butchers and supermarkets. Four were labelled of UK origin, three were from continental Europe; the origin was not specified for two samples. Whole-genome sequencing (WGS), spa typing and the presence of lineage-specific canonical single nucleotide polymorphisms confirmed that they belonged to the livestock-associated clade of clonal complex (CC) 398. Seven (77·8%) isolates were multi-drug resistant. Phylogenetic analyses showed the isolates were diverse, suggesting multiple silent introductions of LA-MRSA into the UK food chain. Two chicken meat isolates belonged to a sub-clade recently reported from human cases in Europe where poultry meat was the probable source. The low levels of MRSA identified (<20 CFU per g) and absence of enterotoxin genes suggest the risk of acquisition of, or food-poisoning due to, LA-MRSA is low. Nevertheless, the MRSA contamination rate is higher than previously estimated; further evaluation of the public health impacts of LA-MRSA is warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a public health concern worldwide, but has only been reported sporadically in the UK. In the largest UK study to date, samples of raw meat at retail sale were examined for both the presence and levels of MRSA. We report the first isolations of CC398 LA-MRSA from poultry meat in the UK including representatives of a particular sub-clade associated with cases of human infection/colonization in Europe. Although levels were low (<20 CFU per g), the contamination rate was higher than previous UK studies. Moreover, whole-genome sequencing revealed multiple independent introductions of LA-MRSA into the UK food chain.
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Contaminación de Alimentos/análisis , Contaminación de Alimentos/legislación & jurisprudencia , Ganado/microbiología , Carne/microbiología , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Animales , Pollos , Inglaterra , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Filogenia , Infecciones Estafilocócicas/microbiología , Porcinos , PavosRESUMEN
Extra-intestinal pathogenic Escherichia coli are a significant cause of urinary tract infection and bacteraemia within the UK. We sought to identify the serogroups of 658 E. coli isolates collected in the UK between January 2011 and March 2012, to better understand the ExPEC population and understand the relevance of serogroups in this pathotype. Isolates were typed and serogroup identified using established phenotypic and molecular methods. Sixty-two serogroups were identified; 54 among urinary isolates and 35 among bloodstream isolates. However, serogroups O25, O6, and O2 dominated both infection types. These serogroups were linked to the major ExPEC STs as follows: ST131-O25, ST73-O6, ST127-O6, and ST95-O2. The serogroup data from this study have increased our understanding of the ExPEC population in the UK, but also highlighted key ST-serogroup relationships within the major ExPEC clones. These data can be used to guide vaccine design and in the development of laboratory diagnostic tests targeting the ExPEC population.
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Técnicas de Tipificación Bacteriana , Infecciones por Escherichia coli/microbiología , Escherichia coli Patógena Extraintestinal/clasificación , Serogrupo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/epidemiología , Bacteriemia/microbiología , Niño , Preescolar , Infecciones por Escherichia coli/epidemiología , Escherichia coli Patógena Extraintestinal/aislamiento & purificación , Femenino , Genotipo , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reino Unido/epidemiología , Infecciones Urinarias/epidemiología , Infecciones Urinarias/microbiología , Adulto JovenRESUMEN
OBJECTIVES: The diazabicyclooctane ß-lactamase inhibitor OP0595 (RG6080) also acts as an antibiotic, targeting PBP2 in Enterobacteriaceae, but this activity is vulnerable to mutational resistance. We used WGS to investigate the basis of this resistance. METHODS: Twenty OP0595-selected mutants, comprising four derived from each of five different Escherichia coli strains, were sequenced on Illumina HiSeq. Reads from each mutant were mapped to the assembled genome of the corresponding parent. A variant-calling file generated with Samtools was parsed to determine genetic alterations. RESULTS: Besides OP0595, the mutants consistently showed decreased susceptibility to mecillinam, which likewise targets PBP2, and grew as stable round forms in the presence of subinhibitory concentrations of OP0595. Among the 20 mutants, 18 had alterations in genes encoding tRNA synthase and modification functions liable to induce expression of the RpoS sigma factor through activation of the stringent response or had mutations suppressing inactivators of RpoS or the stringent response signal-degrading enzyme, SpoT. TolB was inactivated in one mutant: this activates RcsBC regulation and was previously associated with mecillinam resistance. The mechanism of resistance remained unidentified in one mutant. Both the RpoS and RcsBC systems regulate genes of cell division, including ftsAQZ that can compensate for loss or inhibition of PBP2, allowing survival of the challenged bacteria as stable round forms, as seen. CONCLUSIONS: WGS identified the global stringent response signal, entailing induction of RpoS, as the main mediator of mutational resistance to OP0595 in E. coli.
