Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

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.

Estimating the burden of illness caused by domestic waterborne Legionnaires' disease in Canada: 2015-2019.

Legionellosis is a disease caused by the bacterium Legionella that most commonly presents as Legionnaires' disease (LD), a severe form of pneumonia. From 2015 to 2019, an average of 438 LD cases per year were reported in Canada. However, it is believed that the actual number of cases is much higher, since LD may be underdiagnosed and underreported. The purpose of this study was to develop an estimate of the true incidence of illnesses, hospitalizations, and deaths associated with LD in Canada. Values were derived using a stochastic model, based on Canadian surveillance data from 2015 to 2019, which were scaled up to account for underdiagnosis and underreporting. Overall, there were an estimated 1,113 (90% CrI: 737-1,730) illnesses, 1,008 (90% CrI: 271-2,244) hospitalizations, and 34 (90% CrI: 4-86) deaths due to domestically acquired waterborne LD annually in Canada from 2015 to 2019. It was further estimated that only 36% of illnesses and 39% of hospitalizations and deaths were captured in surveillance, and that 22% of illnesses were caused by Legionella serogroups and species other than Legionella pneumophila serogroup 1 (non-Lp1). This study highlights the true burden and areas for improvement in Canada's surveillance and detection of LD.

Water quality influences Legionella pneumophila determination.

Legionellosis is a respiratory disease of public health concern. The bacterium Legionella pneumophila is the etiologic agent responsible for >90% of legionellosis cases in the United States. Legionellosis transmission primarily occurs through the inhalation or aspiration of contaminated water aerosols or droplets. Therefore, a thorough understanding of L. pneumophila detection methods and their performance in various water quality conditions is needed to develop preventive measures. Two hundred and nine potable water samples were collected from taps in buildings across the United States. L. pneumophila was determined using three culture methods: Buffered Charcoal Yeast Extract (BCYE) culture with Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) identification, Legiolert® 10- and 100-mL tests, and one molecular method: quantitative Polymerase Chain Reaction (qPCR) assay. Culture and molecular positive results were confirmed by secondary testing including MALDI-MS. Eight water quality variables were studied, including source water type, secondary disinfectant, total chlorine residual, heterotrophic bacteria, total organic carbon (TOC), pH, water hardness, cold- and hot-water lines. The eight water quality variables were segmented into 28 categories, based on scale and ranges, and method performance was evaluated in each of these categories. Additionally, a Legionella genus qPCR assay was used to determine the water quality variables that promote or hinder Legionella spp. occurrence. L. pneumophila detection frequency ranged from 2 to 22% across the methods tested. Method performance parameters of sensitivity, specificity, positive and negative predictive values, and accuracy were >94% for the qPCR method but ranged from 9 to 100% for the culture methods. Water quality influenced L. pneumophila determination by culture and qPCR methods. L. pneumophila qPCR detection frequencies positively correlated with TOC and heterotrophic bacterial counts. The source water-disinfectant combination influenced the proportion of Legionella spp. that is L. pneumophila. Water quality influences L. pneumophila determination. To accurately detect L. pneumophila, method selection should consider the water quality in addition to the purpose of testing (general environmental monitoring versus disease-associated investigations).

Increased Incidence of Legionellosis after Improved Diagnostic Methods, New Zealand, 2000-2020.

Legionellosis, notably Legionnaires' disease, is recognized globally and in New Zealand (Aotearoa) as a major cause of community-acquired pneumonia. We analyzed the temporal, geographic, and demographic epidemiology and microbiology of Legionnaires' disease in New Zealand by using notification and laboratory-based surveillance data for 2000‒2020. We used Poisson regression models to estimate incidence rate ratios and 95% CIs to compare demographic and organism trends over 2 time periods (2000-2009 and 2010-2020). The mean annual incidence rate increased from 1.6 cases/100,000 population for 2000-2009 to 3.9 cases/100,000 population for 2010-2020. This increase corresponded with a change in diagnostic testing from predominantly serology with some culture to almost entirely molecular methods using PCR. There was also a marked shift in the identified dominant causative organism, from Legionella pneumophila to L. longbeachae. Surveillance for legionellosis could be further enhanced by greater use of molecular typing of isolates.

Legionellosis risk-an overview of Legionella spp. habitats in Europe.

