ABSTRACT
Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.
Subject(s)
DNA, Bacterial , Drinking Water , Legionella , RNA, Ribosomal, 16S , Water Microbiology , Legionella/isolation & purification , Legionella/genetics , Legionella/classification , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , RNA, Ribosomal, 16S/genetics , Drinking Water/microbiology , Polymerase Chain Reaction/methodsABSTRACT
Public bath facilities are a major source of Legionella infections in Japan. In this study, we performed 16S rRNA gene amplicon sequencing to characterize the bacterial community in bath and shower water from public bath facilities, along with chemical parameters, and investigated the effect of the bacterial microbiome on the presence of Legionella species. Although no significant difference in bacterial community richness was observed between bath and shower water samples, there was a remarkable difference in the bacterial community structure between them. Distance-based redundancy analysis revealed that several factors (free residual chlorine, pH, and conductivity) were correlated with the bacterial community in bath water. The most abundant bacterial genera in the samples were Pseudomonas (13.7%) in bath water and Phreatobacter (13.6%) in shower water, as indicated by the taxonomic composition, and the dominant bacteria differed between these environmental samples. Legionella pneumophila was the most frequently detected Legionella species, with additional 15 other Legionella species detected in water samples. In Legionella-positive water samples, several unassigned and uncultured bacteria were enriched together. In addition, the co-occurrence network showed that Legionella was strongly interconnected with two uncultured bacteria. Corynebacterium and Sphingomonas negatively correlated with Legionella species. The present study reveals the ecology of Legionella species, especially their interactions with other bacteria that are poorly understood to date. IMPORTANCE: Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.
Subject(s)
Legionella pneumophila , Legionella , Legionellosis , Microbiota , Humans , Legionella/genetics , RNA, Ribosomal, 16S/genetics , Water , Genes, rRNA , Water Microbiology , Legionella pneumophila/geneticsABSTRACT
Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.
Subject(s)
Disease Outbreaks , Legionellosis , Spectroscopy, Fourier Transform Infrared/methods , Humans , Japan/epidemiology , Legionellosis/epidemiology , Legionellosis/diagnosis , Legionellosis/microbiology , Polymorphism, Single Nucleotide , Bacterial Typing Techniques/methods , Legionella pneumophila/genetics , Legionella pneumophila/isolation & purification , Legionella pneumophila/classification , Legionella/genetics , Legionella/isolation & purification , Legionella/classificationABSTRACT
The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.
Subject(s)
Legionella pneumophila , Legionella , Legionnaires' Disease , Humans , Acetyltransferases/metabolism , Eukaryotic Initiation Factor-3/metabolism , Lysine/metabolism , Prokaryotic Initiation Factor-3/metabolism , Legionella/genetics , Legionella pneumophila/genetics , Protein Biosynthesis , Bacterial Proteins/metabolismABSTRACT
In this study, we compared conventional vacuum filtration of small volumes through disc membranes (effective sample volumes for potable water: 0.3-1.0 L) with filtration of high volumes using ultrafiltration (UF) modules (effective sample volumes for potable water: 10.6-84.5 L) for collecting bacterial biomass from raw, finished, and tap water at seven drinking water systems. Total bacteria, Legionella spp., Legionella pneumophila, Mycobacterium spp., and Mycobacterium avium complex in these samples were enumerated using both conventional quantitative PCR (qPCR) and viability qPCR (using propidium monoazide). In addition, PCR-amplified gene fragments were sequenced for microbial community analysis. The frequency of detection (FOD) of Legionella spp. in finished and tap water samples was much greater using UF modules (83% and 77%, respectively) than disc filters (24% and 33%, respectively). The FODs for Mycobacterium spp. in raw, finished, and tap water samples were also consistently greater using UF modules than disc filters. Furthermore, the number of observed operational taxonomic units and diversity index values for finished and tap water samples were often substantially greater when using UF modules as compared to disc filters. Conventional and viability qPCR yielded similar results, suggesting that membrane-compromised cells represented a minor fraction of total bacterial biomass. In conclusion, our research demonstrates that large-volume filtration using UF modules improved the detection of opportunistic pathogens at the low concentrations typically found in public drinking water systems and that the majority of bacteria in these systems appear to be viable in spite of disinfection with free chlorine and/or chloramine.IMPORTANCEOpportunistic pathogens, such as Legionella pneumophila, are a growing public health concern. In this study, we compared sample collection and enumeration methods on raw, finished, and tap water at seven water systems throughout the State of Minnesota, USA. The results showed that on-site filtration of large water volumes (i.e., 500-1,000 L) using ultrafiltration membrane modules improved the frequency of detection of relatively rare organisms, including opportunistic pathogens, compared to the common approach of filtering about 1 L using disc membranes. Furthermore, results from viability quantitative PCR (qPCR) with propidium monoazide were similar to conventional qPCR, suggesting that membrane-compromised cells represent an insignificant fraction of microorganisms. Results from these ultrafiltration membrane modules should lead to a better understanding of the microbial ecology of drinking water distribution systems and their potential to inoculate premise plumbing systems with opportunistic pathogens where conditions are more favorable for their growth.