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Amdinocilina/farmacología , Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Lactamas/farmacología , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Amdinocilina/metabolismo , Compuestos de Azabiciclo/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Variación Genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Lactamas/metabolismo , Microscopía Electrónica de Transmisión , Mutación , Factor sigma/genética , Inhibidores de beta-Lactamasas/metabolismo , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
OBJECTIVES: Escherichia coli is the most common agent of bacteraemia, bacterial gastroenteritis and urinary tract infections (UTIs). Lineages causing UTIs and gastrointestinal disease are well defined, but less is known about those causing bacteraemia. We therefore investigated the population structure of E. coli from bacteraemia in the UK and Ireland between 2001 and 2010. METHODS: E. coli isolates (nâ=â2166) were submitted to the BSAC Bacteraemia Surveillance Programme from 18 UK and Irish centres from 2001 to 2010. Genotypes were analysed by MLST using the Achtman scheme; MICs, blaCTX-M group and patient demographics were previously determined in the BSAC surveillance. RESULTS: Four hundred and forty-eight STs were identified, but five of these, and their associated clonal complexes (CCs), accounted for 58.4% (1264 of 2166) of isolates: CC73 was the most common (20.7%), followed by CC131 (13.9%), CC95 (11.3%), CC69 (6.9%) and CC12 (5.5%). All these, except CC69 (group D), belong to phylogenetic group B2. CC131 isolates were much more often MDR than other STs were: they rose from 2.9% of isolates in 2001 to 20.5%-20.7% in 2007-08 and then declined to 14.3% in 2010. Resistance rates to cephalosporins, aminoglycosides and fluoroquinolones remained below 10% in other major CCs throughout. CONCLUSIONS: The five most prevalent bacteraemia STs have all been associated previously with UTIs. They dominated in all years, but their proportions fluctuated, most notably for ST131, a globally disseminated high-risk clone that is often MDR.
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Bacteriemia/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Variación Genética , Genotipo , Adolescente , Bacteriemia/epidemiología , Niño , Preescolar , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Femenino , Humanos , Lactante , Recién Nacido , Irlanda/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Prevalencia , Reino Unido/epidemiología , Adulto Joven , beta-Lactamasas/análisisRESUMEN
The multidrug-resistant ST131-O25b clone of Escherichia coli is well established as a significant cause of extra-intestinal infections worldwide. However, there have been only two small regional studies comparing ST131 isolates from the UK. Therefore, we characterized 143 ST131 E. coli (38 urinary, 105 bloodstream) collected between January 2011 and March 2012 from 38 centres located across the UK and Republic of Ireland. Phenotypic and genotypic characterization of clonal isolates revealed high rates of resistance to amoxicillin-clavulanate (56 %), cefotaxime (32 %), ciprofloxacin (79 %), temocillin (69 %, bloodstream isolates only), gentamicin (67 %) and trimethoprim-sulfamethoxazole (59 %). The most frequently detected extended-spectrum beta-lactamase was CTX-M-15 (87 %), predominantly encoded on IncF plasmids, although it was also associated with IncU plasmids in two isolates. The majority of UK ST131 clonal isolates possessed the O25b antigen (97 %) and the H30 fimH allele (92 %), but three serogroups (O19a, O136 and O153) novel to ST131 were identified among our strains. Contrary to previous reports, UK ST131-O16 isolates were typically susceptible to ciprofloxacin and lacked beta-lactamase genes (n = 12/12). In summary, ST131 strains of E. coli circulating in the UK possess characteristic clonal features, but are becoming more diverse than other international ST131 populations.
