Treatment of legionellosis including a single intravenous dose of 1.5 g azithromycin: 18-year experience at a tertiary care hospital.

OBJECTIVES: Due to recent safety concerns regarding fluoroquinolones and the potential medical and economic benefits, we investigated the efficacy of a single intravenous dose of 1.5 g azithromycin for the treatment of pulmonary legionellosis. METHODS: Using a nationwide legionellosis registry for pre-selection, 74 patients admitted from 2000-2018 to a tertiary care hospital owing to pneumonia caused by Legionella pneumophila were retrospectively included in this study. RESULTS: Conventional treatment regimens consisting of fluoroquinolones (n = 20), macrolides (n = 30) or combinations of both (n = 24) and a single intravenous dose of azithromycin (n = 12) have been demonstrated to be equally effective. Single-dose azithromycin treatment was well tolerated and resulted in a shorter hospital stay (P = 0.0464) and shorter antibiotic treatment duration (P = 0.0004) allowing earlier discharge. CONCLUSION: A single intravenous dose of azithromycin might be a valuable treatment alternative for patients with legionellosis.

Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness.

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs' stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.

Bacterial Long-Range Warfare: Aerial Killing of Legionella pneumophila by Pseudomonas fluorescens.

Legionella pneumophila, the causative agent of Legionnaires' disease, is mostly found in man-made water systems and is one of the most closely monitored waterborne pathogens. With the aim of finding natural ways to control waterborne pathogens and thus further reduce the impact of disinfection by-products on human health, some studies have demonstrated the ability of bacteria to kill Legionella through the production of secondary metabolites or antimicrobial compounds. Here, we describe an unexpected growth inhibition of L. pneumophila when exposed to a physically separated strain of Pseudomonas fluorescens, designated as MFE01. Most of the members of the Legionellaceae family are sensitive to the volatile substances emitted by MFE01, unlike other bacteria tested. Using headspace solid-phase microextraction GC-MS strategy, a volatilome comparison revealed that emission of 1-undecene, 2-undecanone, and 2-tridecanone were mainly reduced in a Tn5-transposon mutant unable to inhibit at distance the growth of L. pneumophila strain Lens. We showed that 1-undecene was mainly responsible for the inhibition at distance in vitro, and led to cell lysis in small amounts, as determined by gas chromatography-mass spectrometry (GC-MS). Collectively, our results provide new insights into the mode of action of bacterial volatiles and highlight them as potent anti-Legionella agents to focus research on novel strategies to fight legionellosis. IMPORTANCE Microbial volatile compounds are molecules whose activities are increasingly attracting the attention of researchers. Indeed, they can act as key compounds in long-distance intrakingdom and interkingdom communication, but also as antimicrobials in competition and predation. In fact, most studies to date have focused on their antifungal activities and only a few have reported on their antibacterial properties. Here, we describe that 1-undecene, naturally produced by P. fluorescens, is a volatile with potent activity against bacteria of the genus Legionella. In small amounts, it is capable of inducing cell lysis even when the producing strain is physically separated from the target. This is the first time that such activity is described. This molecule could therefore constitute an efficient compound to counter bacterial pathogens whose treatment may fail, particularly in pulmonary diseases. Indeed, inhalation of these volatiles should be considered as a possible route of therapy in addition to antibiotic treatment.

Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper.

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Source attribution of community-acquired cases of Legionnaires' disease-results from the German LeTriWa study; Berlin, 2016-2019.

