RESUMO
Legionella pneumophila is a human pathogen that causes Legionnaires' disease, a severe form of pneumonia. It can be found in various aquatic environments ranging from cooling towers to ponds. In addition to causing disease in humans, it can also infect free-living amoebae commonly found in various aquatic environments. Once inside a human lung macrophage, it creates a niche called the Legionella-containing vacuole where it can evade phagolysosomal degradation and replicate. During infection, normal cellular functions are hijacked by proteins that are secreted by the pathogen, called bacterial effectors. Here, the structural characterization of the effector LegA15/AnkD is reported. The protein contains an ankyrin-repeat domain followed by a cysteine protease-like (CPL) domain with a putative catalytic triad consisting of His268-Asn290-Cys361. The CPL domain shows similarity to the CE clan in the MEROPS database, which contains ubiquitin-like hydrolases. The C-terminal segment of LegA15, including the CPL domain, shows structural similarity to another effector, LegA3/AnkH, while they share only 12% sequence identity. When expressed in mammalian cells, LegA15 is localized within the cytoplasm, in contrast to LegA3, which localizes to the nucleus.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína Proteases/metabolismo , Legionella/metabolismo , Proteínas de Bactérias/química , Cisteína Proteases/química , Interações Hospedeiro-Patógeno , Legionella/patogenicidade , Conformação Proteica , Domínios ProteicosRESUMO
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
Assuntos
Complexo de Golgi/patologia , Legionella/patogenicidade , Serina/metabolismo , Humanos , UbiquitinaçãoRESUMO
Healthcare-related Legionnaires' disease has a devastating impact on high risk patients, with a case fatality rate of 30-50%. Legionella prevention and control in hospitals is therefore crucial. To control Legionella water colonisation in a hospital setting we evaluated the effect of pipeline improvements and temperature increase, analysing 237 samples over a 2-year period (first year: 129, second year: 108). In the first year, 25.58% of samples were positive for Legionella and 16.67% for amoeba. Assessing the distance of the points analysed from the hot water tank, the most distal points presented higher proportion of Legionella colonisation and lower temperatures (nearest points: 6.4% colonised, and temperature 61.4 °C; most distal points: 50% and temperature 59.1 °C). After the first year, the hot water system was repaired and the temperature stabilised. This led to a dramatic reduction in Legionella colonisation, which was negative in all the samples analysed; however, amoeba colonisation remained stable. This study shows the importance of keeping the temperature stable throughout the circuit, at around 60 °C. Special attention should be paid to the most distal points of the circuit; a fall in temperature at these weak points would favour the colonisation and spread of Legionella, because amoeba (the main Legionella reservoir) are not affected by temperature.
Assuntos
Legionella/patogenicidade , Doença dos Legionários/microbiologia , Doença dos Legionários/prevenção & controle , Microbiologia da Água , Amoeba/patogenicidade , Infecção Hospitalar , Hospitais , Humanos , Legionella/crescimento & desenvolvimento , Doença dos Legionários/epidemiologia , Temperatura , Abastecimento de ÁguaRESUMO
Through coevolution with host cells, microorganisms have acquired mechanisms to avoid the detection by the host surveillance system and to use the cell's supplies to establish themselves. Indeed, certain pathogens have evolved proteins that imitate specific eukaryotic cell proteins, allowing them to manipulate host pathways, a phenomenon termed molecular mimicry. Bacterial "eukaryotic-like proteins" are a remarkable example of molecular mimicry. They are defined as proteins that strongly resemble eukaryotic proteins or that carry domains that are predominantly present in eukaryotes and that are generally absent from prokaryotes. The widest diversity of eukaryotic-like proteins known to date can be found in members of the bacterial genus Legionella, some of which cause a severe pneumonia in humans. The characterization of a number of these proteins shed light on their importance during infection. The subsequent identification of eukaryotic-like genes in the genomes of other amoeba-associated bacteria and bacterial symbionts suggested that eukaryotic-like proteins are a common means of bacterial evasion and communication, shaped by the continuous interactions between bacteria and their protozoan hosts. In this review, we discuss the concept of molecular mimicry using Legionella as an example and show that eukaryotic-like proteins effectively manipulate host cell pathways. The study of the function and evolution of such proteins is an exciting field of research that is leading us toward a better understanding of the complex world of bacterium-host interactions. Ultimately, this knowledge will teach us how host pathways are manipulated and how infections may possibly be tackled.