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Bacteriemia/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Infecciones Urinarias/microbiología , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Bacteriemia/epidemiología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Humanos , Pruebas de Sensibilidad Microbiana , Reino Unido/epidemiología , Infecciones Urinarias/epidemiología , Factores de VirulenciaRESUMEN
Escherichia coli sequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion of E. coli urinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen published E. coli genomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessed in silico by seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515 E. coli isolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominant E. coli STs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n = 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinal E. coli STs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.
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Bacteriemia/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Tipificación de Secuencias Multilocus/métodos , Infecciones Urinarias/microbiología , Antibacterianos/farmacología , Bacteriemia/diagnóstico , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/diagnóstico , Genoma Bacteriano , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Infecciones Urinarias/diagnósticoRESUMEN
Using data from whole-genome projects, an updated multiplex PCR strategy was developed to assign Escherichia coli isolates rapidly to major phylogenetic groups. This assay accommodates sequence variations detected within target sequences, thereby increasing sensitivity and reliability. It was validated using 185 isolates of known sequence types and showed improved congruence with multilocus sequence typing data.
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Técnicas Bacteriológicas/métodos , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Escherichia coli/genética , Humanos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Extended-spectrum beta-lactamases (ESBLs) are an increasingly important cause of resistance in Gram-negative bacteria throughout the world. AIM: We investigated the clinical and molecular epidemiology of infections caused by ESBL-producing Enterobacteriaceae in a UK hospital, to identify the types of ESBL produced and risk factors for acquisition. METHODS: Between July 2008 and June 2009, all patients yielding ESBL-producing Enterobacteriaceae from any clinical specimen were prospectively investigated using a questionnaire. API20E was used for bacterial identification; susceptibility testing and ESBL production were assessed by BSAC disc diffusion and cefpodoxime-clavulanate synergy tests, respectively. Polymerase chain reaction was used to screen a subset of isolates for bla(CTX-M) genes, to assign Escherichia coli isolates to their phylogenetic groups, and to identify members of the uropathogenic ST131 lineage. RESULTS: The overall prevalence of ESBL producers among clinical samples yielding Enterobacteriaceae was 1%; ESBL producers, obtained from 124 patients, were E. coli (N = 105), Klebsiella pneumoniae (N = 12), and others (N = 7). The main risk factors identified include recent antibiotic use (93%) and presence of a urinary catheter (24%). CTX-M group 1 ESBLs dominated (in 59 of 78, 76%, isolates studied). Most E. coli (35 of 56 tested) were phylogroup B2; of these, 23 belonged to the ST131 clone, 12 were phylogroup D, and four each belonged to phylogroups A and B1. CONCLUSION: ESBLs are an uncommon but significant problem in north-west Cambridgeshire. CTX-M-type enzymes were found in 75% of ESBL-positive isolates. All but two patients had at least one recognized risk factor. This study supports the requirement for interventions to reduce inappropriate urinary catheterization and antibiotic prescribing.
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Enterobacteriaceae/enzimología , Infecciones por Escherichia coli/epidemiología , Infecciones por Klebsiella/epidemiología , beta-Lactamasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Análisis por Conglomerados , ADN Bacteriano/genética , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Femenino , Genotipo , Hospitales de Distrito , Humanos , Lactante , Infecciones por Klebsiella/microbiología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Tipificación Molecular , Reacción en Cadena de la Polimerasa , Prevalencia , Encuestas y Cuestionarios , Reino Unido/epidemiología , Adulto Joven , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: The global accumulation of Escherichia coli with CTX-M extended-spectrum ß-lactamases partly reflects the dissemination of clonal lineages, notably ST131 and ST405. More recently, E. coli have emerged that produce NDM carbapenemase. We sought to determine the clonal diversity of E. coli with this enzyme from English hospitals, and to compare them with isolates from Pakistan and India. METHODS: The 18 NDM-positive E. coli were from hospitals in England (nâ=â10), Pakistan (nâ=â7) and India (nâ=â1). Isolates were compared by phylogenetic grouping, multilocus sequence typing and PFGE of XbaI-digested DNA. Isolates were screened by PCR for acquired AmpC genes, bla(CTX-M), and the 16S rRNA methylase genes armA and rmtC. RESULTS: Most of the isolates belonged to phylogenetic groups B1 (nâ=â9) or D (nâ=â7); two were group A and none was group B2. Nine isolates from England and Pakistan belonged to the B1 lineage ST101, with seven of these clustering at >77% similarity by PFGE. Other lineages included ST405 (nâ=â3, group D), ST648 (nâ=â3, group D), the ST23 complex (one each of ST90 and ST410, both group A) and ST156 (nâ=â1, group D). Sixteen of 18 isolates had a group 1 CTX-M gene, 13 had a CIT-type acquired AmpC, and 16 had either or both of armA and rmtC. CONCLUSIONS: The E. coli isolates producing NDM-1 carbapenemase belonged to six sequence types and included diverse clonal lineages. Nevertheless, isolates of B1-ST101 accounted for half the collection, and included isolates from both England and Pakistan. None of the isolates belonged to ST131 or to phylogroup B2.
