Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012-2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.

Methicillin-resistant and methicillin-sensitive Staphylococcus aureus in pork industry workers, Catalonia, Spain.

Background: Methicillin-resistant S. aureus (MRSA) especially ST398, is a zoonotic agent. This study aimed to determine the prevalence of methicillin-susceptible S. aureus (MSSA) and MRSA among workers in the pork production chain. Methods: 659 workers associated with 123 pig farms, livestock transporters, one pig slaughterhouse, pork transporters and 23 pork butcheries were studied for S. aureus recovery, and all isolates were characterized (antibiotic resistance, MLST and spa-typing). Results: The prevalence of S. aureus was 35.5%, 75.6% of isolates being MRSA. The prevalence of MRSA was 68.7% (149/217) among pig farm, 33.9% (19/56) livestock transporters, 2.9% (9/306) slaughterhouse, 0% in pork transporters (0/36) and butchery workers (0/44). Of the 234 S. aureus-positive workers, 100% (149/149) of pig farm workers, 82.6% (19/23) of livestock transporters, and 16.4% (9/55) of slaughterhouse workers carried MRSA isolates (p < 0.001). Of the workers who had contact with live swine, 61.8% (178/288) were S. aureus-positive, MRSA being detected in 96.1% of cases (p < 0.001). The most frequent lineage among MRSA were: ST398 (97.7%; 173/177) and ST1 (1.7%; 3/177); and among MSSA were ST30 (19.2%; 11/57) and ST5 (10.5%; 6/57). The most frequent spa-types among MRSA were t011 (93.8%, 166/177) and t1451 (2.25%, 4/177), and among MSSA: t084 (10.5%, 6/57) and t021 (7.0%, 4/57). All MRSA isolates showed resistance to tetracycline, 92.7% to clindamycin, 81.9% to erythromycin and 40.1% to cotrimoxazole. Conclusions: Pig industry workers having occupational contact with live animals present a high risk of colonization of MRSA, especially by MRSA-ST398. Prevention measures should be intensified in any employment sector involving live animals.

[Low risk of environmental contagion by SARS-CoV-2 in non-sanitary spaces]. / Bajo riesgo de contagio ambiental por SARS-CoV-2 en espacios no sanitarios.

Objective: To study the presence of SARS-CoV-2 on surfaces (high, medium and low contacts) and airs in non-sanitary spaces with high public influx to evaluate the risk of environmental contagion. Method: Surfaces and airs were analysed by RT-qPCR to detect the presence of SARS-CoV-2. Results: A total of 394 surfaces and air samples were obtained from spaces with high public influx such as offices, shopping centres and nursing homes. The virus was not detected in any of the samples analysed. Conclusion: Although we cannot emphatically conclude that there is no risk of environmental infection by SARS-CoV-2 in non-sanitary spaces, we can affirm that the risk is almost non- existent.

Low risk of environmental contagion by SARS-CoV-2 in non-sanitary spaces.

OBJECTIVE: To study the presence of SARS-CoV-2 on surfaces (high, medium and low contact) and airs in non-sanitary spaces with high public influx to evaluate the risk of environmental contagion. METHODS: Surfaces and airs were analysed by RT-qPCR to detect the presence of SARS-CoV-2. RESULTS: 394 surfaces and air samples were obtained from spaces with high public influx such as offices, shopping centres and nursing homes. The virus was not detected in any of the samples analysed. CONCLUSION: Although we cannot emphatically conclude that there is no risk of environmental 27 infection by SARS-CoV-2 in non-sanitary spaces, we can affirm that the risk is almost non- existent.

Clinical and Epidemiological Characteristics of Streptococcus suis Infections in Catalonia, Spain.

Introduction: Streptococcus suis (S. suis) is a human zoonotic pathogen of occupational origin, with infection acquired through contact with live pigs or pig meat. Pig farming is one of Catalonia's biggest industries and as a result this region of Spain has one of the highest density pig populations per km2. The aim of our study was to describe the infections caused by S. suis occurring in that area over a 9-year period. Materials and Methods: A retrospective, multi-center study was carried out by searching records from 15 hospitals in Catalonia for the period between 2010 and 2019. Results: Over the study period altogether nine cases of S. suis infection were identified in five hospitals, with five of these cases occurring in the 2018-2019 period. The mean age of patients was 48 ± 8.9 years and all of them were males. Five patients (55.6%) worked in pig farms. The most frequent manifestation of infection was meningitis (5 cases; 55.6%) followed by septic arthritis (3 cases; 33.3%). None of the patients died at 30 days; nonetheless, 4 developed hearing loss as a long-term complication. Conclusion: The most commonly identified S. suis infection was meningitis. Over 50% of the episodes occurred in the last 2 years and have affected pig farm workers. Further surveillance is needed in order to know its prevalence.

Nondetection of SARS-CoV-2 on high-touch surfaces of public areas next to COVID-19 hospitalization units.

We studied the contamination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the bacterial load of high-touch surfaces located in public areas next to coronavirus disease (COVID-19) hospitalization units. Ninety-two samples were obtained from 46 different high-touch surfaces: 36 sites next to COVID-19 hospitalization units and 10 sites in the cabins of the public elevators. SARS-CoV-2 was not detected at any site, despite high bacterial loads suggested that the studied sites had been frequently touched prior to the sampling.

Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement.

Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2-3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL-1 with a detection limit (LoD) of 4 cfu·mL-1.

Legionella SBT applied directly to respiratory samples as a rapid molecular epidemiological tool.

