Wastewater irrigation increases the abundance of potentially harmful gammaproteobacteria in soils in Mezquital Valley, Mexico

Abstract

Wastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence of sul genes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance of Proteobacteria in rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular, Gammaproteobacteria, including potential pathogens, such as Pseudomonas, Stenotrophomonas, and Acinetobacter spp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with the Gammaproteobacteria (mainly potentially pathogenic Stenotrophomonas strains) and 50% with the Bacilli, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with the Bacilli. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.

FIG 1

Rarefaction curves indicating the observed numbers of OTUs at genetic distances of 3 and 20% within the analyzed soil samples. Sample abbreviations: RS0, rainy season rain-fed soil; RS10, rainy season soil with 10 years of wastewater irrigation; RS85, rainy season soil with 85 years of wastewater irrigation; RS100, rainy season soil with 100 years of wastewater irrigation; DS0, dry season rain-fed soil; DS8, dry season soil with 8 years of wastewater irrigation; DS85, dry season soil with 85 years of wastewater irrigation; DS100, dry season soil with 100 years of wastewater irrigation. Triplicates were analyzed (indicated by “a,” “b,” and “c” in sample names).

FIG 2

Relative abundances of dominant phyla and proteobacterial classes determined for the analyzed soil samples. Sample abbreviations: RS0, rainy season rain-fed soil; RS10, rainy season soil with 10 years of wastewater irrigation; RS85, rainy season soil with 85 years of wastewater irrigation; RS100, rainy season soil with 100 years of wastewater irrigation; DS0, dry season rain-fed soil; DS8, dry season soil with 8 years of wastewater irrigation; DS85, dry season soil with 85 years of wastewater irrigation; DS100, dry season soil with 100 years of wastewater irrigation. Data from analysis of triplicates are illustrated using error bars.

FIG 3

Heat map showing relative abundances of gammaproteobacterial genera as affected by wastewater irrigation during dry as well as rainy season. Triplicates were analyzed (indicated by “a,” “b,” and “c”).

FIG 4

Weighted UniFrac 2D principal coordinate analysis plot for beta diversity analysis. Sample abbreviations: RS0, rainy season rain-fed soil; RS10, rainy season soil with 10 years of wastewater irrigation; RS85, rainy season soil with 85 years of wastewater irrigation; RS100, rainy season soil with 100 years of wastewater irrigation; DS0, dry season rain-fed soil; DS8, dry season soil with 8 years of wastewater irrigation; DS85, dry season soil with 85 years of wastewater irrigation; DS100, dry season soil with 100 years of wastewater irrigation.

FIG 5

(A) Abundances of bacterial classes in isolates from wastewater-irrigated soils. (B) Abundances of different bacterial genera in isolates from wastewater-irrigated soils.

FIG 6

Percentages of antibiotic-resistant isolates from wastewater-irrigated soils and rain-fed soils. CIP, ciprofloxacin (5 μg); Kana, kanamycin (30 μg); SMX, sulfamethoxazole (25 μg); Tet, tetracycline (30 μg); Doxy, doxycycline (30 μg); Gm, gentamicin (10 μg); Amp, ampicillin (25 μg); Sm, streptomycin (25 μg); Oxa, oxacillin (5 μg); Cm, chloramphenicol (30 μg); Van, vancomycin (30 μg); Em, erythromycin (10 μg); ww, wastewater.