An increase in the number of reports of legionellosis in the European Union and the European Economic Area have been recorded in recent years. The increase in cases is significant: from 6947 reports in 2015 to 11,298 in 2019. This is alarming as genus Legionella, which comprises a large group of bacteria inhabiting various aquatic systems, poses a serious threat to human health and life, since more than 20 species can cause legionellosis, with L. pneumophila being responsible for the majority of cases. The ability to colonize diverse ecosystems makes the eradication of these microorganisms difficult. A detailed understanding of the Legionella habitat may be helpful in the effective control of this pathogen. This paper provides an overview of Legionella environments in Europe: natural (lakes, groundwater, rivers, compost, soil) and anthropogenic (fountains, air humidifiers, water supply systems), and the role of Legionella spp. in nosocomial infections, which are potentially fatal for children, the elderly and immunocompromised patients.

New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene.

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes. IMPORTANCELegionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

Effects of climate changes and road exposure on the rapidly rising legionellosis incidence rates in the United States.

Legionellosis is an infection acquired through inhalation of aerosols that are contaminated with environmental bacteria Legionella spp. The bacteria require warm temperature for proliferation in bodies of water and moist soil. The legionellosis incidence in the United States has been rising rapidly in the past two decades without a clear explanation. In the meantime, the US has recorded consecutive years of above-norm temperature since 1997 and precipitation surplus since 2008. The present study analyzed the legionellosis incidence in the US during the 20-year period of 1999 to 2018 and correlated with concurrent temperature, precipitation, solar ultraviolet B (UVB) radiation, and vehicle mileage data. The age-adjusted legionellosis incidence rates rose exponentially from 0.40/100,000 in 1999 (with 1108 cases) to 2.69/100,000 in 2018 (with 9933 cases) at a calculated annual increase of 110%. In regression analyses, the rise correlated with an increase in vehicle miles driven and with temperature and precipitation levels that have been above the 1901-2000 mean since 1997 and 2008, respectively, suggesting more road exposure to traffic-generated aerosols and promotive effects of anomalous climate. Remarkably, the regressions with cumulative anomalies of temperature and precipitation were robust (R2 ≥ 0.9145, P ≤ 4.7E-11), implying possible changes to microbial ecology in the terrestrial and aquatic environments. An interactive synergy between annual precipitation and vehicle miles was also found in multiple regressions. Meanwhile, the bactericidal UVB radiation has been decreasing, which also contributed to the rising incidence in an inverse correlation. The 2018 legionellosis incidence peak corresponded to cumulative effects of the climate anomalies, vast vehicle miles (3,240 billion miles, 15904 km per capita), record high precipitation (880.1 mm), near record low UVB radiation (7488 kJ/m2), and continued above-norm temperature (11.96°C). These effects were examined and demonstrated in California, Florida, New Jersey, Ohio, and Wisconsin, states that represent diverse incidence rates and climates. The incidence and above-norm temperature both rose most in cold Wisconsin. These results suggest that warming temperature and precipitation surplus have likely elevated the density of Legionella bacteria in the environment, and together with road exposure explain the rapidly rising incidence of legionellosis in the United States. These trends are expected to continue, warranting further research and efforts to prevent infection.

LegionellaDB - A Database on Legionella Outbreaks.

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online.

Sit bath systems: A new source of Legionella infection.

Sit Bath Systems (SBSs) are the most common hygiene method for patients who are not self-sufficient. Therefore, the water quality of SBSs in the nosocomial environment plays a fundamental role in controlling infections for both patients and health-care workers. A long-term study on Legionella and Pseudomonas aeruginosa (P. aeruginosa) contamination was performed in SBSs (n = 20) of six Health Care Facilities (HCFs). A total of 254 water samples were analyzed following ISO procedures. The samples were positive for P. aeruginosa (46.85%) and Legionella (53.54%), respectively, both over the directive limits. Legionella isolates were identified as: Legionella pneumophila (L. pneumophila) serogroups 1, 3, and 6 and Legionella non-pneumophila species (L. anisa, L. londiniensis, L. rubrilucens, and L. nagelii). Moreover, the contamination found was studied with respect to median temperature measured (42 °C), from which two groups (A and B) could be distinguished. P. aeruginosa was found in both groups (100% of SBSs), while a higher percentage of Legionella positive samples was found in group A (75% of SBSs), compared to group B (50% of SBSs), showing how Legionella control could be carried out by using temperatures above 42 °C. An analysis of SBS water pipelines, maintenance, and disinfection treatments indicates SBSs as a new source of infection risk for both patients and health-care workers.