Legionnaires' disease (LD) is an atypical pneumonia caused by the inhalation of Legionella. The methods used for the diagnosis of LD are direct culture of respiratory samples and urinary antigen detection. However, the sensitivity of culture is low, and the urinary antigen test is specific only for L. pneumophila sg1. Moreover, as no isolates are obtained, epidemiological studies cannot be performed. The implementation of Nested-sequence-based typing (Nested-SBT) makes it possible to carry out epidemiological studies while also confirming LD, especially in cases caused by non-sg 1. Sixty-two respiratory samples from patients with Legionella clinically confirmed by positive urinary antigen tests were cultured and tested by Nested-SBT, following the European Study Group for Legionella Infections (ESGLI) protocol. Only 2/62 (3.2%) respiratory samples were culture-positive. Amplification and sequencing of Nested-SBT genes were successfully performed in 57/62 samples (91.9%). The seven target genes were characterised in 39/57 (68.4%) respiratory samples, and the complete sequence type (ST) was obtained. The mip gene was the most frequently amplified and sequenced. Nested-SBT is a useful method for epidemiological studies in culture-negative samples, achieving a 28.7-fold improvement over the results of culture studies and reducing the time needed to obtain molecular epidemiological results.

New system for the detection of Legionella pneumophila in water samples.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Population structure of Environmental and Clinical Legionella pneumophila isolates in Catalonia.

Legionella is the causative agent of Legionnaires' disease (LD). In Spain, Catalonia is the region with the highest incidence of LD cases. The characterisation of clinical and environmental isolates using molecular epidemiology techniques provides epidemiological data for a specific geographic region and makes it possible to carry out phylogenetic and population-based analyses. The aim of this study was to describe and compare environmental and clinical isolates of Legionella pneumophila in Catalonia using sequence-based typing and monoclonal antibody subgrouping. A total of 528 isolates were characterised. For data analysis, the isolates were filtered to reduce redundancies, and 266 isolates (109 clinical and 157 environmental) were finally included. Thirty-two per cent of the clinical isolates were ST23, ST37 and ST1 while 40% of the environmental isolates were ST284 and ST1. Although the index of diversity was higher in clinical than in environmental ST isolates, we observed that clinical STs were similar to those recorded in other regions but that environmental STs were more confined to particular study areas. This observation supports the idea that only certain STs trigger cases or outbreaks in humans. Therefore, comparison of the genomes of clinical and environmental isolates could provide important information about the traits that favour infection or environmental persistence.

Antibody test for Legionella pneumophila detection.

Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.

Differential Proteome Between Patient-Related and Non-related Environmental Isolates of Legionella pneumophila.

Molecular epidemiologic studies of Legionella have shown different molecular types coexisting in the same environment, with only one having the ability to trigger an outbreak. We therefore studied the proteome of isolates of these different molecular types in search of the proteins responsible for infection. In this study, we performed a differential proteomic analysis between patient-related and non-patient-related environmental isolates using two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry. Sixty-three spots were observed as being different between the two groups; 31 spots were identified corresponding to 23 different proteins. Patient-related isolates overexpressed proteins associated with metabolism, with enzymes of the tricarboxylic acid cycle and the degradation pathways being the most abundant proteins identified. However, the largest group of non-patient-related proteins was associated with stress response. Furthermore, the MOMP protein was located in different spots depending on their patient-related or non-patient-related origin, suggesting different post-translational modifications. According to these results, different bacterial adaptation pathways are activated in stress conditions which influence their ability to produce infection.

Characterization of unrelated clinical Legionella pneumophila isolates in Catalonia by monoclonal subgrouping and sequence-based typing.

AIM: To characterize the genetic diversity of unrelated Legionella pneumophila clinical isolates in Catalonia and compare with other European regions. METHODS: 95 unrelated isolates were analyzed using monoclonal antibodies and sequence-based typing, 1989-2013. RESULTS: The isolates showed a high diversity (IOD 0.964) with a predominance of some profiles (ST37-Phialdelphia, ST23-Philadelphia and ST1-OLDA). All regions had predominant sequence types (STs) that differed between regions, and only 3% of STs were shared between the three regions. CONCLUSION: L. pneumophila clinical isolates from Catalonia presented a high diversity and can be used in epidemiological surveillance studies. The heterogeneous predominance of STs between European regions suggested a relationship between geographical distribution and virulence of some STs.

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

AIM: To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. MATERIALS & METHODS: Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. RESULTS: The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. CONCLUSION: SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

[Proteomics in infectious diseases]. / Proteómica en enfermedades infecciosas.

Infectious diseases have a high incidence in the population, causing a major impact on global health. In vitro culture of microorganisms is the first technique applied for infection diagnosis which is laborious and time consuming. In recent decades, efforts have been focused on the applicability of "Omics" sciences, highlighting the progress provided by proteomic techniques in the field of infectious diseases. This review describes the management, processing and analysis of biological samples for proteomic research.

A comprehensive proteome of Mycoplasma genitalium.

Mycoplasma genitalium is a human pathogen associated with several sexually transmitted diseases. Proteomic technologies, along with other methods for global gene expression analysis, play a key role in understanding the mechanisms of bacterial pathogenesis and physiology. The proteome of M. genitalium, model of a minimal cell, has been extended using a combination of different proteomic approaches and technologies. The total proteome of this microorganism has been analyzed using gel-based and gel-free approaches, achieving the identification of 85.3% of the predicted ORFs. In addition, a comprehensive analysis of membrane subproteome has been performed. For this purpose, the TX-114 soluble fraction has been analyzed as well as the surface proteins, using cell-surface protein labeling with CyDye. Finally, the serological response of M. genitalium-infected patients and healthy donors has been analyzed to identify proteins that trigger immunological response. Here, we present the most extensive M. genitalium proteome analysis (85.3% of predicted ORFs), a comprehensive M. genitalium membrane analysis, and a study of the human serological response to M. genitalium.