Microbiological hazard identification in river waters used for recreational activities.

Pathogenic bacteria, viruses, and parasites can cause waterborne disease outbreaks. The study of coastal water quality contributes to identifying potential risks to human health and to improving water management practices. The Río de la Plata River, a wide estuary in South America, is used for recreational activities, as a water source for consumption and as a site for sewage discharges. In the present study, as the first step of a quantitative microbial risk assessment of the coastal water quality of this river, a descriptive study was performed to identify the microbial pathogens prevalent in its waters and in the sewage discharged into the river. Two sites, representing two different potential risk scenarios, were chosen: a heavily polluted beach and an apparently safe beach. Conductivity and fecal contamination indicators including enterococci, Escherichia coli, F + RNA bacteriophages, and human polyomaviruses showed high levels. Regarding enterococci, differences between sites were significant (p-values <0.001). 93.3% and 56.5% of the apparently safe beach exceeded the recreational water limits for E. coli and enterococci. Regarding pathogens, diarrheagenic E. coli, Salmonella, and noroviruses were detected with different frequencies between sites. The parasites Cryptosporidium spp. and Giardia duodenalis were frequently detected in both sites. The results regarding viral, bacterial, and parasitic pathogens, even without correlation with conventional indicators, showed the importance of monitoring a variety of microorganisms to determine water quality more reliably and accurately, and to facilitate further studies of health risk assessment. The taxonomic description of microbial pathogens in river waters allow identifying the microorganisms that infect the population living on its shores but also pathogens not previously reported by the clinical surveillance system.

Phylogeography of Rotavirus G8P[8] Detected in Argentina: Evidence of Transpacific Dissemination.

Rotavirus is one of the leading causes of diarrhea in children. In 2018, G8P[8], an unusual association of genotypes, was detected with moderate frequency in symptomatic children in Argen-tina, unlike a previous sporadic identification in 2016. The aim of this study was to analyze the dissemination pattern of the G8P[8]-lineage IV strains detected in Argentina. Nucleotide sequences of the VP7 gene of Argentine G8P[8] strains (2016, 2018 and 2019) were studied by discrete phylodynamic analyses, together with other worldwide relevant G8-lineage IV strains. Bayes Factor (BF) was used to assess the strength of the epidemiological association between countries. Phylodynamic analyses determined an evolutionary rate of 3.7 × 10-3 (HDP95%: 1.4 × 10-3-8.2 × 10-3) substitutions/site/year. Likewise, the most recent common ancestor was 32.2 years old, dating back to 1986 (HDP95% = 1984-1988). The spatiotemporal dynamics analysis revealed South Korea as being the country of origin of the Argentine strains (posterior probability of the ancestral state: 0.8471), which was also evidenced by a significant rate of diffusion from South Korea to Argentina (BF: 55.1). The detection of G8 in South America in 2016-2017 was not related to the cases detected in 2018-2019, revealing a new G8 introduction to the region and supporting a transpacific dissemination.

Dynamics of SARS-CoV-2 in wastewater in three districts of the Buenos Aires metropolitan region, Argentina, throughout nine months of surveillance: A pilot study.

In the current pandemic of COVID-19, sewage surveillance of SARS-CoV-2 genome has been used to complement viral epidemiology in different countries. The aim of this work was to introduce and evaluate this wastewater-based tool in the metropolitan region of Buenos Aires, Argentina. As a pilot study, surveillance of SARS-CoV-2 in wastewater from three districts of this area was performed for more than nine months from June 2020 to April 2021. Viruses present in the samples were concentrated using polyethylene glycol precipitation and quantified using RT-qPCR CDC N1 assay. Virus recovery for SARS-CoV-2 and a potential surrogate, bovine coronavirus Mebus strain, that shares the Betacoronavirus genus and structural characteristics with SARS-CoV-2, were evaluated after concentration and detection procedures. Recovery of both viruses did not differ significantly, with a median for SARS-CoV-2 and BCoV of 0.085 (95% CI: 0.021-0.179) and 0.262 (95% CI: 1.18 × 10-5-0.564) respectively. The concentration of SARS-CoV-2 genome in wastewater ranged from 10 -1 to 10 3 cg/ml, depending on the wastewater treatment plant, type of collection site, viral recovery of the concentration method and the epidemiological situation of the outbreaks. Significant correlations were observed between SARS-CoV-2 concentration in wastewater and reported clinical cases, reinforcing the utility of this approach to monitor the epidemiological status of populations.