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Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , beta-Lactamasas/genética , Infección Hospitalaria/microbiología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Electroforesis en Gel de Campo Pulsado , Inglaterra , Infecciones por Escherichia coli/microbiología , Variación Genética , Humanos , India , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Pakistán , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The emergence of metallo-ß-lactamase (MBL)-producing Enterobacteriaceae is a serious public health concern. Producers have been repeatedly isolated from patients and long-term care facility (LTCF) residents around Bolzano, and we sought to assess their prevalence and clinical impact. All routine Enterobacteriaceae isolates from a Bolzano tertiary-care hospital and associated long-term care facilities in 2008 (n = 5500) were screened for MBLs, with case details reviewed for the source patients. In total, 36 producers were obtained from 29 patients, comprising 14 Escherichia coli, six Klebsiella pneumoniae, four Klebsiella oxytoca, four Citrobacter freundii, two Enterobacter cloacae and two Morganella morganii, as well as single Citrobacter amalonaticus, Enterobacter aerogenes, Providencia stuartii and Proteus mirabilis isolates. All were PCR-positive for bla(VIM) and 25 were PCR-positive for qnrS; 19 non-K. pneumoniae had bla(SHV) and one had bla(CTX-M-group1); 13 were from 12 LTCF residents and 23 were from 17 acute-care patients. All these patients had serious underlying diseases with prolonged hospitalization or LTCF stay; only seven had infections due to the MBL producers, comprising four urinary tract infections, two catheter-related bloodstream infections and one patient with both a surgical site infection and pneumonia. Five patients had more than one MBL-producing organism. Pulsed-field gel electrophoresis identified a cluster of six related E. coli, whereas pairs of K. pneumoniae and C. freundii isolates had >85% profile similarity. Transformants prepared from two isolates were shown to be PCR-positive for bla(VIM), qnrS and bla(SHV); their plasmids gave similar restriction fragment length polymorphism patterns, and bla(VIM-1), qnrS1 and bla(SHV-12) were detected by sequencing.
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Infección Hospitalaria/microbiología , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , beta-Lactamasas/genética , Anciano , Anciano de 80 o más Años , Técnicas de Tipificación Bacteriana , Preescolar , Análisis por Conglomerados , Electroforesis en Gel de Campo Pulsado , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Femenino , Hospitales , Humanos , Lactante , Recién Nacido , Italia , Cuidados a Largo Plazo , Masculino , Persona de Mediana Edad , Tipificación Molecular , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: the emergence of carbapenemases in Enterobacteriaceae is driving a search for therapeutic alternatives. We tested ACHN-490, a sisomicin derivative that evades all plasmid-mediated aminoglycoside-modifying enzymes, against 82 carbapenem-resistant Enterobacteriaceae isolates. Comparators included internationally and locally available aminoglycosides. Methods The isolates variously had KPC (n = 12), SME-1 (n = 1), IMP (n = 13), VIM (n = 5), NDM (n = 17) or OXA-48 (n = 19) carbapenemases, or had combinations of impermeability with AmpC (nâ=â5) or extended-spectrum ß-lactamases (nâ=â10). They included 53 Klebsiella spp., 19 Enterobacter spp., 6 Escherichia coli and 4 others; most were multiresistant. Genes were identified by PCR and sequencing; MICs were measured by CLSI agar dilution. RESULTS: ACHN-490 was active at ≤ 2 mg/L against all 65 isolates with carbapenem resistance mechanisms other than NDM enzyme, mostly with MICs of 0.12-0.5 mg/L; isepamicin was active against 63/65 at ≤ 8 mg/L. In contrast, 35% were resistant to gentamicin at 4 mg/L, 61% to tobramycin at 4 mg/L and 20% to amikacin at 16 mg/L. However, 16 of the 17 isolates with NDM-1 enzyme were resistant to ACHN-490, with MICs ≥ 64 mg/L, and these were cross-resistant to all other human-use