INTRODUCTION: Sources of infection of most cases of community-acquired Legionnaires' disease (CALD) are unknown. OBJECTIVE: Identification of sources of infection of CALD. SETTING: Berlin; December 2016-May 2019. PARTICIPANTS: Adult cases of CALD reported to district health authorities and consenting to the study; age and hospital matched controls. MAIN OUTCOME MEASURE: Percentage of cases of CALD with attributed source of infection. METHODS: Analysis of secondary patient samples for monoclonal antibody (MAb) type (and sequence type); questionnaire-based interviews, analysis of standard household water samples for Legionella concentration followed by MAb (and sequence) typing of Legionella pneumophila serogroup 1 (Lp1) isolates; among cases taking of additional water samples to identify the infectious source as appropriate; recruitment of control persons for comparison of exposure history and Legionella in standard household water samples. For each case an appraisal matrix was filled in to attribute any of three source types (external (non-residence) source, residential non-drinking water (RnDW) source (not directly from drinking water outlet), residential drinking water (RDW) as source) using three evidence types (microbiological results, cluster evidence, analytical-comparative evidence (using added information from controls)). RESULTS: Inclusion of 111 study cases and 202 controls. Median age of cases was 67 years (range 25-93 years), 74 (67%) were male. Among 65 patients with urine typable for MAb type we found a MAb 3/1-positive strain in all of them. Compared to controls being a case was not associated with a higher Legionella concentration in standard household water samples, however, the presence of a MAb 3/1-positive strain was significantly associated (odds ratio (OR) = 4.9, 95% confidence interval (CI) 1.7 to 11). Thus, a source was attributed by microbiological evidence if it contained a MAb 3/1-positive strain. A source was attributed by cluster evidence if at least two cases were exposed to the same source. Statistically significant general source types were attributed by calculating the population attributable risk (analytical-comparative evidence). We identified an external source in 16 (14%) cases, and RDW as source in 28 (25%). Wearing inadequately disinfected dentures was the only RnDW source significantly associated with cases (OR = 3.2, 95% CI 1.3 to 7.8) and led to an additional 8% of cases with source attribution, for a total of 48% of cases attributed. CONCLUSION: Using the appraisal matrix we attributed almost half of all cases of CALD to an infectious source, predominantly RDW. Risk for LD seems to be conferred primarily by the type of Legionella rather than the amount. Dentures as a new infectious source needs further, in particular, integrated microbiological, molecular and epidemiological confirmation.

Antibiotic Resistance of Legionella pneumophila in Clinical and Water Isolates-A Systematic Review.

The current systematic review investigates the antibiotic susceptibility pattern of Legionella pneumophila isolates from the 1980s to the present day, deriving data from clinical and/or water samples from studies carried out all over the world. Eighty-nine papers meeting the inclusion criteria, i.e., "Legionella pneumophila" and "resistance to antibiotics", were evaluated according to pre-defined validity criteria. Sixty articles referred to clinical isolates, and 18 articles reported water-related L. pneumophila isolates, while 11 articles included both clinical and water isolates. Several methods have been proposed as suitable for the determination of MICs, such as the E-test, broth and agar dilution, and disk diffusion methods, in vivo and in vitro, using various media. The E-test method proposed by the European Society of Clinical Microbiology and Infectious Diseases (EUCAST) seems to be the second most frequently used method overall, but it is the preferred method in the most recent publications (2000-2019) for the interpretation criteria. Erythromycin has been proved to be the preference for resistance testing over the years. However, in the last 19 years, the antibiotics ciprofloxacin (CIP), erythromycin (ERM), levofloxacin (LEV) and azithromycin (AZM) were the ones that saw an increase in their use. A decrease in the sensitivity to antibiotics was identified in approximately half of the reviewed articles.

Bacterial co-infections with SARS-CoV-2.

The pandemic coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected millions of people worldwide. To date, there are no proven effective therapies for this virus. Efforts made to develop antiviral strategies for the treatment of COVID-19 are underway. Respiratory viral infections, such as influenza, predispose patients to co-infections and these lead to increased disease severity and mortality. Numerous types of antibiotics such as azithromycin have been employed for the prevention and treatment of bacterial co-infection and secondary bacterial infections in patients with a viral respiratory infection (e.g., SARS-CoV-2). Although antibiotics do not directly affect SARS-CoV-2, viral respiratory infections often result in bacterial pneumonia. It is possible that some patients die from bacterial co-infection rather than virus itself. To date, a considerable number of bacterial strains have been resistant to various antibiotics such as azithromycin, and the overuse could render those or other antibiotics even less effective. Therefore, bacterial co-infection and secondary bacterial infection are considered critical risk factors for the severity and mortality rates of COVID-19. Also, the antibiotic-resistant as a result of overusing must be considered. In this review, we will summarize the bacterial co-infection and secondary bacterial infection in some featured respiratory viral infections, especially COVID-19.

Efficacy of delafloxacin versus moxifloxacin against atypical bacterial respiratory pathogens in adults with community-acquired bacterial pneumonia (CABP): Data from the Delafloxacin Phase 3 CABP Trial.