Assessment of Microbiological Quality of Fresh Vegetables and Oysters Produced in Buenos Aires Province, Argentina.

Fresh vegetables and shellfish are prone to microbial contamination through irrigation or breeding with sewage-polluted waters, as well as by infected food handlers. In this work, we studied the presence of human and bovine polyomaviruses and human norovirus in fresh lettuces, strawberries and oysters produced in Buenos Aires province, Argentina. In oysters, we also investigated F-specific RNA bacteriophages, indicator Escherichia coli (E. coli) and pathogen bacteria of concern (Salmonella spp., Vibrio spp.). Within vegetables, we found viral contamination of human origin given the presence of human-associated polyomaviruses -MCPyV, HPyV6, JCPyV, and SV40- in lettuce and strawberry samples (16 and 10%, respectively), probably coming from irrigation waters and food handling. Among oysters, human (MCPyV, 4.2%) and bovine (BPyV1, 8.4%) polyomaviruses were detected even with low counts of E. coli. Bacteriophages (n = 3) and Salmonella spp. (n = 1) were also found, while Vibrio spp. was not detected. These results may indicate that the contamination in oysters comes from human and animal excreta, probably present in breeding waters. Norovirus was not detected in any food sample. To our knowledge, this is the first description of SV40 in lettuces and MCPyV and BPyV1 in oysters. The detection of different viral contaminants encourages further studies to evaluate the need for including viral indicators in microbiological standards. The identification of possible sources and routes of contamination using viral markers during routine microbiological controls, such as the polyomaviruses used in this work, would be useful to focus attention on the most hazardous stages of the food production chain.

Diversity of ß-lactamase-encoding genes in wastewater: bacteriophages as reporters.

A reservoir of antibiotic resistance genes (ARGs) is present in pathogenic, commensal, and environmental bacteria as well as in mobile genetic elements, including bacteriophages. Wastewater treatment plants (WWTPs) are considered hotspots for the spread of ARGs. The aim of this work was to analyze the diversity of the highly prevalent ARGs blaCTX-M and blaTEM in bacterial and bacteriophage fractions associated with human and animal environments through the study of urban waste and animal residues discharged into WWTPs to provide information about the composition and maintenance of the current resistome in Buenos Aires, Argentina. The results showed that a putative extended-spectrum variant of the blaTEM gene was the most frequently detected, with blaTEM-116 being the most prevalent, while a recently described type, blaTEM-229, was also found. In the bacteriophage fraction, we detected blaCTX-M genes from four out of the five clusters described. The detection of blaCTX- M-9-like and blaCTX-M-25-like genes was unexpected based on surveys of the ARGs from clinical pathogens circulating regionally. The finding of divergent blaCTX-M sequences associated with previously reported environmental genes argues in favor of the natural environment as a reservoir of resistance genes. ARGs were detected in bacteriophages as frequently as in bacterial communities, and furthermore, the blaCTX-M genes were more diverse in the bacteriophage fraction. Bacteriophages might therefore play a role in the spread of ARGs in the environment, but they might also be used as "reporters" for monitoring circulating ARGs.

Viral tools for detection of fecal contamination and microbial source tracking in wastewater from food industries and domestic sewage.