Subject(s)
Azides , Drinking Water , Legionella pneumophila , Legionella , Mycobacterium , Propidium/analogs & derivatives , Drinking Water/microbiology , Mycobacterium/genetics , Water Microbiology , Water Supply , Legionella/geneticsABSTRACT
BACKGROUND: Phigenics Validation Test (PVT) VIABLE® (Viability Identification Assay by Legionella Enrichment) is a method to detect viable Legionella bacteria in building water systems. OBJECTIVE: To evaluate PVT VIABLE against the ISO 11731:2017 Water Quality-Enumeration of Legionella reference method for the detection of viable Legionella species in potable and non-potable water. METHODS: PVT VIABLE was tested for inclusivity (n = 50 strains of Legionella) and exclusivity (n = 30 non-target strains), robustness, and stability. A multi-laboratory instrument variation study was performed to evaluate the PCR data. The matrix study was performed on potable and non-potable water samples inoculated with Legionella pneumophila at a low (n = 20) and high fractional level (n = 5). Samples were analyzed using the PVT VIABLE and confirmed using ISO 11731:2017. RESULTS: Statistical analysis showed no difference between PVT VIABLE and ISO 11731:2017 for 100 mL test portions of potable and non-potable water. PVT VIABLE demonstrated high levels of specificity and sensitivity in the inclusivity and exclusivity study. Results of the robustness and stability studies demonstrated the method was sufficiently robust to handle small method changes and met the method claims of stability. CONCLUSION: PVT VIABLE allows the end user to obtain presumptive results for viable Legionella spp. contamination of potable and non-potable water in 2-3 days. HIGHLIGHTS: PVT VIABLE provides viable Legionella results in 2-3 days versus 10-14 days for traditional spread plating. This novel diagnostic tool also differentiates between Legionella pneumophila sg1, Legionella pneumophila sg2-15, and Legionella spp. without the need for additional confirmation steps as outlined in ISO 11731:2017.
Subject(s)
Legionella pneumophila , Legionella , Legionella/genetics , Water Microbiology , Polymerase Chain ReactionABSTRACT
The intracellular pathogen Legionella pneumophila translocates more than 300 effector proteins into its host cells. The expression levels of the genes encoding these effectors are orchestrated by an intricate regulatory network. Here, we introduce LelA, the first L. pneumophila LysR-type transcriptional regulator of effectors. Through bioinformatic and experimental analyses, we identified the LelA target regulatory element and demonstrated that it directly activates the expression of three L. pneumophila effectors (legL7, legL6, and legU1). We further found that the gene encoding LelA is positively regulated by the RpoS sigma factor, thus linking it to the known effector regulatory network. Examination of other species throughout the Legionella genus revealed that this regulatory element is found upstream of 34 genes encoding validated effectors, putative effectors, and hypothetical proteins. Moreover, ten of these genes were examined and found to be activated by the L. pneumophila LelA as well as by their orthologs in the corresponding species. LelA represents a novel type of Legionella effector regulator, which coordinates the expression of both adjacently and distantly located effector-encoding genes, thus forming small groups of co-regulated effectors.
Subject(s)
Legionella pneumophila , Legionella , Legionella/genetics , Legionella/metabolism , Bacterial Proteins/metabolism , Legionella pneumophila/metabolism , Sigma Factor/genetics , Sigma Factor/metabolism , Regulatory Sequences, Nucleic AcidABSTRACT
Legionella is an important waterborne pathogen that causes legionellosis. Public baths are considered the primary cause of legionellosis infection in Japan. We investigated the prevalence and genetic distribution of 338 Legionella spp. isolates from 81 public bath facilities, including 35 hot springs and 46 other facilities, through annual periodic surveillance in Kobe, Japan, from 2016 to 2021. In addition, the genotypes of nine clinical strains of unknown infectious source from the same period were compared to those of bathwater isolates. We elucidated the differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths. Legionella israelensis, L. londiniensis, and L. micdadei colonized hot springs along with L. pneumophila. The minimum spanning tree analysis based on multiple-locus variable number tandem repeat analysis (MLVA) also identified four major clonal complexes (CCs) in L. pneumophila SG1 and found that CC1 of the four CCs is a specific novel genotype with the lag-1 gene in hot springs. The same MLVA genotypes and sequence types as those of the clinical strains were not present among the strains isolated from bath water. Thus, our surveillance is useful for estimating the sources of legionellosis infection in Japan and developing prevention strategies.
