Isolates Of Legionella Pneumophila Serogroup Research Articles

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.

Legionella pneumophila serogroup 1 in a birthing pool

This report describes a risk assessment and subsequent actions following isolation of Legionella pneumophila serogroup 1 in the water supply to a birthing pool during a planned maintenance programme. A literature search for cases of neonatal legionellosis identified 24 reports of cases among babies aged <2 months, two of which were associated with water births. On this basis, the pool was closed until Legionella spp. were undetectable. Control proved difficult as hyperchlorination failed, and a filter fitted to the thermostatic mixer tap supplying the pool slowed filling so much that additional taps were required to achieve a satisfactory flow rate.

Distribution of Legionella species from environmental water sources of public facilities and genetic diversity of L. pneumophila serogroup 1 in South Korea.

A total of 560 Legionella species were isolated from environmental water sources from public facilities from June to September 2008 throughout South Korea. The distribution of Legionella isolates was investigated according to geographical region, facility type, and sample type. The genetic diversity of 104 isolates of Legionella pneumophila serogroup 1 (sg 1) was analyzed by sequence-based typing (SBT). L. pneumophila was distributed broadly throughout Korea, accounting for 85.0% of the isolates, and L. pneumophila sg 1 predominated in all of the public facilities except for the springs. Legionella anisa and Legionella bozemanii predominated among non-L. pneumophila species (48.1% and 21.0%, respectively). The second most dominant strain differed depending on the facility type: L. anisa was the second most dominant strain in the buildings (10.8%), L. pneumophila sg 5 in public baths (21.6%), L. pneumophila sg 6 in factories (12.0%), and L. pneumophila sg 7 in hospitals (13.1%). In the SBT analysis, 104 L. pneumophila sg 1 isolates were differentiated into 26 sequence types (STs) and categorized into 3 clonal groups (CGs) and 10 singleton STs via the eBURST V3 program. ST1, a potential founder of major CG1, was commonly distributed (48.1%). The dominant ST in hot water was ST-K1 (7, 12, 17, 3, 35, 11, 11), which was designated in this study (36.1%). The second most dominant strain differed depending on the type of facility from which the samples were obtained. The unique allelic profile of ST-K1, obtained from hot water, was not found in the European Working Group for Legionella Infections (EWGLI) SBT database.

Nosocomial Legionnaires' disease caused by Legionella pneumophila serogroup 6: Implication of the sequence-based typing method (SBT)

Sequence-based typing (SBT) was used to determine the allelic profiles of 3 sporadic clinical isolates as well as 7 environmental isolates of Legionella pneumophila serogroup 6, isolated at the Karolinska Hospital during 2004. The clinical isolates were cultured from patients with nosocomial Legionnaires' disease (LD), while the environmental isolates were cultured from potable water sources of the hospital wards in the close vicinity of the 3 patients being investigated. The genes sequenced for the construction of the SBT profile included flaA, pilE, asd, mip, mompS and proA, in this pre-determined order and the allelic profile of the 10 isolates was identical (3, 13, 1, 28, 14, 9). Furthermore, 2 of the isolates, 1 clinical and 1 environmental, were analysed using the amplified fragment length polymorphism analysis (AFLP). The AFLP genotype of both isolates was congruent. Eight of 9 control L. pneumophila serogroup 6 isolates had the same SBT profile as the study isolates. We conclude that the environmental strain isolated from our hospital's drinking water is indistinguishable genotypically from the 3 clinical isolates of Legionella. However, this genotype of L. pneumophila is geographically widespread. Thus, results of genotyping must be evaluated in conjunction with the clinical and epidemiological data.

Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates ofLegionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Clinical and Environmental Isolates of Legionella pneumophila Serogroup 1 Cannot Be Distinguished by Sequence Analysis of Two Surface Protein Genes and Three Housekeeping Genes

We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.