Alternative indicators may be more suitable than thermotolerant coliform bacteria to assess enteric virus pollution in environmental waters and their removal from wastewaters. In this study, F-specific RNA bacteriophages (F-RNAPh) showed to be potential viral indicators of fecal contamination when they were quantified from domestic and food-industrial effluents containing human, chicken, swine or bovine wastes. In addition, they showed to be resistant to the primary and secondary treatments of the wastewater treatment plants. The viable F-RNAPh count showed correlation with viable thermotolerant coliforms but also with human polyomaviruses (HPyV) quantified by a new molecular method. In domestic effluents, F-RNAPh and HPyV indicators significantly correlated with a human viral pathogen, norovirus, while the bacterial indicator did not, being then better predictors of the behavior of enteric pathogenic viruses. In addition, we assessed human, bovine and fowl microbial source tracking markers, based on the molecular detections of human polyomavirus, bovine polyomavirus, and fowl adenovirus, respectively. The techniques implemented extend the range of viruses detected, since they target different viral types simultaneously. These markers could be applied when multiple source pollution is suspected, contributing to making decisions on public health interventions.

Phylodynamics of Merkel-cell polyomavirus and human polyomavirus 6: A long-term history with humans.

New human polyomaviruses have been described in the last years, including the Merkel-cell polyomavirus (MCPyV; Human polyomavirus 5) and the Human polyomavirus 6 (HPyV6). Although their infection is usually asymptomatic, in immunocompromised host can cause life-threatening pathologies, such as the Merkel cell carcinoma, an aggressive skin neoplasia associated to the MCPyV. Despite being prevalent viruses in population, epidemiological data from South America are scarce, as well as the characterization of the viral types circulating and their origin. The aims of this work were to describe MCPyV and HPyV6 from environmental samples with different geographical origin and to analyze their phylogenetic and evolutionary histories, particularly for MCPyV. Partial and complete genome sequences were obtained from sewage samples from Argentina, Uruguay and Spain. A total number of 87 sequences were obtained for MCPyV and 33 for HPyV6. Phylogenetic analysis showed that MCPyV sequences distributed according to their geographic origin in Europe/North America, Africa, Asia, South America and Oceania groups, suggesting that viral diversification might have followed human migrations across the globe. In particular, viruses from Argentina associated with Europe/North America and South America genotypes, whereas those from Uruguay and Spain also grouped with Africa genotype, reflecting the origin of the current population in each country, which could arrive not only during ancient human migration but also during recent migratory events. In addition, the South American group presented a high level of clusterization, showing internal clusters that could be related to specific locations, such as French Guiana and Brazil or the Southern region into South America, such as Argentina and Uruguay, suggesting a long term evolutionary process in the region. Additionally, in this work, we carried out the first analysis about the evolutionary history of MCPyV trough the integration of phylogenetic, epidemiological and historical data. Since a strong association is observed between the phylogenetic relationships and the origin of the sampled population, this analysis was based on the hypothesis of co-divergence between the virus and human populations. This analysis resulted in a substitution rate of 5.1 × 10-8 s/s/y (∼5.1% of divergence per million years) for the complete genome of MCPyV, which is in the range of those estimated for other double-stranded DNA viruses. Regarding HPyV6, a South American group with clusterization was observed (sequences from Uruguay). Meanwhile, sequences from Argentina grouped with European ones (France and Spain) and remained separated from those isolated in China, USA or Australia. The analysis of viruses from the environment allowed us to deep characterize prevalent infections in different geographic regions, reveling that viruses circulating in each population reflected its origin and that there are specific lineages associated with South America.

Comparison of internal process control viruses for detection of food and waterborne viruses.

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.

High diversity of human polyomaviruses in environmental and clinical samples in Argentina: Detection of JC, BK, Merkel-cell, Malawi, and human 6 and 7 polyomaviruses.