Assuntos
Coevolução Biológica/genética , Interações entre Hospedeiro e Microrganismos/genética , Legionella/genética , Mimetismo Molecular , Proteínas de Bactérias/genética , Genoma Bacteriano , Humanos , Legionella/patogenicidade , Fatores de Virulência/genéticaRESUMO
Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.
Assuntos
Acanthamoeba castellanii/fisiologia , Bdellovibrio/isolamento & purificação , Legionella/isolamento & purificação , Microbiota/fisiologia , Oxigênio/metabolismo , Acanthamoeba castellanii/genética , Bdellovibrio/genética , Bdellovibrio/fisiologia , DNA/isolamento & purificação , Legionella/genética , Legionella/patogenicidade , Legionella/fisiologia , Lagoas/microbiologia , Lagoas/parasitologia , RNA Ribossômico 16S/química , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , VirulênciaRESUMO
Intracellular bacterial pathogens harbour genes, the closest homologues of which are found in eukaryotes. Regulator of chromosome condensation 1 (RCC1) repeat proteins are phylogenetically widespread and implicated in protein-protein interactions, such as the activation of the small GTPase Ran by its cognate guanine nucleotide exchange factor, RCC1. Legionella pneumophila and Coxiella burnetii, the causative agents of Legionnaires' disease and Q fever, respectively, harbour RCC1 repeat coding genes. Legionella pneumophila secretes the RCC1 repeat 'effector' proteins LegG1, PpgA and PieG into eukaryotic host cells, where they promote the activation of the pleiotropic small GTPase Ran, microtubule stabilisation, pathogen vacuole motility and intracellular bacterial growth as well as host cell migration. The RCC1 repeat effectors localise to the pathogen vacuole or the host plasma membrane and target distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself. Coxiella burnetii translocates the RCC1 repeat effector NopA into host cells, where the protein localises to nucleoli. NopA binds to Ran GTPase and promotes the nuclear accumulation of Ran(GTP), thus pertubing the import of the transcription factor NF-κB and innate immune signalling. Hence, divergent evolution of bacterial RCC1 repeat effectors defines the range of Ran GTPase cycle targets and likely allows fine-tuning of Ran GTPase activation by the pathogens at different cellular sites.
Assuntos
Evolução Biológica , Coxiella burnetii/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucléolo Celular/enzimologia , Coxiella burnetii/genética , Coxiella burnetii/patogenicidade , Ativação Enzimática , Genes Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Legionella/genética , Legionella/metabolismo , Legionella/patogenicidade , Legionella pneumophila/genética , Doença dos Legionários/microbiologia , Transporte Proteico , Febre Q/microbiologia , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
Type 1 secretion systems (T1SSs) are broadly distributed among bacteria and translocate effectors with diverse function across the bacterial cell membrane. Legionella pneumophila, the species most commonly associated with Legionellosis, encodes a T1SS at the lssXYZABD locus which is responsible for the secretion of the virulence factor RtxA. Many investigations have failed to detect lssD, the gene encoding the membrane fusion protein of the RtxA T1SS, in non-pneumophila Legionella, which has led to the assumption that this system is a virulence factor exclusively possessed by L. pneumophila. Here we discovered RtxA and its associated T1SS in a novel Legionella taurinensis strain, leading us to question whether this system may be more widespread than previously thought. Through a bioinformatic analysis of publicly available data, we classified and determined the distribution of four T1SSs including the RtxA T1SS and four novel T1SSs among diverse Legionella spp. The ABC transporter of the novel Legionella T1SS Legionella repeat protein secretion system shares structural similarity to those of diverse T1SS families, including the alkaline protease T1SS in Pseudomonas aeruginosa. The Legionella bacteriocin (1-3) secretion systems T1SSs are novel putative bacteriocin transporting T1SSs as their ABC transporters include C-39 peptidase domains in their N-terminal regions, with LB2SS and LB3SS likely constituting a nitrile hydratase leader peptide transport T1SSs. The LB1SS is more closely related to the colicin V T1SS in Escherichia coli. Of 45 Legionella spp. whole genomes examined, 19 (42%) were determined to possess lssB and lssD homologs. Of these 19, only 7 (37%) are known pathogens. There was no difference in the proportions of disease associated and non-disease associated species that possessed the RtxA T1SS (p = 0.4), contrary to the current consensus regarding the RtxA T1SS. These results draw into question the nature of RtxA and its T1SS as a singular virulence factor. Future studies should investigate mechanistic explanations for the association of RtxA with virulence.
