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. 2024 Oct 8:15:1452861.
doi: 10.3389/fmicb.2024.1452861. eCollection 2024.

A rapid and reliable method for early Legionella pneumophila identification and characterization in support of the epidemiology study

Valentina Monistero  1   2 , Nadia Vicari  3 , Paola Prati  3 , Roldano Bragoni  3 , Alessandra Gazzola  4 , Lorenza Sala  4 , Antonio Maisano  4 , Paolo Moroni  1   2 , Valerio Bronzo  1   2 , Mario Vittorio Luini  5 , Bianca Castiglioni  5 , Paola Cremonesi  5
Affiliations

A rapid and reliable method for early Legionella pneumophila identification and characterization in support of the epidemiology study

Valentina Monistero et al. Front Microbiol. .

Abstract

Introduction: Legionnaires' disease is a severe pneumonia predominantly caused by Legionella pneumophila (Lp), whose major reservoirs are artificial water systems. As most human infections are caused by L. pneumophila serogroup 1 (Lp1), a reliable method for Lp distinction can be crucial for bacterial spread prevention. As the ability to withstand in environments and to cause the waterborne disease is strongly related to specific genes, the identification of virulent strains can be of great relevance to implement water environmental monitoring and to contain harmful outbreaks to public health. We aimed to test an assay for Lp identification among different Legionella species, and to determine the serogroups. Additionally, we investigated the carriage of virulence and antimicrobial resistance genes.

Methods: A total of 90 Legionella spp. isolates identified by phenotypic tests were subjected to the designed quantitative PCR assay targeting specific mip for Lp, wzm for Lp1, pvcA and ahpD for biofilm production. Eleven serogroups were investigated in all our isolates tested positive for mip gene, subsequently analyzed for 12 virulence and 8 antimicrobial resistance genes.

Results: Only the 70 Lp isolates were positive for mip. Out of 27 Lp isolates belonging to serogroup 1 based on agglutination test, 23 (85.2%) carried wzm. The presence of ahpD and pvcA was found in 94.3 and 98.6% of Lp isolates, respectively. By multiplex PCR, all 23 wzm-positive strains were confirmed as serogroup 1 that was the most predominant (33%). At least one virulence gene was detected in all Lp isolates. The most frequent gene was ispE (100%), followed by issD (96%), icmK and enhC (93%), cpxA (91%), rtxA2 (74%), lvhB8-B9 (61%), and prpA (54%). The other genes were less diffused in Lp strains (rtxA1, 44%; lvhB3-B4, 47%; pvcB, 27%; lvrE, 24%). Of the macrolide resistance genes, the ereA was found in 84% of Lp strains, while only 14 (20%) harbored the lpeAB among the efflux pump genes.

Conclusion: The assays validated in this study enable the simultaneous Lp and Lp1 detection. The differentiation of Lp strains according to their virulence properties could be useful to predict the bacterial ability to survive and to cause the disease.

Keywords: Legionella pneumophila; Legionnaires’ disease; TaqMan quantitative PCR; antimicrobial resistance; multiplex PCR serotyping; serogroup; virulence factor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Distribution of serogroups (SG) in 58 Legionella pneumophila isolates from Lombardia (A) and Emilia-Romagna (B), based on a mPCR serotyping assay.
See this image and copyright information in PMC

References

    1. Arrigo I., Galia E., Fasciana T., Diquattro O., Tricoli M. R., Serra N., et al. . (2022). Four-year environmental surveillance program of Legionella spp. in one of Palermo's largest hospitals. Microorganisms 10:764. doi: 10.3390/microorganisms10040764, PMID: - DOI - PMC - PubMed
    1. Behets J., Declerck P., Delaedt Y., Creemers B., Ollevier F. (2006). Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples. J. Microb. Methods 68, 137–144. doi: 10.1016/j.mimet.2006.07.002, PMID: - DOI - PubMed
    1. Benitez A. J., Winchell J. M. (2013). Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1. J. Clin. Microbiol. 51, 348–351. doi: 10.1128/JCM.02510-12, PMID: - DOI - PMC - PubMed
    1. Benitez A. J., Winchell J. M. (2016). Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay. Diagn. Microbiol. Infect. Dis. 84, 298–303. doi: 10.1016/j.diagmicrobio.2016.01.007, PMID: - DOI - PMC - PubMed
    1. Buse H. Y., Lu J., Ashbolt N. J. (2015). Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes. Appl. Environ. Microbiol. 81, 630–639. doi: 10.1128/AEM.03394-14, PMID: - DOI - PMC - PubMed

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