New human polyomaviruses have been recently described. The aim of this work was to detect and characterize human polyomaviruses circulating in Argentina by recovering viruses from environmental and sewage samples and evaluating their potential role as viral indicators of human waste contamination. Analysis was performed in a wider context including viruses from clinical samples from an immunocompromised population. River water and sewage samples were analyzed as a strategy to study the molecular epidemiology of viruses excreted by millions of people. Samples belonged to the Matanza-Riachuelo River (2005-2006: n=25 and 2012: n=20) and sewage from Buenos Aires city and suburbs (2011 and 2013: n=24). Viral detection was performed by PCR and the amplified viral genomes were characterized by phylogenetic analysis. Polyomaviruses were detected in 95.8% of sewage samples, identifying BKPyV (87.5%), JCPyV (83.3%), MCPyV (8.3%) and HPyV6 (8.3%). Besides, one sample collected in 2009 resulted positive for HPyV7. In 2005-2006, polyomaviruses were detected in 84.0% of river water samples, with the highest detection for MCPyV (52.0%), followed by BKPyV (44.0%), JCPyV (20.0%) and MWPyV (4.0%). In 2012, polyomaviruses were detected in 85.0% of river samples, finding JCPyV (85.0%), BKPyV (75.0%), MCPyV (25.0%) and HPyV6 (25.0%). Also, polyomaviruses, including JCPyV, BKPyV and MCPyV, were detected in 63.2% of urine samples from patients infected with HIV (n=19). Characterization indicated the coexistence of different genotypes and variants for each virus, particularly in sewage. MCPyV sequences (the only sequences from Argentina) formed a monophyletic group with the single sequence available for South America (French Guiana). The high level of detection and viral diversity found by environmental surveillance, which involved the characterization of viruses not previously described in South America, reinforces the usefulness of this approach to monitor viral contamination and describe the viral epidemiology in the general population.

Influence of overlapping genes on the evolution of human hepatitis B virus.

The aim of this work was to analyse the influence of overlapping genes on the evolution of hepatitis B virus (HBV). A differential evolutionary behaviour among genetic regions and clinical status was found. Dissimilar levels of conservation of the different protein regions could derive from alternative mechanisms to maintain functionality. We propose that, in overlapping regions, selective constraints on one of the genes could drive the substitution process. This would allow protein conservation in one gene by synonymous substitutions while mechanisms of tolerance to the change operate in the overlapping gene (e.g. usage of amino acids with high-degeneracy codons, differential codon usage and replacement by physicochemically similar amino acids). In addition, differential selection pressure according to the HBeAg status was found in all genes, suggesting that the immune response could be one of the factors that would constrain viral replication by interacting with different HBV proteins during the HBeAg(-) stage.

Evaluation of concentration efficiency of the Pseudomonas aeruginosa phage PP7 in various water matrixes by different methods.

Enteric viruses monitoring in surface waters requires the concentration of viruses before detection assays. The aim of this study was to evaluate different methods in terms of recovery efficiencies of bacteriophage PP7 of Pseudomonas aeruginosa, measured by real-time PCR, using it as a viral control process in water analysis. Different nucleic acid extraction methods (silica-guanidinium thiocyanate, a commercial kit (Qiagen Viral RNA Kit) and phenol-chloroform with alcohol precipitation) exhibited very low recovery efficiencies (0.08-4.18 %), being the most efficient the commercial kit used for subsequent experiments. To evaluate the efficiency of three concentration methods, PBS (as model for clean water) and water samples from rivers were seeded to reach high (HC, 10(6) pfu ml(-1)) and low concentrations (LC, 10(4) pfu ml(-1)) of PP7. Tangential ultrafiltration proved to be more efficient (50.36 ± 12.91, 17.21 ± 9.22 and 12.58 ± 2.35 % for HC in PBS and two river samples, respectively) than adsorption-elution with negatively charged membranes (1.00 ± 1.34, 2.79 ± 2.62 and 0.05 ± 0.08 % for HC in PBS and two river samples, respectively) and polyethylene glycol precipitation (15.95 ± 7.43, 4.01 ± 1.12 and 3.91 ± 0.54 %, for HC in PBS and two river samples, respectively), being 3.2-50.4 times more efficient than the others for PBS and 2.7-252 times for river samples. Efficiencies also depended on the initial virus concentration and aqueous matrixes composition. In consequence, the incorporation of an internal standard like PP7 along the process is useful as a control of the water concentration procedure, the nucleic acid extraction, the presence of inhibitors and the variability of the recovery among replicas, and for the calculation of the sample limit of detection. Thus, the use of a process control, as presented here, is crucial for the accurate quantification of viral contamination.

New perspectives on the evolutionary history of hepatitis B virus genotype F.

Hepatitis B virus (HBV) is a globally distributed human pathogen. The aim of this work was to analyze the evolutionary history of HBV genotype F, emphasizing on the study of subgenotypes prevalent in the Southern area of South America. Complete genomes of HBV genotype F from 36 samples from Argentina and Chile were sequenced and analyzed by phylogenetic and Bayesian coalescent methods along with sequences obtained from GenBank database. The phylogeography separated not only Central American from South American isolates but also revealed that different subgenotypes are distributed in constrained although not exclusive areas of the continent. The result obtained with time-stamped complete genomes failed to explain the wide geographical distribution and the clustering observed in this genotype. Conversely, the use of Bayesian coalescent analyses with substitution rates as priors, instead of the co-estimation of tMRCA and substitution rate, allowed us to propose a far origin for the HBV genotype F based on the phylogeographical and epidemiological data.

Molecular characterization of hepatitis B virus genotype A from Argentina and Brazil.

To study the diversification of hepatitis B virus genotype A in Latin America, we analyzed seven new Argentinian isolates and published sequences from Argentina and Brazil and other countries from the region. We found that the European subgenotype A2 prevailed in most of the countries except for Brazil, where the African subgenotype A1 predominated. A2 isolates did not differ significantly from the GenBank sequences, whereas some A1 isolates carried, concomitantly, amino acids characteristic of the subgenotypes A3 (R(501) in P protein) and A2 (D(355) in P/T(54) in preS2). This combination is absent in the A1 subgenotype around the world. We discuss the origin, distribution and introduction of those subgenotypes in the Americas.

Intra-host evolutionary dynamics of hepatitis C virus E2 in treated patients.

Hepatitis C virus (HCV) displays high genetic diversity. Inter-host sequence variability may mainly reflect a neutral drift evolution. In contrast, intra-host evolution may be driven by an adaptive selection to host responses to infection. Here, HCV E2 intra-host evolution in two patients during the course and follow-up of successive treatments with IFN-alpha and IFN-alpha/ribavirin was investigated. Phylogenetic analyses suggested that adaptive pressures prompt a continuous selection of viral variants derived from the previous ones (intra-lineage evolution) and/or a swapping of viral lineages during the course of the infection (inter-lineage evolution). Selection would act not only on the phenotypic features of hypervariable region 1 (HVR1) but also on those of the flanking regions. The pressures operate mainly at the amino acid level, but they also appeared to act on nucleotide sequences. Moreover, HVR1 heterogeneity seemed to be strongly constrained. This work contributes to the knowledge of HCV intra-host evolution during chronicity.

Evolutionary study of HVR1 of E2 in chronic hepatitis C virus infection.

Hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) genome was directly sequenced from 12 chronically infected patients who had not responded to interferon (IFN) treatment. Due to the quasispecies nature of HCV circulating genomes, serum samples from four patients showing different evolutionary characteristics were further analysed. Serial samples from each patient were taken before, soon after and 14-23 months after a 6 month IFN treatment. HVR1 from each sample was amplified, cloned and the clones sequenced. For each patient, a phylogenetic analysis of the clones was performed and quasispecies complexity and genetic distances were calculated. The amino acid sequences and predicted antigenic profiles were analysed. The pre-treatment samples of the different patients presented dissimilar genetic quasispecies composition. For three of the patients, we showed that, regardless of the complexity or diversity of the viral populations before treatment, they evolved towards genetic diversification following selective pressure. Once the environment became stable, the entire population tended towards homogeneity. The fourth patient represented a case where different components of the quasispecies coexisted for long periods without replacement. We propose herein that the evolution of HVR1 of E2 is more likely to be directed by selection of clearly different subpopulations (modification of quasispecies equilibrium) than by a continuous mechanism related to the successive accumulation of point mutations. The prevalence of a quasispecies shift mechanism was revealed by the cloning analysis during the follow-up period of the evolutionary process.

El virus de hepatitis A: variabilidad y restricciones / Hepatitis A virus: variability and restrictions

Hepatitis A virus (HAV) is a hepatotropic agent that causes endemic infections in different regions of the world. The analysis of different strains has revealed the existence of a single antigenic type. However, the virus has shown genetic diversity, not only among different isolates ­which group 7 genotypes­ but also within a single isolate that exhibited a heterogeneous population. The analysis of such variability shows greater reliability when long regions of the genome are studied. The mechanisms responsible for the genomic variation involve point mutations due to errors of the viral polimerase and recombination among different strains, favoured by the co-circulation of different genotypes. It is herein sustained that, despite the antigenic variation, there exist structural constraints on the capside proteins of HAV that allow the virus to preserve its antigenic structure without modifications. However, viral variants with modifications related to antigenic sites were found, either as part of the mixture of variants of a heterogeneous population or as the predominant sequence of a clinical isolate. The study of genomic variations, the evolutionary constraints and the mechanisms involved would contribute to predict the characteristics of future isolates and to define the sanitary control policies.

El virus de hepatitis A (HAV) es un patógeno con tropismo hepático que causa infecciones endémicas en distintas regiones del mundo. Los estudios de distintas cepas determinaron la existencia de un único tipo antigénico. Sin embargo, el virus muestra diversidad genética, no sólo entre distintos aislamientos ­que conforman 7 genotipos­ sino también dentro de un único aislamiento, que exhibió heterogeneidad poblacional. El análisis de esa variabilidad reúne mayor confiabilidad frente a regiones extensas del genoma. Los mecanismos responsables de la variación genómica involucran mutaciones puntuales por errores de la polimerasa viral y recombinación entre virus distintos, favorecida por la cocirculación de diferentes genotipos. Se postula que, a pesar de la variación genética, existen limitaciones estructurales en las proteínas de cápside del HAV que le permiten mantener su estructura antigénica sin modificaciones. Sin embargo, se encontraron variantes virales con modificaciones vinculadas a sitios antigénicos, ya sea como parte de la mezcla de variantes de una población heterogénea o como secuencia predominante de un aislamiento clínico. El estudio de las variaciones genómicas, las restricciones evolutivas y los mecanismos involucrados podríancontribuir a predecir las características de futuros aislamientos ya definir las políticas sanitarias de control.

Evolutionary history of Hepatitis B virus genotype F: an in-depth analysis of Argentine isolates.

A revised analysis on the evolutionary history of hepatitis B virus (HBV) genotype F is herein presented with the incorporation of two new complete genomes from Argentina. The study of the phylogenetic-tree topology, genetic distances, and amino acid mutations confirmed with high reliability the existence of four different genetic clusters of this genotype. Argentine isolates were located in two groups of viruses that showed a great inner homogeneity but, interestingly, divergence between them was in the order of that existing among groups from different locations. Although the origin of these two viral populations is not clear, they do not seem to derive from each other, therefore the existence of at least two founder viral populations in Argentina is a more acceptable explanation.

Genetic characterization of hepatitis A virus isolates from Buenos Aires, Argentina.

The Hepatitis A virus (HAV) has been classified in seven different genotypes, which include human (I, II, III, and VII) and simian (IV, V, and VI) groups. The sequence analysis of HAV strains contributes to the molecular epidemiology of the virus. Although the infection with HAV is endemic in Argentina and vaccination is being implemented in this country, using both IA and IB strains, there are very few data on the genotypes of the circulating viruses. On the basis of the sequences of 20 isolates collected in Buenos Aires during a 2-year period (extended to 3 years by two additional specimens), we observed the presence of a single sub-genotype, IA, but with a high genetic diversity. We analyzed the VP1-2A junction and also the VP3-VP1 region. Most of the Argentine isolates grouped in at least two clusters. One of these was related to South American strains, thus suggesting a co-circulation of related isolates in neighbor countries. The other cluster was composed only of Argentine specimens. Other sequences were more scattered along the phylogenetic tree. However, we demonstrated that a consistent genetic relatedness of sequences could only be inferred on the basis of a more extensive sequencing of each isolate.