Subject(s)
Legionella pneumophila , Legionella , Legionellosis , Humans , Legionella pneumophila/genetics , Japan/epidemiology , Prevalence , Water Microbiology , Legionella/genetics , Legionellosis/epidemiologyABSTRACT
BACKGROUND: Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes, and springs, as well as in engineered water systems where they can potentially lead to human disease outbarks. Legionella are considered to be predominantly freshwater organisms with a limited ability to proliferate in saline environments. Exposure of Legionella to high sodium concentrations inhibits growth and virulence of laboratory strains, particularly under elevated temperatures. Nonetheless, Legionella have been identified in some saline environments where they likely interact with various protozoan hosts. In this work, we examine how these selection pressures, sodium and grazing, help shape Legionella ecology within natural environments. Utilizing Legionella-specific primers targeting a variable region of the Legionella 16S rRNA gene, we characterized Legionella abundance, diversity, and community composition in natural spring clusters of varying sodium concentrations, focusing on high sodium concentrations and elevated temperatures. RESULTS: We observed the highest abundance of Legionella in spring clusters of high salinity, particularly in combination with elevated temperatures. Legionella abundance was strongly related to sodium concentrations. The Legionella community structure in saline environments was characterized by relatively low diversity, compared to spring clusters of lower salinity. The community composition in high salinity was characterized by few dominant Legionella genotypes, not related to previously described species. Protozoan microbial community structure and composition patterns resembled those of Legionella, suggesting a common response to similar selection pressures. We examined Legionella co-occurrence with potential protozoan hosts and found associations with Ciliophora and Amoebozoa representatives. CONCLUSIONS: Our results indicate that selection forces in saline environments favor a small yet dominant group of Legionella species that are not closely related to known species. These novel environmental genotypes interact with various protozoan hosts, under environmental conditions of high salinity. Our findings suggest that alternative survival mechanisms are utilized by these species, representing mechanisms distinct from those of well-studied laboratory strains. Our study demonstrate how salinity can shape communities of opportunistic pathogens and their hosts, in natural environments, shedding light on evolutionary forces acting within these complex environments. Video Abstract.
Subject(s)
Legionella , Humans , Legionella/genetics , RNA, Ribosomal, 16S/genetics , Ecology , Water Microbiology , Fresh WaterABSTRACT
For microbiological management of water quality, it is important to identify bacteria and to understand the community structure. To analyze the community structure during water purification and distribution, we selected a distribution system in which water from other water treatment facilities was not mixed with the target water. Changes in the bacterial community structure during treatment and distribution processes in a slow filtration water treatment facility were analyzed using 16S rRNA gene amplicon sequencing with a portable sequencer MinION. The microbial diversity was reduced by chlorination. The genus level diversity increased during distribution and this diversity was maintained through to the terminal tap water. Yersinia and Aeromonas were dominant in the intake water, and Legionella was dominant in the slow sand filtered water. Chlorination greatly reduced the relative abundance of Yersinia, Aeromonas, and Legionella, and these bacteria were not detected in the terminal tap water. Sphingomonas, Starkeya and Methylobacterium became dominant in the water after chlorination. These bacteria could be used as important indicator bacteria to provide useful information for microbiological control in drinking water distribution systems.
Subject(s)
Drinking Water , Legionella , Water Purification , RNA, Ribosomal, 16S/genetics , Bacteria/genetics , Legionella/genetics , High-Throughput Nucleotide SequencingABSTRACT
Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.
Subject(s)
Acanthamoeba , Amoeba , Legionella pneumophila , Legionella , Legionella/genetics , Amoeba/microbiology , Water , Legionella pneumophila/genetics , Acanthamoeba/microbiology , DNA , Hospitals , Water MicrobiologyABSTRACT
Extracellular vesicles (EVs or exosomes) are well described for bacterial pathogens associated with our gastrointestinal system, and more recently as a novel mechanism for environmental persistence, dissemination and infection for human enteric viruses. However, the roles played by EVs in the ancient arms race that continues between amoebae and one of their prey, Legionella pneumophila, is poorly understood. At best we know of intracellular vesicles of amoebae containing a mix of bacterial prey species, which also provides an enhanced niche for bacteriophage infection/spread. Free-living amoeba-associated pathogens have recently been recognized to have enhanced resistance to disinfection and environmental stressors, adding to previously understood (but for relatively few species of) bacteria sequestered within amoebal cysts. However, the focus of the current work is to review the likely impacts of large numbers of respiratory-sized EVs containing numerous L. pneumophila cells studied in pure and biofilm systems with mixed prey species. These encapsulated pathogens are orders of magnitude more resistant to disinfection than free cells, and our engineered systems with residual disinfectants could promote evolution of resistance (including AMR), enhanced virulence and EV release. All these are key features for evolution within a dead-end human pathogen post lung infection. Traditional single-hit pathogen infection models used to estimate the probability of infection/disease and critical environmental concentrations via quantitative microbial risk assessments may also need to change. In short, recognizing that EV-packaged cells are highly virulent units for transmission of legionellae, which may also modulate/avoid human host immune responses. Key data gaps are raised and a previous conceptual model expanded upon to clarify where biofilm EVs could play a role promoting risk as well as inform a more wholistic management program to proactively control legionellosis.
Subject(s)
Amoeba , Extracellular Vesicles , Legionella pneumophila , Legionella , Sepsis , Humans , Legionella/genetics , Water , Amoeba/microbiologyABSTRACT
Extrapulmonary manifestations of infection with Legionella species, of which 24 may cause disease in humans, are very rare. Here, we describe a case of a 61-year-old woman with no history of immunosuppression presenting with pain and swelling of her index finger after a prick by rose thorns during gardening. Clinical examination showed fusiform swelling of the finger with mild redness, warmth, and fever. The blood sample revealed a normal white blood cell count and a slight increase in C-reactive protein. Intraoperative observation showed extensive infectious destruction of the tendon sheath, while the flexor tendons were spared. Conventional cultures were negative, while 16S rRNA PCR analysis identified Legionella longbeachae that also could be isolated on buffered charcoal yeast extract media. The patient was treated with oral levofloxacin for 13 days, and the infection healed quickly. The present case report, with a review of the literature, indicates that Legionella species wound infections may be underdiagnosed due to the requirement for specific media and diagnostic methods. It emphasizes the need for heightened awareness of these infections during history taking and clinical examination of patients presenting with cutaneous infections.
Subject(s)
Legionella longbeachae , Legionella , Legionellosis , Wound Infection , Humans , Female , Middle Aged , Legionella longbeachae/genetics , Legionella/genetics , RNA, Ribosomal, 16S/genetics , Legionellosis/diagnosisABSTRACT
AIMS: The ISO 11731 norm, published in 2017, describes a method to identify and enumerate Legionella based exclusively on the confirmation of presumptive colonies by subculturing them on BCYE and BCYE-cys agar (BCYE agar without L-cysteine). METHODS AND RESULTS: Despite this recommendation, our laboratory has kept confirming all presumptive Legionella colonies by combining the subculture method with the latex agglutination and polymerase chain reaction (PCR) assays. Here, we show that the ISO 11731:2017 method adequately performs in our laboratory according to ISO 13843:2017. We compared the performance of the ISO method in detecting Legionella in typical and atypical colonies (n = 7156) from health care facilities (HCFs) water samples to that of our combined protocol, and we found a 2.1% false positive rate (FPR), underscoring the importance of combining agglutination test and PCR with subculture to achieve optimal confirmation. Lastly, we estimated the water system disinfection cost for HCFs (n = 7), which due to false positive results, would display Legionella values exceeding the threshold of risk acceptance established by the Italian guidelines. CONCLUSIONS: Overall, this large-scale study indicates that the ISO 11731:2017 confirmation method is error-prone, leading to significant FPRs, and higher costs for HCFs due to remedial actions on their water systems.
Subject(s)
Legionella pneumophila , Legionella , Legionella/genetics , Culture Media , Agar , Water Microbiology , WaterABSTRACT
Introduction: Legionnaires' Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal. Methods: Minimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. Results: Fluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline. Discussion: MIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella.