OBJECTIVES: To report atypical pathogens from clinical trial data comparing delafloxacin to moxifloxacin in the treatment of adults with community-acquired bacterial pneumonia (CABP). METHODS: Multiple diagnostic methods were employed to diagnose atypical infections including culture, serology, and urinary antigen. RESULTS: The microbiological intent-to-treat (MITT) population included 520 patients; 30% had an atypical bacterial pathogen identified (156/520). Overall, 13.1% (68/520) had a monomicrobial atypical infection and 2.3% (12/520) had polymicrobial all-atypical infections. Among patients with polymicrobial infections, Streptococcus pneumoniae was the most frequently occurring co-infecting organism and Chlamydia pneumoniae was the most frequently occurring co-infecting atypical organism. For Mycoplasma pneumoniae and Legionella pneumophila, serology yielded the highest number of diagnoses. Delafloxacin and moxifloxacin had similar in vitro activity against M. pneumoniae and delafloxacin had greater activity against L. pneumophila. Two macrolide-resistant M. pneumoniae isolates were recovered. No fluoroquinolone-resistant M. pneumoniae were isolated. The rates of microbiological success (documented or presumed eradication) at test-of-cure were similar between the delafloxacin and moxifloxacin groups. There was no evidence of a correlation between minimum inhibitory concentration (MIC) and outcome; a high proportion of favorable outcomes was observed across all delafloxacin baseline MICs. CONCLUSIONS: Delafloxacin may be considered a treatment option as monotherapy for CABP in adults, where broad-spectrum coverage including atypical activity is desirable.

Persistent Legionnaires' Disease and Associated Antibiotic Treatment Engender a Highly Disturbed Pulmonary Microbiome Enriched in Opportunistic Microorganisms.

Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections.IMPORTANCE The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance.

In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts.

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages.

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires' Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

In Vitro and Intracellular Activities of Omadacycline against Legionella pneumophila.

Omadacycline is an aminomethylcycline antibiotic with in vitro activity against pathogens causing community-acquired bacterial pneumonia (CABP). This study investigated the activity of omadacycline against Legionella pneumophila strains isolated between 1995 and 2014 from nosocomial or community-acquired respiratory infections. Omadacycline exhibited extracellular activity similar to comparator antibiotics; intracellular penetrance was found by day 3 of omadacycline exposure. These results support the utility of omadacycline as an effective antibiotic for the treatment of CABP caused by L. pneumophila.

Photocatalytic biocidal effect of copper doped TiO2 nanotube coated surfaces under laminar flow, illuminated with UVA light on Legionella pneumophila.

Legionella pneumophila can cause a potentially fatal form of humane pneumonia (Legionnaires' disease), which is most problematic in immunocompromised and in elderly people. Legionella species is present at low concentrations in soil, natural and artificial aquatic systems and is therefore constantly entering man-made water systems. The environment temperature for it's ideal growth range is between 32 and 42°C, thus hot water pipes represent ideal environment for spread of Legionella. The bacteria are dormant below 20°C and do not survive above 60°C. The primary method used to control the risk from Legionella is therefore water temperature control. There are several other effective treatments to prevent growth of Legionella in water systems, however current disinfection methods can be applied only intermittently thus allowing Legionella to grow in between treatments. Here we present an alternative disinfection method based on antibacterial coatings with Cu-TiO2 nanotubes deposited on preformed surfaces. In the experiment the microbiocidal efficiency of submicron coatings on polystyrene to the bacterium of the genus Legionella pneumophila with a potential use in a water supply system was tested. The treatment thus constantly prevents growth of Legionella pneumophila in presence of water at room temperature. Here we show that 24-hour illumination with low power UVA light source (15 W/m2 UVA illumination) of copper doped TiO2 nanotube coated surfaces is effective in preventing growth of Legionella pneumophila. Microbiocidal effects of Cu-TiO2 nanotube coatings were dependent on the flow of the medium and the intensity of UV-A light. It was determined that tested submicron coatings have microbiocidal effects specially in a non-flow or low-flow conditions, as in higher flow rates, probably to a greater possibility of Legionella pneumophila sedimentation on the coated polystyrene surfaces, meanwhile no significant differences among bacteria reduction was noted regarding to non or low flow of medium.

Investigation of potential pathogenicity of Willaertia magna by investigating the transfer of bacteria pathogenicity genes into its genome.

Willaertia magna c2c maky is a thermophilic amoeba closely related to the genus Naegleria. This free-living amoeba has the ability to eliminate Legionella pneumophila, which is an amoeba-resisting bacterium living in an aquatic environment. To prevent the proliferation of L. pneumophila in cooling towers, the use of W. magna as natural biocide has been proposed. To provide a better understanding of the W. magna genome, whole-genome sequencing was performed through the study of virulence factors and lateral gene transfers. This amoeba harbors a genome of 36.5 megabases with 18,519 predicted genes. BLASTp analyses reported protein homology between 136 W. magna sequences and amoeba-resistant microorganisms. Horizontal gene transfers were observed based on the basis of the phylogenetic reconstruction hypothesis. We detected 15 homologs of N. fowleri genes related to virulence, although these latter were also found in the genome of N. gruberi, which is a non-pathogenic amoeba. Furthermore, the cytotoxicity test performed on human cells supports the hypothesis that the strain c2c maky is a non-pathogenic amoeba. This work explores the genomic repertory for the first draft genome of genus Willaertia and provides genomic data for further comparative studies on virulence of related pathogenic amoeba, N. fowleri.

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.

Epidemiological surveillance and wild-type MIC distribution of Legionella pneumophila in north-western Spain. 2003-2016 / Vigilancia epidemiológica y distribución de CMIs de cepas salvajes de Legionella pneumophila en el noroeste de España. 2003-2016

PURPOSE: To perform epidemiological surveillance of Legionella pneumophila in recreational swimming pools in the city of Valladolid (Spain), an area with a continental climate and low incidence of legionella-associated infections. Additionally, wild-type minimum inhibitory concentration (MIC) distributions for eight antibiotics commonly used for the treatment of legionellosis were calculated from the isolates obtained. METHODS: Twelve recreational pools were enrolled between June 2003 and December 2016 and 7221 water samples were taken from three different points of the water network (tank, tap and shower). Legionella culture was performed according to ISO 11731 and 11731-2 standards. MICs of antibiotics were obtained by a gradient test. RESULTS: 1.44% of the water samples were positive for L. pneumophila. 60 strains (57.69%) were isolated from showers, 26 (25.00%) from tanks and 18 (17.31%) from taps. L. pneumophila counts were < 100 CFU/L in 75 samples (72.12%), 100-1000 CFU/L in 17 (16.35%) and > 1000 CFU/L in 12 (11.54%). The MIC90 values obtained were for Rifampicin 0.125 mg/L; Trimethoprim-Sulfamethoxazole 0.25mg/L; Azithromycin and Levofloxacin 0.5 mg/L; Clarithromycin and Ciprofloxacin 1.0mg/L; Doxycycline and Tigecycline 4.0 mg/L. CONCLUSIONS: The use of showers in recreational pools can become a potential pathway for exposure to L. pneumophila, even in cold climates. The wild-type MIC distributions presented in this article may be useful for a better detection of antibiotic resistance and can contribute to improvements in the choice of the antibiotic treatment of legionellosis

PROPÓSITO: Realizar la vigilancia epidemiológica de Legionella pneumophila en piscinas recreacionales de Valladolid (España), un área con clima continental y baja incidencia de legionelosis. La distribución de las CMIs de ocho antibióticos usados en la legionelosis fue calculada a partir de los aislados obtenidos. MÉTODOS: Se incluyeron doce piscinas recreacionales entre junio 2003-diciembre 2016. 7.221 muestras de agua fueron tomadas en tres puntos de la red (vaso, grifo y ducha). El cultivo de legionela se realizó acorde a las normas ISO 11731 y 11731-2. Las CMIs de los antibióticos se obtuvieron mediante un método en gradiente. RESULTADOS: 1,44% de las muestras proporcionaron crecimiento de L. pneumophila. 60 cepas (57,69%) se aislaron en duchas, 26 (25,00%) en vasos y 18 (17,31%) en grifos. Los recuentos de L. pneumophila fueron < 100 UFC/L en 75 muestras (72,12%), 100-1.000 UFC/L en 17 (16,35%) y > 1.000 UFC/L en 12 (11,54%). Las CMI90 obtenidas fueron para rifampicina 0,125 mg/L; trimetoprim-sulfametoxazol 0,25 mg/L; azitromicina y levofloxacino 0,5 mg/L; clarithromicina y ciprofloxacino 1,0 mg/L; doxiciclina y tigeciclina 4, 0mg/L. CONCLUSIONES: El uso de las duchas en piscinas recreacionales puede convertirse en una vía potencial para la exposición a L. pneumophila, incluso en climas fríos. Las CMIs presentadas en este artículo son útiles para la detección de la resistencia a antibióticos y pueden mejorar la elección del tratamiento antibiótico de la legionelosis

Antibiotic Resistance and Azithromycin Resistance Mechanism of Legionella pneumophila Serogroup 1 in China.