Assuntos
Proteínas de Bactérias/genética , Proteínas Hemolisinas/genética , Legionella/genética , Legionelose/genética , Sistemas de Secreção Tipo I/genética , Transportadores de Cassetes de Ligação de ATP/genética , Membrana Celular/genética , Biologia Computacional , Escherichia coli/genética , Genoma Bacteriano/genética , Humanos , Legionella/patogenicidade , Legionella pneumophila/genética , Legionelose/microbiologia , Análise de Sequência , Fatores de Virulência/genética , Sequenciamento Completo do GenomaRESUMO
CONTEXT: The Veterans Health Administration requires implementation of Legionella prevention policy in potable water systems at Department of Veterans Affairs (VA) medical facilities across the United States and territories. PROGRAM: The Veterans Health Administration Central Office program offices with expertise in engineering and clinical aspects of Legionella prevention policy have provided joint, structured on-site assistance to VA medical facilities for consultation on policy implementation. Site visits included review of facility documentation and data, discussions with staff, touring of buildings, and development of recommendations. IMPLEMENTATION: Information obtained from on-site consultative assistance provided to VA medical facilities from December 2012 through January 2018 was reviewed to identify engineering and clinical challenges and lessons from implementation of Legionella prevention policy in VA health care buildings. Fifteen consultative site visits were conducted during this period regarding implementation of Legionella prevention and validation of effectiveness. EVALUATION: It was found that implementation of Legionella prevention policy in potable water systems was complex and practices varied for each building. Common implementation challenges included capability of applying engineering controls, water stagnation, and assessment of health care association of Legionella cases. Process challenges included routine verification of actions, methods for assessing environmental validation data, and documentation of requirements. It was found that consistent and data-driven implementation of policy is crucial for an effective program. DISCUSSION: Guidance and standards documents in the community for Legionella prevention in building water systems are often general in nature, but implementation requires specific decisions and routine assessments and modifications to optimize outcomes. This real-world review of challenges and lessons from a large health care system with a detailed primary Legionella prevention policy informs future development of guidance and policy, both within and external to VA, and can provide insight to other health care facilities planning to implement practices for water safety.
Assuntos
Política de Saúde/tendências , Controle de Infecções/métodos , Doença dos Legionários/prevenção & controle , Humanos , Controle de Infecções/normas , Controle de Infecções/estatística & dados numéricos , Legionella/patogenicidade , Doença dos Legionários/epidemiologia , Melhoria de Qualidade , Estados Unidos/epidemiologia , United States Department of Veterans Affairs/organização & administração , United States Department of Veterans Affairs/estatística & dados numéricosRESUMO
Legionella-related disease is caused by an intracellular bacteria mainly living in water. Contamination results from inhalation of Legionella sp containing aerosolized water. Main risk factors are tobacco, immunodeficiency, and advanced age. Antigenuria is the cornerstone of the diagnosis. Immunocompromised patients, more commonly infected with non pneumophilaLegionella, present negative antigenuria, and culture and PCR are essential for the diagnosis. Legionnaires' disease may be severe, especially in elderly and/or immunocompromised patients. Mortality rate varies from 10 % in the general population to 50 % in intensive care. Treatment is based on macrolides or fluoroquinolones. Antibiotic resistance is very rare.