Culture Proven Legionella Pneumophila Pneumonia in a HIV-infected Patient: Case Report and Review

We report the first culture proven case of Legionella pneumonia in a HIV-positive patient in Italy. The laboratory diagnosis was obtained by isolation of Legionella pneumophila serogroup 1, serology, urinary antigen detection and PCR. Culture first allowed diagnosis of the infection, that probably would have been unrecognized. Since Legionellosis in HIV-positive patients with respiratory symptoms is rare and difficult to confirm, we strongly suggest that all available laboratory tests, and particularly culture, should be performed. A review of literature on culture proven cases is also provided.

A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence

Abstract The majority of clinical isolates of Legionella pneumophila serogroup 1 produce lipopolysaccharide (LPS) that reacts with monoclonal antibody (MAb) 3/1. By using a negative cell sorting method, we isolated a spontaneous LPS mutant from L. pneumophila serogroup 1 strain Corby that lost reactivity with this MAb. The mutant contained a single nucleotide exchange in position 169 of the lag-1 gene that encodes an O-acetyltransferase that is responsible for O-acetylation of the L. pneumophila O-repeat unit (legionaminic acid). This mutation resulted in a single amino acid exchange in a highly conserved motif present in many O-acetyltransferase-like proteins. RT-PCR analysis revealed that the mutant lag-1 gene was transcribed, but the resulting protein lacked O-acetyltransferase activity. Chemical analysis of the mutant LPS revealed that it lacked 8- O-acetyl groups in legionaminic acid. In addition, the mutant failed to produce high-molecular-weight long-chain O-polysaccharide. Complementation of the mutant with the wild-type lag-1 gene restored reactivity with MAb 3/1 and the chemical structure of the wild-type LPS. Strain Corby and its MAb 3/1-negative mutant were indistinguishable in their serum resistance characteristics, and in uptake and intracellular multiplication in Acanthamoeba castellanii and macrophages.

Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis.

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

Subinhibitory concentrations of antimicrobial agents reduce the uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 cells by altering the expression of virulence-associated antigens.

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

Isolation of Legionella pneumophila serogroup 1 from human feces with use of amebic cocultures.

Isolation of Legionella pneumophila serogroup 1 from human feces with use of amebic cocultures.

Isolation of Legionella pneumophila Serogroup 5 from Empyema Following Esophageal Perforation: Source of the Organism and Mode of Transmission

A patient undergoing esophageal dilatation for carcinoma of the esophagus suffered esophageal perforation and development of an empyema. Culture of pleural fluid yielded multiple organisms, including Legionella pneumophila serogroup 5. Epidemiologic investigation showed that the source of L pneumophila was a tap used by the nursing personnel to fill patients' water pitchers. Whole-cell restriction endonuclease analysis of DNA from the clinical and environmental isolates of L pneumophila serogroup 5 yielded identical patterns. Our findings suggest that L pneumophila was acquired by the patient at least 12 h prior to the procedure causing the esophageal perforation and empyema, suggesting that the organism can persist in an infectious form in the upper aerodigestive tract.

Wide distribution of a 36-megadalton plasmid among clinical and environmental Spanish isolates ofLegionella pneumophila serogroup 1

With the eventual goal of characterizingLegionella pneumophila serogroup 1 plasmids at the molecular level, we have analyzed the plasmid contents of 78 clinical and environmental Spanish isolates. After selection of a suitable alkaline lysis method, we detected plasmids with approximate molecular weights of 25, 36, 40, 61, 80, 85, 90, and 95 megadalton (MDal). Several factors (i.e., wide temporal and geographic distribution, high frequency in both clinical and environmental isolates, and apparent high copy number after subculturing) make the 36 MDal type IA plasmid an appropriate plasmid for further molecular studies.

Common Epitope on Urinary Antigen Derived from Different Legionella pneumophila Serogroup 1 Strains Recognized by a Monoclonal Antibody

Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

Phenotypic variation amongst genotypically homogeneousLegionella pneumophilaserogroup 1 isolates: implications for the investigation of outbreaks of Legionnaires' disease

One hundred and seventy-nine isolates of Legionella pneumophila serogroup 1, obtained from a site associated with an outbreak of Legionnaires' disease, were examined by monoclonal antibody subgrouping, restriction fragment length polymorphism typing, restriction endonuclease analysis and plasmid content. Nine distinct phenotypes were detected but at the genotypic level all strains were closely related. The data presented indicate that phenotypic variation of a single parent strain can occur within an environmental site. The implications of these findings are discussed in relation to the investigation of outbreaks of Legionnaires' disease.

