New insights into the epidemiology of Listeria monocytogenes – A cross-sectoral retrospective genomic analysis in the Netherlands (2010–2020)

IntroductionListeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling.MethodsWe investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient’s age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category.ResultsWe confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10  years.DiscussionTaken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden

Occurrence and Genetic Diversity of Uncultured Legionella spp. in Drinking Water Treated at Temperatures below 15°C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 × 10(3) to 7.8 × 10(5) cells liter(−1) and were significantly higher in SW treated with multiple barriers at 4°C than in GW treated at 9 to 12°C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(−1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15°C

Identification of Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers

Free-living N2-fixing Frankia strains isolated from Casuarina sp. were investigated for genomic polymorphism. We used six 10-mer oligonucleotides as single arbitrary primers (AP) for the polymerase chain reaction (PCR) in order to amplify random DNA fragments in the genome of free-living Frankia strains. Agarose-gels of the amplified genomic DNA revealed that two of the six arbitrary primers showed polymorphism in the eight different Frankia genomes. Analysis of the AP-PCR products showed 9 polymorphic bands ranging from 4.1-0.60 kb. We conclude that single arbitrary primers can be used to amplify genomic DNA, and that polymorphism can be detected between the amplification products of the different Frankia genomes

Concentration and Diversity of Uncultured Legionella spp. in Two Unchlorinated Drinking Water Supplies with Different Concentrations of Natural Organic Matter ▿ †

Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8 ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 102 cells liter−1 in supply A and 2.5 (± 1.6) × 103 cells liter−1 in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity) were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration

Identification of Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers

Free-living N2-fixing Frankia strains isolated from Casuarina sp. were investigated for genomic polymorphism. We used six 10-mer oligonucleotides as single arbitrary primers (AP) for the polymerase chain reaction (PCR) in order to amplify random DNA fragments in the genome of free-living Frankia strains. Agarose-gels of the amplified genomic DNA revealed that two of the six arbitrary primers showed polymorphism in the eight different Frankia genomes. Analysis of the AP-PCR products showed 9 polymorphic bands ranging from 4.1-0.60 kb. We conclude that single arbitrary primers can be used to amplify genomic DNA, and that polymorphism can be detected between the amplification products of the different Frankia genomes.</p

Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test▿ †

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C

Flow-through real time bacterial biosensor for toxic compounds in water

A flow-through fiber-optic-based bacterial monitoring system for online monitoring of toxic pollutants in water has been developed. Two bacterial strains containing fusions of recA (DNA damage) and grpE (heat-shock) promoters to the lux operon (CDABE) were immobilized on a fiber optic and tested for their ability to detect pollutants in flowing tap water and surface water. Conditions for running the system for 24 h were optimized and first experiments with the system show (1-h) response times and response heights similar to the previous static systems. Responses were related to the doses and the sensitivity is good (comparable to static systems), but needs to be increased to be able to monitor whether also the low guideline values are exceeded by pollutants. 24-h measurements in tap water demonstrate the ability of the device to run for such a time period, but in river water loss of functionality of the bacteria was observed. This flow-through fiber-optic-based monitoring system has proven to be a useful next step in the development of a simple on-line real time sensor for relevant human toxicants in flowing water.</p

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter-1) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter-1) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter-1 and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter-1

Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences▿ †

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa