Characterization of Legionella pneumophila Populations by Multilocus Variable Number of Tandem Repeats (MLVA) Genotyping from Drinking Water and Biofilm in Hospitals from Different Regions of the West Bank

Abstract

The West Bank can be considered a high-risk area for Legionnaires’ disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS.

Cytotoxicity, Intracellular Replication, and Contact-Dependent Pore Formation of Genotyped Environmental Legionella pneumophila Isolates from Hospital Water Systems in the West Bank, Palestine

“…This study included 60 L. pneumophila environmental strains isolated from eight hospitals in the West Bank, Palestine ( Table 1 and Supplementary Material Table S1 ). Genotyping was conducted using multilocus variable number of tandem repeat analysis using 13 loci—MLVA-13 (MLVA-12 plus 1 loci unique for the MLVA-8 scheme) designated as (MLVA-8(12) [ 38 , 45 ] assigning the majority of the strains to three genotypes (Gt4(17), Gt6(18), and Gt10(93)) and four clonal complexes (VACC1, VACC2, VACC5 and VACC11) [ 38 ]. Table 1 describes the details of the environmental L. pneumophila isolates used for this study.…”

“…Multi locus variable number of tandem repeats (VNTR) analysis (MLVA) using 13 loci designated as MLVA-8(12) was successfully used to assess the genetic diversity among L. pneumophila isolates. VNTRs consist of relatively short DNA fragments repeated in tandem and can vary in copy number among strains [ 38 , 39 , 40 , 41 , 42 ]. Recent publications demonstrated that the majority of clinically relevant strains can be grouped into a limited number of Clonal Complexes (CCs) defined by MLVA, called VNTR analysis CC (VACC) [ 38 , 41 ].…”

“…VNTRs consist of relatively short DNA fragments repeated in tandem and can vary in copy number among strains [ 38 , 39 , 40 , 41 , 42 ]. Recent publications demonstrated that the majority of clinically relevant strains can be grouped into a limited number of Clonal Complexes (CCs) defined by MLVA, called VNTR analysis CC (VACC) [ 38 , 41 ]. MLVA can be used to complement recommended Sequence-Based Typing (SBT) and gain insights into the clonal structure of L. pneumophila populations.…”

“…MLVA can be used to complement recommended Sequence-Based Typing (SBT) and gain insights into the clonal structure of L. pneumophila populations. Many studies have used MLVA for the genotyping of L. pneumophila strains [ 38 , 39 , 40 , 41 , 42 ]. They showed the high correspondence between MLVA genotypes and STs with an important increase in resolution when applying MLVA, which is relevant for understanding clonal populations.…”

“…The current study on virulence traits is based on the results of two former studies [ 38 , 43 ]. In comprehensive two-year surveillance, DWDS of eight hospitals across the West Bank were analyzed with respect to L. pneumophila occurrence by culture and PCR, and environmental parameters.…”