Legionella is one of the most important waterborne pathogens that can lead to both outbreaks and sporadic cases. The majority of travel-associated Legionnaires' disease (TALD) cases are contracted during hotel stays. The aim of this study was to evaluate the prevalence and genetic diversity of Legionella spp. in hotel water supply systems in Latvia. In total, 834 hot water samples were collected from the water systems of 80 hotels in Latvia. At least one Legionella spp. positive sample was detected in 47 out of 80 hotels (58.8%). Overall, 235 out of 834 samples (28.2%) were Legionella spp. positive. The average hot water temperature in Latvian hotels was 49.8 °C. The most predominant L. pneumophila serogroup (SG) was SG3 which was found in 113 (49.8%) positive samples from 27 hotels. For 79 sequenced L. pneumophila isolates, 21 different sequence types (ST) were obtained, including 3 new types-ST2582, ST2579, and ST2580. High Legionella contamination and high genetic diversity were found in the hotel water supply systems in Latvia, which, together with the insufficient hot water temperature, may indicate that the lack of regulation and control measures may promote the proliferation of Legionella.

Background: Biofilms, when formed on the surfaces of water pipes, can be responsible for a wide range of water quality and operational problems. We sought to assess the bacterial and free-living protozoa (FLP) diversity, in relation to the presence of Legionnaire's disease-causing bacteria Legionella pneumophila ( L. pneumophila ) in 45 biofilms of hot water distribution system pipes of apartment buildings in Riga, the capital city of Latvia. Results: 16S rRNA amplicon sequencing (metataxonomics) revealed that each biofilm contained 224 rather evenly distributed bacterial genera and that most common and most abundant were two genera, completely opposites in terms of their oxygen requirements: the obligately anaerobic Thermodesulfovibrio and the strictly aerobic Phenylobacterium . Water temperature and north-south axis (i.e., different primary water sources) displayed the most significant effect on the inter-sample variations, allowing us to re-construct three sub-networks (modules) of co-occurring genera, one involving (potentially FLP-derived) Legionella spp. Pangenome-based functional profile predictions suggested that all three may be dominated by pathways related to the development and maintenance of biofilms, including quorum sensing and nutrient transport, as well as the utilization of various energy sources, such as carbon and nitrogen. In our 18S rRNA amplicon sequencing data, potential hosts of L. pneumophila were detected in 11 out of 12 biofilm samples analyzed, however, in many cases, their relative abundance was very low (<1%). By validating our findings using culture-based methods, we detected L. pneumophila (serogroups 2, 3, 6 and 9) in nine (20%) biofilms, whereas FLP (mostly Acanthamoeba, Vahlkampfidae and Vermamoeba spp.) were present in six (~13%) biofilms. In two biofilms, L. pneumophila and its potential hosts were detected simultaneously, using culture-based methods. Conclusions: Overall, our study sheds light on the community diversity of hot water biofilms and predicts how several environmental factors, such as water temperature and source might shape it.

Rapid identification of methicillin-resistant Staphylococcus could ensure appropriate medical care. A total of 409 Staphylococcus spp. strains were used to develop a reliable MALDI-TOF method for species identification. We tested twelve S. aureus strains to compare three different sample preparation methods and the reproducibility of the methicillin-resistant m/z 2414 ± 2 indicator peak with direct method in triplicate. A total of 65 Staphylococcus spp. strains (including 37 methicillin-resistant strains) from clinical and hospital environment isolates were used to confirm the presence of phenol-soluble modulin (PSM-mec) peptide. All 272 S. aureus strains from 409 samples were correctly identified at species level by MALDI-TOF. The samples prepared by three methods gave spectra with differences in the intensities and presence of certain peaks. The PSM-mec peak was not visible after the extraction method. The peak m/z 2414 ± 2 was only detected in 61% of the methicillin-resistant strains and in none of the methicillin-sensitive strains. The peak reproducibility for the five analyzed S. aureus strains showing the peak at m/z 2414 ± 2 was 87%. The delta-toxin was observed in 49 out of 65 samples regardless of methicillin susceptibility, as well as in all the samples exhibiting the PSM-mec peak. The peak m/z 2414 ± 2 is specific to methicillin-resistant strains carrying the mecA gene, but the absence of peak m/z 2414 ± 2 does not exclude the possibility of resistance to methicillin. Thus, implementing MALDI-TOF analysis in routine laboratory work, especially with clinical samples, would in many cases provide rapid warning about the presence of methicillin-resistant strains.

Background and Objectives:Legionella is one of the most important water-related pathogens. Inside the water supply systems and the biofilms, Legionella interact with other bacteria and free-living amoeba (FLA). Several amoebas may serve as hosts for bacteria in aquatic systems. This study aimed to investigate the co-occurrence of Legionella spp. and FLA in drinking water supply systems. Materials and Methods: A total of 268 water samples were collected from apartment buildings, hotels, and public buildings. Detection of Legionella spp. was performed in accordance with ISO 11731:2017 standard. Three different polymerase chain reaction (PCR) protocols were used to identify FLA. Results: Occurrence of Legionella varied from an average of 12.5% in cold water samples with the most frequent occurrence observed in hot water, in areas receiving untreated groundwater, where 54.0% of the samples were Legionella positive. The occurrence of FLA was significantly higher. On average, 77.2% of samples contained at least one genus of FLA and, depending on the type of sample, the occurrence of FLA could reach 95%. In the samples collected during the study, Legionella was always isolated along with FLA, no samples containing Legionella in the absence of FLA were observed. Conclusions: The data obtained in our study can help to focus on the extensive distribution, close interaction, and long-term persistence of Legionella and FLA. Lack of Legionella risk management plans and control procedures may promote further spread of Legionella in water supply systems. In addition, the high incidence of Legionella-related FLA suggests that traditional monitoring methods may not be sufficient for Legionella control.

Background: Campylobacter is the main cause of bacterial gastroenteritis worldwide. The main transmission route is through consumption of food contaminated with Campylobacter species or contact with infected animals. In Latvia, the prevalence of campylobacteriosis is reported to be low (4.6 cases per 100,000 population in 2016). Aim: To determine prevalence, species spectrum and antimicrobial resistance (AMR) of Campylobacter spp. in Latvia, using data from various livestock and human clinical samples. Methods: We analysed data of Campyloba cter microbiological monitoring and AMR (2008 and 2014-16) in Latvia. Data from broilers, poultry, pigs, calves and humans were used to determine prevalence of Campylobacter. Additionally, 45 different origin isolates (22 human) were sequenced on the Illumina MiSeq platform; for each isolate core genome multilocus sequence typing was used and relevant antimicrobial resistance mechanisms were identified. Results: Overall, Campylobacter prevalence in was 83.3% in pigs, 50.2% in broilers, 16.1% in calves and 5.3% in humans; C. jejuni was the predominant species in all sources except pigs where C. coli was main species. High level of resistance in Campylobacter were observed against fluoroquinolones, tetracycline and streptomycin, in most of sequenced isolates genetic determinants of relevant AMR profiles were identified. Conclusions: In Latvia, prevalence of Campylobacter in livestock is high, especially in pigs and broilers; prevalence in poultry and humans were lower than in other European countries. AMR analysis reveals increase of streptomycin and tetracycline resistant broiler origin C. jejuni strains. WGS demonstrates a high compliance between resistance phenotype and genotype for quinolones and tetracyclines.