The application of molecular and immunologic techniques to study the epidemiology of Legionella pneumophila serogroup 1.

We applied monoclonal antibody typing and restriction endonuclease analysis of plasmid DNA to study 28 clinical and 35 environmental (potable water) isolates of Legionella pneumophila serogroup 1 from three hospitals in Iowa between 1981 and 1986. Monoclonal antibody typing employed a panel of seven antibodies and delineated eight different subtypes. Plasmids were present in 57% of the isolates including 12 of 28 (43%) clinical and 25 of 35 (69%) potable water isolates. The plasmids ranged in size from 28 to 98 kilobase pairs and comprised eight distinct subtypes by restriction endonuclease analysis with Eco RI. Combination of monoclonal antibody and restriction endonuclease subtyping (composite subtyping) revealed 19 different composite subtypes of Legionella pneumophila serogroup 1. The most common composite subtype, 09:04, comprised 29% (18 of 63) of the isolates and was only found in clinical and potable water samples from a single pavilion in hospital A during an outbreak of Legionella pneumophila serogroup 1 pneumonia. Aside from this cluster the diversity of composite subtypes of Legionella pneumophila serogroup 1 observed in clinical and potable water sources over the 5-year period was striking. The combination of monoclonal antibody and restriction endonuclease typing resulted in improved strain delineation and a more useful use of epidemiologic markers for Legionella pneumophila serogroup 1.

Atypical legionellosis: Isolation of Legionella pneumophila serogroup 1 from a patient with aspiration pneumonia

It has been suspected that the number of Legionella pneumophila infections is substantially higher than commonly reported, due to subclinical infections. We report a patient where the diagnosis of a legionella infection was made only because of a large-scale surveillance programme for legionellosis. The case demonstrates the possibility of legionellas being involved in lung disease though not necessarily causing overt legionellosis.

Correlation of subtypes of Legionella pneumophila defined by monoclonal antibodies with epidemiological classification of cases and environmental sources.

Twenty-five clinical isolates of Legionella pneumophila serogroup I were classified as epidemic or sporadic on epidemiological grounds. All were isolated from patients at the Medical Center Hospital of Vermont (MCHV) in Burlington between 1977 and 1983. These coded isolates were examined with a panel of eight monoclonal antibodies to serogroup 1 and an indirect microimmunofluorescent test. Strains from 14 of the 15 MCHV epidemic cases had one staining pattern (A); isolates from eight of nine MCHV sporadic (nosocomial) cases had a different pattern (B). One sporadic strain was of staining pattern A, and in one epidemic case an isolate of each pattern (A and B) was obtained. The only case that was clearly community-acquired displayed a third staining pattern (C). Four isolates from the cooling tower considered the most likely source for the epidemic strains had an antigenic reaction identical to isolates in epidemic cases, whereas three strains from the potable water system displayed an antigenic pattern identical to isolates in sporadic cases.

Molecular epidemiology of Legionella pneumophila serogroup 1.

The DNA of patient and environmental isolates of Legionella pneumophila serogroup 1 was analyzed by restriction endonuclease cleavage. The electrophoretic patterns of the DNA digests of isolates from a group of patients with Legionnaires disease acquired in a hospital were indistinguishable from one another and were identical to the DNA pattern of a strain isolated from the hot water supply of the hospital. On the other hand, they were easily differentiated from strains isolated from patients and hot water supplies in other hospitals in the same city. The homogeneity of populations of L. pneumophila serogroup 1 colonizing plumbing systems was also investigated by DNA restriction endonuclease analysis in three hospitals. We distinguished two subtypes in one hospital; the two other hospitals had homogeneous populations. Restriction endonuclease digest analysis of L. pneumophila serogroup 1 DNA enables subtyping and appears to be a useful method for examining the epidemiology of outbreaks of Legionnaires disease.