Distribution of Legionella species and serogroups in patients with culture-confirmed Legionella pneumonia.

Legionella species are consistently identified as some of the most common causative agents of severe community-acquired pneumonia (CAP) or nosocomial pneumonia. Although the number of reported Legionella infection cases is gradually increasing in Japan, most cases are diagnosed by a urinary antigen test, which identifies only L. pneumophila serogroup 1. Therefore, assessment of pneumonia-causing Legionella species and serogroups would be important. The Japan Society for Chemotherapy Legionella committee has collected the isolates and clinical information on cases of sporadic community-acquired Legionella pneumonia throughout Japan. Between December 2006 and March 2019, totally 140 sporadic cases were identified, in which L. pneumophila was the most frequently isolated species (90.7%) followed by L. bozemanae (3.6%), L. dumofii (3.6%), L. micdadei (1.4%), and L. longbeachae (0.7%). Among 127 isolates of L. pneumophila, 111 isolates were of serogroup 1, two of serogroup 2, four of serogroup 3, one of serogroup 4, one of serogroup 5, seven of serogroup 6, and one was of serogroup 10. We also assessed in vitro activity of antibiotics against these isolates and showed that quinolones and macrolides have potent anti-Legionella activity. Our study showed that pneumonia-causing Legionella species and serogroup distribution was comparable to that reported in former surveillances. L. pneumophila was the most common etiologic agent in patients with community-acquired Legionella pneumonia, and L. pneumophila serogroup 1 was the predominant serogroup.

Whole genome sequence analysis reveals the broad distribution of the RtxA type 1 secretion system and four novel putative type 1 secretion systems throughout the Legionella genus.

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.

Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes.

Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.

Quorum sensing modulates the formation of virulent Legionella persisters within infected cells.

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.

Genetic diversity of Legionella pcs and pmtA genes and the effect of utilization of choline by Legionella spp. on induction of proinflammatory cytokines.

Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6).

The clinical presentation of Legionella arthritis reveals the mode of infection and the bacterial species: case report and literature review.

BACKGROUND: While Legionella is a common cause of pneumonia, extrapulmonary infections like arthritis are scarce. Here, we describe a case of monoarthritis due to Legionella bozemanii, with no history of pneumonia. We provide a literature review of the 9 previously published Legionella arthritis and highlight a dichotomous epidemiology suggesting different physiopathological pathways leading to joint infection. CASE PRESENTATION: A 56-year old woman under immunosuppressive treatment by oral and intra-articular corticosteroids, methotrexate, and tocilizumab for an anti-synthetase syndrome was hospitalized for worsening pain and swelling of the left wrist for 3 days. Clinical examination showed left wrist synovitis and no fever. The arthritis occurred a few days after an accidental fall on wet asphalt responsible for a cutaneous wound followed by a corticosteroid intra-articular injection. Due to both the negativity of conventional culture of articular fluid and suspicion of infection, 16S rRNA and specific PCRs were performed leading to the identification of L. bozemanii. Legionella-specific culture of the articular fluid was performed retrospectively and isolated L. bozemanii. The empiric antibiotic therapy was switched for oral levofloxacin and rifampin and the patient recovered after a 12-week treatment. CONCLUSION: We report a case of L. bozemanii monoarthritis in an immunosuppressed woman, following a fall on wet asphalt and intra-articular corticosteroid injection. The review of the literature found that the clinical presentation reveals the mode of infection and the bacterial species. Monoarthritis more likely occurred after inoculation in patients under immunosuppressive therapy and were associated with non-Legionella pneumophila serogroup 1 (Lp1) strains that predominate in the environment. Polyarthritis were more likely secondary legionellosis localizations after blood spread of Lp1, the most frequently found in pneumonia. In both settings, 16S rRNA and Legionella-specific PCR were key factors for the diagnosis.

Impact of Chlorine and Chloramine on the Detection and Quantification of Legionella pneumophila and Mycobacterium Species.

