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Free-living amoebae (FLA) are phagocytic protists that play crucial roles in microbial communities as significant microbial grazers. However, our current knowledge of their diversity, ecology, and population genetic structures is marginal due to the shallow and biased sampling of ecosystems and the use of few, poorly resolving molecular markers. Thirty-two FLA were isolated from soil and water samples collected across representative ecosystems of the State of Morelos in Central Mexico, including the drinking water distribution system (DWDS) from the state capital. We classified our isolates as members of Acanthamoeba, Vermamoeba, Naegleria, and Tetramitus by 18S ribosomal DNA (rDNA) sequencing. Vermamoeba isolates were recovered exclusively from the DWDS samples. In contrast, Naegleria strains displayed a broad distribution in soil and water samples across the natural ecosystems. We used a combination of phylogenetic and population genetic analyses of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (COI) sequences from our isolates and a comprehensive set of reference sequences to analyze the currently known diversity of Naegleria spp. Significant associations were uncovered between the most prevalent lineages of Naegleria and Vermamoeba and broad ecological and geographical variables at regional and global levels. The population structure and cryptic diversity within the Naegleria galeacystis-Naegleria americana and Vermamoeba vermiformis species complexes were thoroughly analyzed. Our results prove that the genus Vermamoeba, which was previously thought to consist of only one species, actually encompasses at least seven widely distributed species, as indicated by consistent evidence from Bayesian phylogenetics, two species-delimitation programs, and population genetics analyses. IMPORTANCE Our study sheds new light on the population genetic structure of V. vermiformis and diverse Naegleria species. Using improved molecular markers and advanced analytical approaches, we discovered that N. americana, previously considered a single species, actually contains multiple distinct lineages, as revealed by COI sequencing. These lineages are highly differentiated, with little gene flow between them. Our findings demonstrate that the genus Vermamoeba holds multiple cryptic species, requiring a significant taxonomic revision in light of multilocus sequence analyses. These results advance our understanding of the ecology, molecular systematics, and biogeography of these genera and species complexes at both regional and global scales. This study has significant implications for diagnosing amoebal infections and evaluating health risks associated with FLA in domestic and recreational waters.

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RESUMO Este artigo modela um sistema de distribuição de água (SDA) real com um grande número de vazamentos para determinar se existe diferença entre a vazão perdida pelo SDA predita pela equação de FAVAD e pela equação geral. Foi estudado o efeito da área e da quantidade dos vazamentos não visíveis, da área dos vazamentos potencialmente detectáveis, da carga de pressão e do coeficiente de descarga sobre a vazão perdida do SDA usando planejamento fatorial. Os resultados mostram que os três fatores significativos em ordem decrescente são: a área de vazamentos potencialmente detectáveis, a interação entre a área de vazamentos potencialmente detectáveis e a carga de pressão e a área de vazamentos de fundo. A quantidade de vazamentos não visíveis e o coeficiente de descarga não influenciam a diferença entre a equação de FAVAD e a geral, nem como fator principal tampouco como interação com outros fatores.

ABSTRACT This paper models a real water distribution system (WDS) with a large number of leaks to determine if there is a difference between the flow lost by the WDS predicted by the FAVAD and the GENERAL equations. The effect of the area and the amount of non-visible/background leaks, the area of potentially detectable leaks, the pressure load and the discharge coefficient on the leakage flow rate in a WDS were studied using factorial design. The results show that the three significant factors in descending order are: the area of potentially detectable leaks, the interaction between the area of potentially detectable leaks and the head pressure and the area of background leaks. The amount of background leaks and the discharge coefficient do not influence the difference between the FAVAD and the GENERAL equations, neither as a main factor nor as an interaction with other factors.

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Background: There is evidence that tap water is the vehicle through which nontuberculous mycobacteria (NTM) infect or colonize the human body. The objective of this study was to determine the presence and diversity of NTM in the water distribution system of Bahía Blanca city, Argentina (sites S2/S3) and in the dike that supplies water to it (S1). Methods: Culture-dependent method, biochemical tests, and molecular method (16S rRNA sequencing gene) were combined to detect and identify NTM. Results: NTM were isolated in 51.6% (64/124) of all the samples analyzed. Mycobacterium gordonae was the most frequently isolated organism (15/64) in all samples analyzed, followed by Mycobacterium peregrinum and Mycobacterium frederiksbergense. Significant differences were found in the residual chlorine values between sampling S2 and S3. In both sites, maximum counts were recorded but they did not correlate with low chlorine values. A concentration higher than 500 colony-forming unit/L of NTM was never found, which can be attributed to the negative effect caused by decontamination methods being a point to consider for the recovery of NTM. In 46.9% (30/64) of samples, both methods coincided in the identification, and the obtained sequences presented ≥99% identity. Identification at the species level was achieved in 50% (32/64) of the isolates. Nearly 17.2% (11/64) of the isolates showed a similarity <99%. Conclusions: It should be taken into account that sequencing of the 16S rRNA gene and biochemical tests are useful for the identification of several species, but it is necessary to incorporate other genes (hsp 65 and rpo B) to obtain accurate identification.

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RESUMO

Filamentous fungi in drinking water distribution systems are known to (a) block water pipes; (b) cause organoleptic biodeterioration; (c) act as pathogens or allergens and (d) cause mycotoxin contamination. Yeasts might also cause problems. This study describes the occurrence of several fungal species in a water distribution system supplied by groundwater in Recife-Pernambuco, Brazil. Water samples were collected from four sampling sites from which fungi were recovered by membrane filtration. The numbers in all sampling sites ranged from 5 to 207 colony forming units (CFU)/100 mL with a mean value of 53 CFU/100 mL. In total, 859 isolates were identified morphologically, with Aspergillus and Penicillium the most representative genera (37% and 25% respectively), followed by Trichoderma and Fusarium (9% each), Curvularia (5%) and finally the species Pestalotiopsis karstenii (2%). Ramichloridium and Leptodontium were isolated and are black yeasts, a group that include emergent pathogens. The drinking water system in Recife may play a role in fungal dissemination, including opportunistic pathogens.

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