Legionnaires' disease, caused by Legionella pneumophila, has been treated primarily with antibiotics. However, few reports have been published on antibiotic-resistant Legionella in China. Our aim was to determine the azithromycin resistance mechanism of L. pneumophila serogroup 1 in China. The sensitivities of 149 L. pneumophila serogroup 1 strains, isolated from clinical cases or environmental water in China from 2002 to 2016, to five antibiotics, including erythromycin, azithromycin, levofloxacin, moxifloxacin, and rifampin, were evaluated. The mechanisms of the resistance of L. pneumophila serogroup 1 to azithromycin were studied. The expression levels of efflux pump gene lpeAB and the MIC of azithromycin-resistant strains in the presence and absence of the efflux pump inhibitor carbonyl cyanide-chlorophenylhydrazone (CCCP) were determined. All 149 strains were sensitive to erythromycin, levofloxacin, moxifloxacin, and rifampin, among which 25 of the strains exhibited azithromycin resistance. These 25 strains, including strains of sequence type 1 (ST1), ST144, ST150, ST154, and ST629, were screened. Expression of lpeAB was responsible for the reduced azithromycin susceptibility in all 25 of these strains. The phenotypes of 25 strains with virulence were linked by evaluating the intracellular growth ability in mouse macrophage J774 cells. Among the 25 strains, 60% were more virulent than the ATCC 33152 reference strain. The results determined in our study represent data supporting the further study of the antibiotic sensitivity of L. pneumophila strains in China.

Bactericidal efficacies of food additive grade calcium hydroxide toward Legionella pneumophila.

Food additive grade calcium hydroxide (FdCa(OH)2) in the solution of 0.17% was evaluated for its bactericidal efficacies toward Legionella pneumophila with or without sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine, at room temperature (RT) (25°C ± 2°C) and 42°C, either with or without 5% fetal bovine serum (FBS). Besides, FdCa(OH)2 in different concentration solutions were prepared in field water samples (hot spring and bath tab water) and evaluated for their bactericidal efficacies at 42°C. FdCa(OH)2 (0.17%) inactivated the L. pneumophila to the undetectable level (<2.6 log CFU/ml) within 5 min and 3 min, respectively, at RT and 42°C, with 5% FBS. At RT and 42°C, NaOCl inactivated L. pneumophila to the undetectable level within 5 min, without 5% FBS, but with 5% FBS, it could only inactivate this bacterium effectively (≥3 log reductions). Conversely, at RT and 42°C, the mixture of 0.17% FdCa(OH)2 and 200 ppm NaOCl could inactivate L. pneumophila to the undetectable level, respectively, within 3 min and 1 min, even with 5% FBS, and it was elucidated that FdCa(OH)2 has a synergistic bactericidal effect together with NaOCl. FdCa(OH)2 0.05% solution prepared in hot spring water could inactivate L. pneumophila to the undetectable within 3 min at 42°C. So, FdCa(OH)2 alone could show nice bactericidal efficacy at 42°C, even with 5% FBS, as well as in field water samples.

Antibiotic susceptibility of environmental Legionella pneumophila isolated in India.

Aim: Data are limited regarding the antibiotic susceptibility of Legionella pneumophila in India. The aim of this study was to determine the drug susceptibility of environmental L. pneumophila isolates in India for antibiotics commonly used in clinical practice for Legionnaires' disease treatment. Materials & methods: The activities of seven antibiotics against 46 environmental isolates of L. pneumophila were evaluated by using E-test on buffered charcoal yeast extract-α agar. Results: Among the L. pneumophila isolates tested, no tendency toward drug resistance was observed. Rifampicin was the most potent drug followed by levofloxacin, while doxycycline and tetracycline were found to be the less active agents. Conclusion: Susceptibility testing of Legionella environmental isolates could be beneficial to notify resistance to antibiotics in the environment before it becomes evident in clinical strains.