Assuntos
Legionella/patogenicidade , Legionelose , Doença dos Legionários , Idoso , Antibacterianos/uso terapêutico , Diagnóstico Diferencial , Surtos de Doenças , Humanos , Hospedeiro Imunocomprometido , Legionelose/diagnóstico , Legionelose/epidemiologia , Legionelose/etiologia , Legionelose/terapia , Doença dos Legionários/diagnóstico , Doença dos Legionários/epidemiologia , Doença dos Legionários/etiologia , Doença dos Legionários/terapia , Reação em Cadeia da Polimerase , Fatores de RiscoRESUMO
The facultative intracellular bacterium Legionella pneumophila replicates in environmental amoebae and in lung macrophages, and causes Legionnaires' disease. Here we show that L. pneumophila reversibly forms replicating and nonreplicating subpopulations of similar size within amoebae. The nonreplicating bacteria are viable and metabolically active, display increased antibiotic tolerance and a distinct proteome, and show high virulence as well as the capacity to form a degradation-resistant compartment. Upon infection of naïve or interferon-γ-activated macrophages, the nonreplicating subpopulation comprises ca. 10% or 50%, respectively, of the total intracellular bacteria; hence, the nonreplicating subpopulation is of similar size in amoebae and activated macrophages. The numbers of nonreplicating bacteria within amoebae are reduced in the absence of the autoinducer synthase LqsA or other components of the Lqs quorum-sensing system. Our results indicate that virulent, antibiotic-tolerant subpopulations of L. pneumophila are formed during infection of evolutionarily distant phagocytes, in a process controlled by the Lqs system.
Assuntos
Legionella/patogenicidade , Legionelose/microbiologia , Macrófagos/microbiologia , Percepção de Quorum , Amoeba/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Espaço Intracelular/microbiologia , Legionella/crescimento & desenvolvimento , Camundongos , Viabilidade Microbiana , Proteoma/metabolismo , Vacúolos/microbiologia , VirulênciaRESUMO
The UK Cellular Microbiology Network held its inaugural conference in February 2019. This stimulating day of scientific exchange will be the first of many, its organisers hope.
Assuntos
Legionella/metabolismo , Infecções Pneumocócicas/imunologia , Infecções por Salmonella/metabolismo , Staphylococcus aureus/metabolismo , Tuberculose/imunologia , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Celular , Legionella/genética , Legionella/patogenicidade , Técnicas Microbiológicas/instrumentação , Técnicas Microbiológicas/métodos , Infecções por Salmonella/genética , Staphylococcus aureus/patogenicidade , Tuberculose/enzimologia , Tuberculose/genética , Tuberculose/metabolismo , Reino UnidoRESUMO
Legionnaires' disease (LD) is an atypical pneumonia caused by the inhalation of Legionella. The methods used for the diagnosis of LD are direct culture of respiratory samples and urinary antigen detection. However, the sensitivity of culture is low, and the urinary antigen test is specific only for L. pneumophila sg1. Moreover, as no isolates are obtained, epidemiological studies cannot be performed. The implementation of Nested-sequence-based typing (Nested-SBT) makes it possible to carry out epidemiological studies while also confirming LD, especially in cases caused by non-sg 1. Sixty-two respiratory samples from patients with Legionella clinically confirmed by positive urinary antigen tests were cultured and tested by Nested-SBT, following the European Study Group for Legionella Infections (ESGLI) protocol. Only 2/62 (3.2%) respiratory samples were culture-positive. Amplification and sequencing of Nested-SBT genes were successfully performed in 57/62 samples (91.9%). The seven target genes were characterised in 39/57 (68.4%) respiratory samples, and the complete sequence type (ST) was obtained. The mip gene was the most frequently amplified and sequenced. Nested-SBT is a useful method for epidemiological studies in culture-negative samples, achieving a 28.7-fold improvement over the results of culture studies and reducing the time needed to obtain molecular epidemiological results.
