Lp1 Isolates Research Articles

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains.

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

TNF-α response in macrophages depends on clinical Legionella pneumophila isolates genotypes

ABSTRACT Legionnaires’ Disease (LD) is a severe pneumonia mainly caused in Europe by Legionella pneumophila serogroup 1 (Lp1). Sequence-based typing methods reveal that some sequence types (ST) are overrepresented in clinical samples such as ST1 and ST47, suggesting that some strains are more fit for infection than others. In the present study, a collection of 108 Lp1 clinical isolates were used to evaluate the strain-dependent immune responses from human macrophages. Clinical Lp1 isolates induced differential TNFα secretion from macrophages. ST1 isolates induced a significantly higher TNF-α secretion than non-ST1, whereas ST47 isolates induced a significantly lower TNF-α secretion than non-ST47 isolates. ST1 isolates induced a significantly higher cell death than ST47 isolates evaluated by lactate dehydrogenase activity (cytotoxicity) and caspase-3 activity (apoptosis). Treatment of macrophages with anti-TNF-α antibodies significantly reduced the cell death in macrophages infected with ST1 or ST47 strains. The TNF-α secretion was neither explained by a differential bacterial replication nor by the number or type (bystander or infected) of TNF-α producing cells following infection but by a differential response from macrophages. The Paris ST1 reference strain elicited a significantly higher TNF-α gene transcription and a higher induction of NF-κB signaling pathway than the Lorraine ST47 reference strain. Clinical Lp1 isolates induce a diverse immune response and cell death, which could be related to the genotype. The two predominant sequence-types ST1 and ST47 trigger opposite inflammatory response that could be related to the host susceptibility.

Genome Sequencing Links Persistent Outbreak of Legionellosis in Sydney (New South Wales, Australia) to an Emerging Clone of Legionella pneumophila Sequence Type 211.

The city of Sydney, Australia, experienced a persistent outbreak of Legionella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source and guide public health actions, the genomes of clinical and environmental Lp1 isolates recovered over 7 weeks were examined. A total of 48 isolates from human cases and cooling towers were sequenced and compared using single-nucleotide polymorphism (SNP)-based core-genome multilocus sequencing typing (MLST) and pangenome approaches. All three methods confirmed phylogenetic relatedness between isolates associated with outbreaks in the Central Business District (CBD) in March and May and those in suburb 1. These isolates were designated the "main cluster" and consisted of isolates from two patients from the CBD March outbreak, one patient and one tower isolate from suburb 1, and isolates from two cooling towers and three patients from the CBD May outbreak. All main cluster isolates were sequence type 211 (ST211), which previously has only been reported in Canada. Significantly, pangenome analysis identified mobile genetic elements containing a unique type IV A F-type secretion system (T4ASS), which was specific to the main cluster, and cocirculating clinical strains, suggesting a potential mechanism for increased fitness and persistence of the outbreak clone. Genome sequencing enabled linking of the geographically dispersed environmental sources of infection among the spatially and temporally coinciding cases of legionellosis in a highly populated urban setting. The discovery of a unique T4ASS emphasizes the role of genome recombination in the emergence of successful Lp1 clones.IMPORTANCE A new emerging clone has been responsible for a prolonged legionellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linked two outbreaks thought to be unrelated and confirmed the outliers. These findings led to the resampling and subsequent identification of the source, guiding public health actions and bringing the outbreak to a close. Significantly, the outbreak clone was identified as sequence type 211 (ST211). Our study reports this ST in the Southern Hemisphere and presents a description of ST211 genomes from both clinical and environmental isolates. A unique mobile genetic element containing a type IV secretion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1 ST42 isolates that were cocirculating at the time of the outbreak.

