Prevalence and seasonal dynamics of blaCTX-M antibiotic resistance genes and fecal indicator organisms in the lower Lahn River, Germany

Abstract

Antibiotic-resistant bacteria represent an emerging global health problem and are frequently detected in riverine environments. Analyzing the occurrence of corresponding antibiotic-resistant genes in rivers is of public interest as it contributes towards understanding the origin and dissemination of these emerging microbial contaminants via surface water. This is critical for devising strategies to mitigate the spread of resistances in the environment. Concentrations of blaCTX-M antibiotic resistance genes were quantified weekly over a 12-month period in Lahn River surface water at two sampling sites using quantitative real-time PCR. Gene abundances were statistically assessed with regard to previously determined concentrations of fecal indicator organisms Escherichia coli, intestinal enterococci and somatic coliphages, as well as influential environmental factors. Similar seasonal patterns and strong positive correlations between fecal indicators and blaCTX-M genes indicated identical sources. Accordingly, linear regression analyses showed that blaCTX-M concentrations could largely be explained by fecal pollution. E. coli provided the best estimates (75% explained variance) at the upstream site, where proportions of blaCTX-M genes in relation to fecal indicator organisms were highest. At this site, rainfall proved to be more influential, hinting at surface runoff as an emission source. The level of agricultural impact increased from downstream to upstream, linking increasing blaCTX-M concentrations after rainfall events to the degree of agricultural land use. Exposure assessment revealed that even participants in non-swimming recreational activities were at risk of incidentally ingesting blaCTX-M genes and thus potentially antibiotic resistant bacteria. Considering that blaCTX-M genes are ubiquitous in Lahn River and participants in bathing and non-bathing water sports are at risk of exposure, results highlight the importance of microbial water quality monitoring with an emphasis on antibiotic resistance not only in designated bathing waters. Moreover, E. coli might serve as a suitable estimate for the presence of respective antibiotic resistant strains.

Fig 1. Distribution of bla_CTX-M genes and FIO in Lahn River throughout the study period.

Log₁₀-transformed concentrations of FIO E. coli, intestinal enterococci, somatic coliphages and bla_CTX-M genes (A) at site 1 and (B) at site 2 are shown in relation to global solar irradiance and discharge.

Fig 2. Boxplots of bla_CTX-M and FIO concentrations in Lahn River surface water samples throughout the sampling period at sites 1 and 2.

(A) Concentrations of total annual FIO and bla_CTX-M genes. (B) Concentrations of annual bla_CTX-M genes normalized to E. coli concentrations.

Fig 3. PCA biplot of bla_CTX-M genes, FIO and environmental parameters.

Site 1 data are indicated by squares, site 2 data are indicated by triangles. Data are grouped by seasons (cellipses); (circle = correlation circle, chla: chlorophyll-a, cond: conductivity, CTX_M: bla_CTX-M genes, dis: discharge, EC: E. coli, ENT: intestinal enterococci, gsi3: 3-day-sum of global solar irradiance, NH4N: ammonium-nitrogen, oxy: oxygen, rain4: 4-day-sum of rainfall, SC: somatic coliphages, temp: water temperature, turb: turbidity).

Fig 4. PCA biplot of bla_CTX-M genes, FIO and environmental parameters with correlation circle and contributions indicated by color gradient.

Data are shown individually for (A) site 1 and (B) site 2 (Abbreviations: rain4: 4-day-sum of rainfall; temp: water temperature; cond: conductivity; gsi3: 3-day-sum of global solar irradiance; chla: chlorophyll-a; dis: discharge; CTX_M: bla_{CTX_M} genes; EC: E. coli; ENT: intestinal enterococci; SC: somatic coliphages; turb: turbidity; oxy: oxygen content; NH4N: ammonium-nitrogen; contrib: contribution).

Fig 5. Boxplots of fecal indicator and ARG concentrations.

(A) low flow periods (discharge < median of 18.8 m³/s) versus high flow periods (discharge > median of 18.8 m³/s) (B) in dry periods (4d-sum of rainfall = 0 mm) versus wet periods (4d-sum of rainfall > 0 mm) and (C) in periods with low rainfall (4d-sum of rainfall < 5mm) versus high rainfall (4d-sum of rainfall > 5 mm).

Fig 6

Linear model equations and measured and modeled bla_CTX-M ARG concentrations during the sampling period at (A) site 1 and (B) site 2.

Fig 7. Modeled vs. observed concentrations of bla_CTX-M ARG for each individual sampling site.