Assuntos
Legionella pneumophila/patogenicidade , Legionella/patogenicidade , Doença dos Legionários/parasitologia , Epidemiologia Molecular/métodos , Análise de Sequência de DNA/métodos , Alelos , Humanos , Legionella/isolamento & purificação , Legionella pneumophila/isolamento & purificação , Técnicas de Diagnóstico MolecularRESUMO
Legionella pneumophila causes a severe form of pneumonia known as Legionnaires' disease especially in patients with impaired cellular immune response. In order to prevent the disease, immunogenicity and the level of the induction of protective immunity from the recombinant peptidoglycan-associated lipoprotein (rPAL) against Legionella pneumophila in BALB/c mice was examined. Mice immunized with (rPAL) rapidly increased an antibody response in serum and also displayed a strong activation of both innate and adaptive cell-mediated immunity as determined by antigen-specific splenocyte proliferation, an early production of pro-inflammatory cytokines in the serum and in the splenocyte cultures. Infection with a primary sublethal does of Legionella pneumophila serogroup 1, strain paris, caused resistance to a lethal challenge infection in the animals with 100% survival rate. However, mice treated with rPAL survived with 60% rate in 10 days after a lethal i.v challenge with L. pneumophila. All of the control animals receiving PBS died within 24â¯h. The present study indicates that recombinant protein PAL of Legionella pneumophila is strongly immunogenic and capable to elicit early innate and adaptive immune responses and lasting immunity against a lethal dose of Legionella pneumophila challenge. Antigenic characterization and immune protection of recombinant protein PAL would be of considerable value in comprehension the immune-pathogenesis of the disease and in development possible vaccine against the Legionella.
Assuntos
Vacinas Bacterianas/imunologia , Imunidade , Legionella pneumophila/imunologia , Doença dos Legionários/prevenção & controle , Lipoproteínas/imunologia , Peptidoglicano/imunologia , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologia , Animais , Formação de Anticorpos , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Imunidade Celular , Imunidade Inata , Imunização , Legionella/imunologia , Legionella/patogenicidade , Legionella pneumophila/genética , Doença dos Legionários/imunologia , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Peptidoglicano/genética , Taxa de Sobrevida , Vacinas Sintéticas/genéticaRESUMO
Gram-negative bacterium Legionella is able to proliferate intracellularly in mammalian host cells and amoeba, which became known in 1976 since they caused a large outbreak of pneumonia. It had been reported that different strains of Legionella pneumophila, Legionella micdadei, Legionella longbeachae, and Legionella feeleii caused human respiratory diseases, which were known as Pontiac fever or Legionnaires' disease. However, the differences of the virulence traits among the strains of the single species and the pathogenesis of the two diseases that were due to the bacterial virulence factors had not been well elucidated. L. feeleii is an important pathogenic organism in Legionellae, which attracted attention due to cause an outbreak of Pontiac fever in 1981 in Canada. In published researches, it has been found that L. feeleii serogroup 2 (ATCC 35849, LfLD) possess mono-polar flagellum, and L. feeleii serogroup 1 (ATCC 35072, WRLf) could secrete some exopolysaccharide (EPS) materials to the surrounding. Although the virulence of the L. feeleii strain was evidenced that could be promoted, the EPS might be dispensable for the bacteria that caused Pontiac fever. Based on the current knowledge, we focused on bacterial infection in human and murine host cells, intracellular growth, cytopathogenicity, stimulatory capacity of cytokines secretion, and pathogenic effects of the EPS of L. feeleii in this review.