Antimicrobial Activity of Solithromycin against Clinical Isolates of Legionella pneumophila Serogroup 1

The activity of solithromycin was evaluated against clinical Legionella pneumophila serogroup 1 (Lp1) isolates (n = 196) collected in Ontario, Canada, from 1980 to 2011. Its in vitro activity was compared to that of azithromycin (AZM) using the broth microdilution method. Solithromycin had a MIC50 of ≤0.015 μg/ml and a MIC90 of 0.031 μg/ml, making its activity at least 8-fold to 32-fold higher than that of AZM (MIC50 and MIC90, 0.125 μg/ml and 1 μg/ml, respectively). Ninety-nine percent of the isolates had MICs for solithromycin ranging from ≤0.015 μg/ml to 0.031 μg/ml, whereas 83.6% of the isolates showed MICs for AZM ranging from 0.062 μg/ml to 0.25 μg/ml. Interestingly, 96.7% (30 out of 31 clinical isolates) identified with higher AZM MICs (0.5 μg/ml to 2 μg/ml) belonged to the clinically prevalent sequence type 1. To investigate the intracellular activity of solithromycin, in vitro invasion assays were also performed against a subset of representative Lp1 isolates internalized within human lung epithelial cells. Solithromycin and AZM both inhibited growth of all intracellular Lp1 isolates at 1× or 8× MICs, displaying bacteriostatic effects, as would be expected with protein synthesis inhibitor rather than bactericidal activity. Solithromycin demonstrated the highest in vitro and intracellular potency against all Lp1 isolates compared to AZM. Given the rapid spread of resistance mechanisms among respiratory pathogens and the reported treatment failures in legionellosis, the development of this new fluoroketolide, already in phase 3 oral clinical studies, constitutes a promising alternative option for the treatment of legionellosis.

Prevalence of sequence types among clinical and environmental isolates of Legionella pneumophila serogroup 1 in the United States from 1982 to 2012.

Since the establishment of sequence-based typing as the gold standard for DNA-based typing of Legionella pneumophila, the Legionella laboratory at the Centers for Disease Control and Prevention (CDC) has conducted routine sequence-based typing (SBT) analysis of all incoming L. pneumophila serogroup 1 (Lp1) isolates to identify potential links between cases and to better understand genetic diversity and clonal expansion among L. pneumophila bacteria. Retrospective genotyping of Lp1 isolates from sporadic cases and Legionnaires' disease (LD) outbreaks deposited into the CDC reference collection since 1982 has been completed. For this study, we compared the distribution of sequence types (STs) among Lp1 isolates implicated in 26 outbreaks in the United States, 571 clinical isolates from sporadic cases of LD in the United States, and 149 environmental isolates with no known association with LD. The Lp1 isolates under study had been deposited into our collection between 1982 and 2012. We identified 17 outbreak-associated STs, 153 sporadic STs, and 49 environmental STs. We observed that Lp1 STs from outbreaks and sporadic cases are more similar to each other than either group is to environmental STs. The most frequent ST for both sporadic and environmental isolates was ST1, accounting for 25% and 49% of the total number of isolates, respectively. The STs shared by both outbreak-associated and sporadic Lp1 included ST1, ST35, ST36, ST37, and ST222. The STs most commonly found in sporadic and outbreak-associated Lp1 populations may have an increased ability to cause disease and thus may require special attention when detected.

A Recurrent Outbreak of Nosocomial Legionnaires' Disease Detected by Urinary Antigen Testing: Evidence for Long-Term Colonization of a Hospital Plumbing System

AbstractBackground:In 1994, a hospital reported an increase in nosocomial legionnaires' disease after implementing use of a rapid urinary antigen test for Legionella pneumophila serogroup 1 (Lp-1). This hospital was the site of a previous nosocomial legionnaires' disease outbreak during 1980 to 1982.Methods:Infection control records were reviewed to compare rates of nosocomial pneumonia and the proportion of cases attributable to legionnaires' disease during the 1994 outbreak period with those during the same period in 1993. Water samples were collected for Legionella culture from the hospital's potable water system and cooling towers, and isolates were subtyped by monoclonal antibody (MAb) testing and arbitrarily primed polymerase chain reaction (AP-PCR).Results:Nosocomial pneumonia rates were similar from April through October 1993 and April through October 1994: 5.9 and 6.6 per 1,000 admissions, respectively (rate ratio [RR], 1.1; P=.56); however, 3.2% of nosocomial pneumonias were diagnosed as legionnaires' disease in 1993, compared with 23.9% in 1994 (RR, 9.4; P<.001). In 1994, most legionnaires' disease cases were detected by the urinary antigen testing alone. MAb testing and AP-PCR demonstrated identical patterns among Lp-1 isolates recovered from a patient's respiratory secretions, the hospital potable water system, and stored potable water isolates from the 1980 to 1982 outbreak.Conclusions:There may have been persistent transmission of nosocomial legionnaires' disease at this hospital that went undiscovered for many years because there was no active surveillance for legionnaires' disease. Introduction of a rapid urinary antigen test improved case ascertainment. Legionella species can be established in colonized plumbing systems and may pose a risk for infection over prolonged periods.

A Recurrent Outbreak of Nosocomial Legionnaires' Disease Detected by Urinary Antigen Testing Evidence for Long-Term Colonization of a Hospital Plumbing System

In 1994, a hospital reported an increase in nosocomial legionnaires' disease after implementing use of a rapid urinary antigen test for Legionella pneumophila serogroup 1 (Lp-1). This hospital was the site of a previous nosocomial legionnaires' disease outbreak during 1980 to 1982. Infection control records were reviewed to compare rates of nosocomial pneumonia and the proportion of cases attributable to legionnaires' disease during the 1994 outbreak period with those during the same period in 1993. Water samples were collected for Legionella culture from the hospital's potable water system and cooling towers, and isolates were subtyped by monoclonal antibody (MAb) testing and arbitrarily primed polymerase chain reaction (AP-PCR). Nosocomial pneumonia rates were similar from April through October 1993 and April through October 1994: 5.9 and 6.6 per 1,000 admissions, respectively (rate ratio [RR], 1.1; P=.56); however, 3.2% of nosocomial pneumonias were diagnosed as legionnaires' disease in 1993, compared with 23.9% in 1994 (RR, 9.4; P<.001). In 1994, most legionnaires' disease cases were detected by the urinary antigen testing alone. MAb testing and AP-PCR demonstrated identical patterns among Lp-1 isolates recovered from a patient's respiratory secretions, the hospital potable water system, and stored potable water isolates from the 1980 to 1982 outbreak. There may have been persistent transmission of nosocomial legionnaires' disease at this hospital that went undiscovered for many years because there was no active surveillance for legionnaires' disease. Introduction of a rapid urinary antigen test improved case ascertainment. Legionella species can be established in colonized plumbing systems and may pose a risk for infection over prolonged periods.

Epidemic Legionnaires' disease two decades later: old sources, new diagnostic methods.

In July 1995 we investigated a pneumonia outbreak in a Pennsylvania town. We conducted epidemiological and molecular microbiological studies to determine the outbreak source and interrupt transmission of disease. Legionnaires' disease (LD) was quickly identified by urine antigen testing, and a newly developed immunohistochemical stain confirmed nosocomial transmission to a hospital inpatient. LD was confirmed in 22 patients. Case-patients were more likely than controls to have been within 1,000 feet of the hospital (matched odds ratio, 21.0; 95% confidence interval, 2.9-368) during the 2 weeks prior to illness. Legionella pneumophila serogroup 1 (Lp-1) was isolated from hospital cooling towers (CTs) and rooftop air samples but not from hospital potable water or community CTs. Hospital CT and air Lp-1 isolates matched all five patient isolates by monoclonal antibody, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis subtyping. Strategies to prevent LD must include minimizing transmission from CTs.

Community outbreak of Legionnaires' disease: an investigation confirming the potential for cooling towers to transmit Legionella species.

In August and September 1993, we investigated an outbreak of legionnaires' disease in Fall River, Massachusetts, that involved 11 persons; the attack rate was highest in Flint, a community of Fall River. All cases were infected with Legionella pneumophila serogroup 1 (Lp-1). A case-control study revealed that cases were more likely than matched controls to have visited sites in neighborhood A of Flint. Environmental sampling in Flint found that four of nine aerosol-producing devices sampled contained legionellae; only two, conjoined cooling towers on building A, contained Lp-1. Three independent methods of subtyping--monoclonal antibody subtyping, arbitrary primer polymerase chain reaction, and pulsed-field gel electrophoresis--revealed that Lp-1 isolates from three cases with culture-positive legionnaires' disease matched those from the cooling towers on building A. Water samples from the homes of cases with culture-positive legionnaires' disease contained no legionellae. The results of this epidemiologic and laboratory investigation indicate that the cooling towers on building A were the source of the outbreak of legionnaires' disease and confirm the importance of cooling towers in the transmission of legionnaires' disease.