RESUMO
Antimicrobial resistance is one of the major health problems we face in the 21st century. Nowadays we cannot understand global health without the interdependence between the human, animal and environmental dimensions. It is therefore logical to adopt a "One Health" approach to address this problem. In this review we show why a collaboration of all sectors and all professions is necessary in order to achieve optimal health for people, animals, plants and our environment.
Assuntos
Antibacterianos , Saúde Única , Animais , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Saúde GlobalRESUMO
Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.
Assuntos
Antibacterianos , Infecções por Escherichia coli , Antibacterianos/farmacologia , Biologia Computacional/métodos , Farmacorresistência Bacteriana/genética , Escherichia coli , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Bacterial antimicrobial resistance (AMR) is constantly evolving and horizontal gene transfer through plasmids plays a major role. The identification of plasmid characteristics and their association with different bacterial hosts provides crucial knowledge that is essential to understand the contribution of plasmids to the transmission of AMR determinants. Molecular identification of plasmid and strain genotypes elicits a distinction between spread of AMR genes by plasmids and dissemination of these genes by spread of bacterial clones. For this reason several methods are used to type the plasmids, e.g. PCR-based replicon typing (PBRT) or relaxase typing. Currently, there are 28 known plasmid types in Enterobacteriaceae distinguished by PBRT. Frequently reported plasmids [IncF, IncI, IncA/C, IncL (previously designated IncL/M), IncN and IncH] are the ones that bear the greatest variety of resistance genes. The purpose of this review is to provide an overview of all known AMR-related plasmid families in Enterobacteriaceae, the resistance genes they carry and their geographical distribution.
Assuntos
Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Transferência Genética Horizontal , Genes Bacterianos , Plasmídeos/análise , Plasmídeos/classificação , Enterobacteriaceae/classificação , Genótipo , HumanosRESUMO
Objectives: The mobile colistin resistance gene mcr-1 has been identified worldwide in human and animal sources, while its occurrence in the environment is still largely unknown. The aim of this study was to investigate the presence of mcr-1 -harbouring Enterobacteriaceae in water samples obtained from rivers and waste water treatment plants in the area of Barcelona, Spain. Methods: The presence of mcr-1 was detected by PCR. Bacterial identification was performed via MALDI-TOF MS. Resistance to colistin was determined by a broth dilution method. The epidemiological relationship between the positive isolates was assessed with PFGE and ST was determined by MLST. Plasmid characterization was performed by transformation experiments, antimicrobial susceptibility testing and incompatibility group PCR. Results: Thirty MDR isolates bearing mcr-1 , 29 Escherichia coli (ST632 and ST479) and 1 Klebsiella pneumoniae (ST526), were identified in sewage from two different waste water treatment plants, whereas the gene was not found in river water. All isolates, including the K. pneumoniae , harboured bla CTX-M-55 and bla TEM-1 . mcr-1 was in all cases associated with an IncI2 plasmid, which only conferred resistance to colistin. mcr-1 was harboured by two predominant E. coli clones that were found in both waste water treatment plants. Conclusions: This study showed a high occurrence of mcr-1 in the sewage of Barcelona, mainly due to the dissemination of two E. coli pulsotypes that are circulating in the population. The presence of mcr-1 in the environment is a cause for concern, and suggests high prevalence of mcr-1 in the community.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Esgotos/microbiologia , Microbiologia da Água , Animais , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Escherichia coli/química , Escherichia coli/classificação , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/classificação , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/análise , Plasmídeos/classificação , Reação em Cadeia da Polimerase , Espanha , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transformação BacterianaRESUMO
We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame DeltaactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA-dependent virulence regulon, including actA, are highly expressed in vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the DeltaactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA* bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro) or prfA* DeltaactA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.
Assuntos
Proteínas de Bactérias/fisiologia , Listeria monocytogenes/fisiologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Células COS , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , DNA Bacteriano , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Células HeLa , Humanos , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/metabolismo , Listeria monocytogenes/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura/métodos , Dados de Sequência Molecular , Mutagênese , Fatores de Terminação de Peptídeos , Transativadores , Células VeroRESUMO
The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.
Assuntos
Colesterol Oxidase/genética , Colesterol Oxidase/metabolismo , Rhodococcus equi/enzimologia , Rhodococcus equi/genética , Alelos , Sequência de Aminoácidos , Animais , Colesterol Oxidase/química , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Troca Genética , Escherichia coli/enzimologia , Hemólise , Dados de Sequência Molecular , Mutagênese Insercional , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rhodococcus equi/patogenicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ovinos , Streptomyces/enzimologia , VirulênciaRESUMO
The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.
Assuntos
Proteínas de Bactérias/genética , Listeria/patogenicidade , Listeriose/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Humanos , Listeria/genética , Virulência/genéticaRESUMO
As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.
Assuntos
Listeria/genética , Listeria/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos/genética , Evolução Molecular , Humanos , Listeriose/microbiologia , Dados de Sequência Molecular , Família Multigênica , Virulência/genéticaRESUMO
We describe here the fourth listerial membrane-damaging virulence factor, a sphingomyelinase C (SMase) that is produced specifically by the ruminant pathogen Listeria ivanovii. Its coding gene, smcL, is a monocistron expressed independently of PrfA. The smcL product, SmcL, is highly similar to the staphylococcal beta-toxin and is responsible for the differential hemolytic properties of L. ivanovii (bizonal hemolysis and CAMP-like reaction with R. equi). The role of SmcL in virulence was assessed by gene disruption and complementation. Our data show that SmcL mediates disruption of the membrane of primary phagosomes, thereby promoting bacterial intracellular proliferation. They also suggest that SmcL may play a role in host tropism. smcL is located in LIPI-2, a novel 18-kb pathogenicity island which also contains a cluster of internalin genes. LIPI-2 is unstable, L. ivanovii-specific and required for full virulence in mice and lambs.
Assuntos
Membrana Celular/efeitos dos fármacos , Listeria/patogenicidade , Esfingomielina Fosfodiesterase/toxicidade , Sequência de Aminoácidos , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Listeria/genética , Camundongos , Dados de Sequência Molecular , Fagossomos/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Transcrição Gênica , VirulênciaRESUMO
The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-shaped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (beta-toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock-out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel-shaped CAMP-like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane-damaging activity that, together with PlcA and PlcB, may act in concert with the pore-forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5' end of smcL is contiguous with the internalin locus i-inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.
Assuntos
Listeria/genética , Esfingomielina Fosfodiesterase/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bovinos , Divisão Celular , Linhagem Celular , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Hemólise , Listeria/enzimologia , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Fatores de Terminação de Peptídeos , Fagocitose , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/química , Transativadores/genética , VirulênciaRESUMO
Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.