Subject(s)
Legionella pneumophila , Legionella , Legionnaires' Disease , Humans , Legionella/genetics , Levofloxacin/pharmacology , Portugal , Azithromycin/pharmacology , Clarithromycin/pharmacology , Doxycycline , Legionella pneumophila/genetics , Anti-Bacterial Agents/pharmacology , Fluoroquinolones/pharmacology , Ciprofloxacin/pharmacology , Microbial Sensitivity TestsABSTRACT
Background: Legionella pneumonia, rhabdomyolysis, and acute kidney injury are called the Legionella triad, which is rare and associated with a poor outcome and even death. Early diagnosis and timely treatment are essential for these patients. Case presentation: A 63-year-old man with cough, fever, and fatigue was initially misdiagnosed with common bacterial infection and given beta-lactam monotherapy but failed to respond to it. Conventional methods, including the first Legionella antibody test, sputum smear, and culture of sputum, blood, and bronchoalveolar lavage fluid (BALF) were negative. He was ultimately diagnosed with a severe infection of Legionella pneumophila by metagenomics next-generation sequencing (mNGS). This patient, who had multisystem involvement and manifested with the rare triad of Legionella pneumonia, rhabdomyolysis, and acute kidney injury, finally improved after combined treatment with moxifloxacin, continuous renal replacement therapy, and liver protection therapy. Conclusion: Our results showed the necessity of early diagnosis of pathogens in severe patients, especially in Legionnaires' disease, who manifested with the triad of Legionella pneumonia, rhabdomyolysis, and acute kidney injury. mNGS may be a useful tool for Legionnaires' disease in limited resource areas where urine antigen tests are not available.
Subject(s)
Acute Kidney Injury , Legionella , Legionnaires' Disease , Pneumonia , Rhabdomyolysis , Male , Humans , Middle Aged , Legionnaires' Disease/complications , Legionnaires' Disease/diagnosis , Legionnaires' Disease/microbiology , Acute Kidney Injury/diagnosis , Acute Kidney Injury/etiology , Legionella/genetics , Rhabdomyolysis/diagnosis , Rhabdomyolysis/complications , High-Throughput Nucleotide SequencingABSTRACT
To replicate within host cells, bacterial pathogens must acquire host-derived nutrients while avoiding degradative antimicrobial pathways. Fundamental insights into bacterial pathogenicity have been revealed by bacteria of the genus Legionella, which naturally parasitize free-living protozoa by establishing a membrane-bound replicative niche termed the Legionella-containing vacuole (LCV). Biogenesis of the LCV and intracellular replication rely on rapid evasion of the endocytic pathway and acquisition of host-derived nutrients, much of which is mediated by bacterial effector proteins translocated into host cells by a Dot/Icm type IV secretion system. Billions of years of co-evolution with eukaryotic hosts and broad host tropism have resulted in expansion of the Legionella genome to accommodate a massive repertoire of effector proteins that promote LCV biogenesis, safeguard the LCV from endolysosomal maturation, and mediate the acquisition of host nutrients. This minireview is focused on the mechanisms by which an ancient intracellular pathogen leverages effector proteins and hijacks host cell biology to obtain essential host-derived nutrients and prevent lysosomal degradation.
Subject(s)
Legionella pneumophila , Legionella , Legionella/genetics , Legionella/metabolism , Legionella pneumophila/metabolism , Vacuoles/microbiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Lysosomes/metabolism , Nutrients , Host-Pathogen InteractionsABSTRACT
Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.
Subject(s)
Environmental Monitoring , Hospitals , Water Microbiology , Water Supply , Environmental Monitoring/methods , Italy , Microbiological Techniques/standards , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Legionella/genetics , Legionella/isolation & purification , Sequence Analysis, DNAABSTRACT
Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.
Subject(s)
Legionella pneumophila , Legionella , Legionnaires' Disease , Humans , Legionella pneumophila/genetics , Legionella pneumophila/metabolism , Legionella/genetics , Legionnaires' Disease/metabolism , Macrophages/microbiologyABSTRACT
The reported rate of legionellosis is increasing in Aotearoa New Zealand (NZ) with most cases community-acquired, sporadic (non-outbreak) and without an identifiable source. This analysis used two datasets to describe the environmental sources that contribute to Legionella in NZ, based on linkages with outbreaks and sporadic clinical cases, and analysis of environmental testing data. These findings highlight the need for enhanced environmental investigation of clinical cases and outbreaks. There is also a need for systematic surveillance testing of high-risk source environments to support more rigorous controls to prevent legionellosis.