aminoglycosides tested. Their behaviour was associated with ArmA and RmtC 16S rRNA methylases. Apramycin (a veterinary aminoglycoside) retained its full activity, with MICs of 4-8 mg/L versus strains with armA or rmtC, though resistance was seen in one Klebsiella pneumoniae with AAC(3)-IV (MIC ≥ 256 mg/L). CONCLUSIONS: ACHN-490 has potent activity versus carbapenem-resistant isolates, except those also harbouring 16S rRNA methylases; isepamicin is also widely active, though less potent than ACHN-490. Evasion of 16S rRNA methylases by apramycin is noteworthy and may provide a starting point for future aminoglycoside development.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Carbapenémicos/farmacología , Infecciones por Enterobacteriaceae/microbiología , Enterobacteriaceae/efectos de los fármacos , Sisomicina/análogos & derivados , Resistencia betalactámica , Enterobacteriaceae/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Sisomicina/farmacología , beta-Lactamasas/genética , ARNt Metiltransferasas/genéticaRESUMEN
Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas , Eritromicina/biosíntesis , Genes Bacterianos , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Secuencia de Bases , Deshidrogenasas de Carbohidratos/genética , Cartilla de ADN/genética , Eritromicina/química , Eritromicina/metabolismo , Escherichia coli/genética , Expresión Génica , Prueba de Complementación Genética , Vectores Genéticos , Glicosilación , Glicosiltransferasas/genética , Oxidorreductasas/genética , Plásmidos/genética , Saccharopolyspora/enzimologíaRESUMEN
Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3, 4-isomerase.
Asunto(s)
Eritromicina/análogos & derivados , Genes Bacterianos , Oleandomicina/biosíntesis , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Streptomyces antibioticus/genética , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , ADN Bacteriano/genética , Eritromicina/biosíntesis , Prueba de Complementación Genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Isomerasas/genética , Isomerasas/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Familia de Multigenes , Plásmidos/genética , Saccharopolyspora/crecimiento & desarrollo , Streptomyces antibioticus/crecimiento & desarrollo , Streptomyces antibioticus/metabolismo , Transformación BacterianaRESUMEN
The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5' of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic beta-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.
Asunto(s)
Eritromicina/metabolismo , Genes Fúngicos , Familia de Multigenes , Saccharopolyspora/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Modelos Químicos , Datos de Secuencia Molecular , MutagénesisRESUMEN
The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.
Asunto(s)
Proteínas Bacterianas , Eritromicina , Glicosiltransferasas/genética , Oxidorreductasas Intramoleculares/genética , Complejos Multienzimáticos/genética , Oxidorreductasas/genética , Saccharopolyspora/genética , Secuencia de Aminoácidos , Amino Azúcares/metabolismo , Clonación Molecular , Hexosas/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The matrix protein of vesicular stomatitis virus (VSV) plays a pivotal role in viral assembly. We previously demonstrated the ability of M protein to self-associate at low salt concentrations. Now, we show the ability of M protein to polymerize in the presence of ZnCl2 in a nucleation-dependent manner. Analysis of kinetics revealed that the nuclei are probably made of three or four molecules of M. These results are consistent with the idea that in vitro self association of M protein is not due to amorphous aggregation but rather reflects an intrinsic ability of M to polymerize. Using attenuated total reflectance Fourier transform infrared spectroscopy, we showed that M polymerization is associated with an increase in the beta-sheet content of the protein. We propose a model explaining both the apparent M protein solubility in infected cells and how M polymerization could promote viral assembly. Data available for other negative strand viruses suggest that M polymerization may be the general basis of viral assembly.