Potable water can be a source of transmission for legionellosis and nontuberculous mycobacterium (NTM) infections and diseases. Legionellosis is caused largely by Legionella pneumophila, specifically serogroup 1 (Sg1). Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus are three leading species associated with pulmonary NTM disease. The estimated rates of these diseases are increasing in the United States, and the cost of treatment is high. Therefore, a national assessment of water disinfection efficacy for these pathogens was needed. The disinfectant type and total chlorine residual (TClR) were investigated to understand their influence on the detection and concentrations of the five pathogens in potable water. Samples (n = 358) were collected from point-of-use taps (cold or hot) from locations across the United States served by public water utilities that disinfected with chlorine or chloramine. The bacteria were detected and quantified using specific primer and probe quantitative-PCR (qPCR) methods. The total chlorine residual was measured spectrophotometrically. Chlorine was the more potent disinfectant for controlling the three mycobacterial species. Chloramine was effective at controlling L. pneumophila and Sg1. Plotting the TClR associated with positive microbial detection showed that an upward TClR adjustment could reduce the bacterial count in chlorinated water but was not as effective for chloramine. Each species of bacteria responded differently to the disinfection type, concentration, and temperature. There was no unifying condition among the water characteristics studied that achieved microbial control for all. This information will help guide disinfectant decisions aimed at reducing occurrences of these pathogens at consumer taps and as related to the disinfectant type and TClR.IMPORTANCE The primary purpose of tap water disinfection is to control the presence of microbes. This study evaluated the role of disinfectant choice on the presence at the tap of L. pneumophila, its Sg1 serogroup, and three species of mycobacteria in tap water samples collected at points of human exposure at locations across the United States. The study demonstrates that microbial survival varies based on the microbial species, disinfectant, and TClR.

Environmental surveillance and spatio-temporal analysis of Legionella spp. in a region of northeastern Italy (2002-2017).

Legionella spp. are considered an important cause of potentially preventable morbidity and mortality, making environmental surveillance a crucial component of risk assessment plans. In this work, 20,319 water samples were collected in 3,983 environmental surveys during a 16-year period by ARPA, the Regional Agency for Environmental Protection, Friuli Venezia Giulia, and the results were studied to better understand the diffusion mechanisms of Legionella. The data showed a strong seasonal signal, a prevalence of L. pneumophila serogroup 2-15 in most environments (63% of positive samples), a prevalence of L. pneumophila serogroup 1 in swimming pool-associated environments (82% of positive samples), a persistent presence of Legionella in hospitals and a recurrent presence of Legionella in other facilities such as hotels, possibly years after interventions, highlighting the difficulty of eradicating the bacteria. Retrospective spatio-temporal analyses on geocoded historical data were carried out with SaTScan using an ordinal model with risk as a covariate to identify potential clusters with an excess of cases in the higher-risk categories. Although no outbreaks occurred during the period of study, such analyses identified spatially restricted zones with unusual contamination, which sometimes were also areas in which several surveys triggered by notifications of clinical cases were performed. Simulations of periodic prospective analyses permitted the assessment of the efficacy of the method in early detection of such clusters. The proposed method may be a useful tool in environmental surveillance, prevention and control of Legionella.

[Legionella and Legionellosis]. / Legionella und Legionellose.

Diagnosed and reported Legionella pneumonias are slightly increasing during recent years. This might at least partially be due to more frequently used diagnostic tests.In severe pneumonia, nucleic acid amplification based methods should supplement the L. pneumophila serogroup-1 urinary antigen test because of their improved spectrum and sensitivity.Recent in vitro data suggest enhanced efficacy of levofloxacin when compared to macrolides. This complements recent clinical cohort data. Thus levofloxacin (750-1000 mg/d) is regarded the treatment of choice for confirmed legionellosis. Second line options are azithromycin or moxifloxacin. Treatment duration of 7 days should be sufficient in most cases.Development of resistance is rare and no routine resistance testing is necessary.

Legionella co-infection in HIV-associated pneumonia.

Due to poor diagnostics and increased co-infections, HIV-associated Legionella infections are underreported. We aimed to retrospectively determine the frequency of Legionella infections in bronchoalveolar lavage (BAL) from HIV-associated pneumonia patients hospitalized in Medellin, Colombia, between February 2007 and April 2014. Although culture was negative, 17 BAL (36%) were positive for Legionella by quantitative polymerase chain reaction, most of which were in the Mycobacterium tuberculosis or Pneumocystis jirovecii co-infected patients, and included L. anisa (n = 6), L. bozemanae (n = 4), L. pneumophila (n = 3), and L. micdadei (n = 2). All L. bozemanae and L. micdadei associated with Pneumocystis, while all L. pneumophila associated with M. tuberculosis. Legionella probable cases had more complications and higher mortality rates (P = 0.02) and were rarely administered empirical anti-Legionella therapy while in hospital. Clinicians should be aware of the possible presence of Legionella in HIV and M. tuberculosis or P. jirovecii co-infected patients.