Assuntos
Citocinas/metabolismo , Legionella/patogenicidade , Doença dos Legionários/microbiologia , Doença dos Legionários/patologia , Polissacarídeos Bacterianos/metabolismo , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Humanos , Legionella/crescimento & desenvolvimento , Camundongos , Polissacarídeos Bacterianos/toxicidade , Virulência , Fatores de Virulência/toxicidadeAssuntos
Interações Hospedeiro-Patógeno , Legionella/fisiologia , Legionella/patogenicidade , Doença dos Legionários/patologia , Doença dos Legionários/fisiopatologia , Fatores de Virulência/metabolismo , Acanthamoeba/microbiologia , Pesquisa Biomédica/tendências , Análise Mutacional de DNA , Legionella/genética , Viabilidade Microbiana , Virulência , Fatores de Virulência/genética , Microbiologia da ÁguaRESUMO
In 2017, we surveyed 101 SHEA Research Network hospitals regarding Legionnaires' disease (LD). Of 29 respondents, 94% have or are developing a water management plan with varying characteristics and personnel engaged. Most LD diagnostic testing is limited to urine antigen testing. Many opportunities to improve LD prevention and diagnosis exist.
Assuntos
Infecção Hospitalar/prevenção & controle , Desinfecção/métodos , Legionella/isolamento & purificação , Doença dos Legionários/diagnóstico , Doença dos Legionários/prevenção & controle , Abastecimento de Água , Infecção Hospitalar/transmissão , Hospitais , Humanos , Legionella/patogenicidade , Doença dos Legionários/transmissão , Doença dos Legionários/urina , Medição de Risco , Inquéritos e Questionários , Estados UnidosRESUMO
Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.
Assuntos
Acanthamoeba/microbiologia , Dictyostelium/microbiologia , Modelos Animais de Doenças , Legionella/metabolismo , Doença dos Legionários/microbiologia , Doença dos Legionários/veterinária , Acanthamoeba castellanii/microbiologia , Amoeba/microbiologia , Animais , Autofagia , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Evolução Molecular , GTP Fosfo-Hidrolases , Interações Hospedeiro-Patógeno/fisiologia , Legionella/patogenicidade , Legionella pneumophila/metabolismo , Macrófagos/microbiologia , Fosfatidilinositóis/metabolismo , Proteômica , Sistemas de Secreção Tipo IV/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4â¯h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.
Assuntos
Bactérias/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Pneumonia Bacteriana/diagnóstico , Pneumonia por Mycoplasma/diagnóstico , Infecções Respiratórias/diagnóstico , Adolescente , Bactérias/genética , Bactérias/patogenicidade , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/isolamento & purificação , Chlamydophila pneumoniae/patogenicidade , DNA Bacteriano/genética , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Legionella/genética , Legionella/isolamento & purificação , Legionella/patogenicidade , Masculino , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/economia , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/isolamento & purificação , Mycoplasma pneumoniae/patogenicidade , Pneumonia Bacteriana/microbiologia , Pneumonia por Mycoplasma/microbiologia , Kit de Reagentes para Diagnóstico , Infecções Respiratórias/microbiologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Legionella infections are among the most important waterborne infections with constantly increasing numbers of cases in industrialized countries, as a result of aging populations, rising numbers of immunocompromised individuals and increased need for conditioned water due to climate change. Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. In the past two decades, the relevance of such viable but non-culturable (VBNC) legionellae has been controversially discussed, and whether VBNC legionellae can directly infect human macrophages, the primary targets of Legionella infections, remains unclear. In this study, it was demonstrated for the first time that several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efficacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic biofilms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques. Thus, further quantitative research is needed in order to determine, whether and how many starved VBNC Legionella cells are able to cause disease in humans.