Profiling of Bacterial Communities of Hospital Wastewater Reveals Clinically Relevant Genera and Antimicrobial Resistance Genes

Abstract

In Mexico, hospital wastewater (HWW) is a source of chemical and microbiological contamination, and it is released into the municipal sewage system without prior treatment. This water may contain pathogenic bacteria and antimicrobial resistance genes, which represent a risk to Public Health and the environment. So far, there are no studies that analyse this problem comprehensively, relating bacterial population structures, chemical contaminants, and seasonality. The aim of this work was to seasonally characterise the bacterial communities of HWW, including clinically relevant bacteria and resistance genes in Hospital Juárez de México (HJM), and to evaluate the impact of physicochemical factors on their composition. A one-year observational, cross-sectional study was conducted at five HWW discharge points of HJM. Fourteen physicochemical parameters were determined by using standard methodologies, and statistical differences between discharges and seasons were evaluated. Bacterial communities were analysed by targeted amplicon sequencing of the V3-V4 region of the 16S rRNA gene. In addition, the presence of eight antimicrobial resistance genes of local epidemiological importance was assessed. Data were analysed using alpha and beta diversity indices, principal component analysis, and multivariate statistical tests. HWW showed high taxonomic diversity, with Proteobacteria, Firmicutes, and Bacteroidetes standing out. Clinically relevant bacteria were identified in 73.3% of the analyses, with Enterobacter and Escherichia-Shigella predominating. Total and dissolved solids, temperature, nitrate, and pH significantly influenced the bacterial composition of HWW. Seven out of the eight genes evaluated were identified, with bla_KPC, bla_OXA-40, and mcr-1 being the most frequent, showing significant seasonal differences. This study underlines the microbiological and chemical complexity of HWW, highlighting the impact of clinically relevant bacteria and antimicrobial resistance genes on Public Health. The findings emphasise the need to implement hospital waste management programmes and ideally specific treatment plants to minimise the associated risks and protect the environment and human health.

Keywords: V3-V4 hypervariable; antimicrobial resistance; bacteria; hospital wastewater.

Figure 1

(a) Aerial map of HJM with the discharge points (D1 to D5) of HWW; (b). Geographical coordinates of their location; (c) volume of HWW released in m³ per discharge (per second and 24 h); (d) hospital services related to each of the discharge sites. The discharge points (D1 to D5) are indicated by the red marking symbol.

Figure 2

Physicochemical parameters of HWW from HJM. (A) Standard quality parameters (temperature, chlorine, and pH), (B) chemical equilibrium (acidity, alkalinity, and hardness), (C) electrochemical (electrical conductivity), (D) solid loading (total, suspended, and dissolved), (E) oxidation and organic quality (dissolved O₂ and chemical O₂ demand (CO₂D)), and (F) macronutrients (NO⁻³ and P${\mathrm{O}}_4^{-2}$). X-axis: 1–5: Discharge 1 to 5. Significance level: * (0.01), ** (0.001), *** (0.0001), and **** (0.00001).

Figure 3

Variation in taxonomic diversity and annual relative abundance (at the phylum and genus levels) of bacterial communities released through the HWW of HJM: (A) phylum level and (B) genus level. HWW: hospital wastewater, DW: domestic wastewater, TW: treated wastewater, and WW: water well of the Cutzamala and Nezahualcóyotl system.

Figure 4

Characterisation of the clinically relevant bacteria (at genus level) released from the HWW of HJM. (A) Alluvial diagram of the release of clinically relevant bacteria by season and discharge; (B,C). ANOVA and Tukey’s post hoc tests on the release of clinically relevant bacteria by discharge point and seasonality. D1–D5: Discharge points.

Figure 5

Antimicrobial resistance genes in HWW at HJM. (A) Alluvial plot of the detection of resistance genes by season and discharge point (D1 to D5); (B,C) ANOVA and Tukey’s post hoc tests in the detection of resistance genes by discharge and seasonality. Significance level: * (0.01), ** (0.001), *** (0.0001), and **** (0.00001).

Figure 6

Relative abundance and average taxonomic diversity of HWW from HJM and other water sources. (A,B) Phylum. (C,D) Genus. HWW: hospital wastewater, DW: domestic wastewater, TW: treated wastewater, WW: water well of the Cutzamala and Nezahualcóyotl system, and D1 to D5: hospital discharge points.

Figure 7

Alpha diversity of HWW from HJM. (A) Rarefaction curves, (B) the Chao1 index, (C) Pielou’s evenness, (D) Fisher, (E) Gini–Simpson, (F) inverse Simpson, and (G) Shannon. HWW: hospital wastewater, DW: domestic wastewater, TW: treated wastewater, WW: water well of the Cutzamala and Nezahualcóyotl system. Statistical significance p < 0.05.

Figure 8

Beta diversity of HWW from HJM by weighted PCoA (A) and unweighted (B) UniFrac. HWW: hospital wastewater, DW: domestic wastewater, TW: treated wastewater, WW: water well of the Cutzamala and Nezahualcóyotl system. Statistical significance: p < 0.05.

Figure 9

Seasonal influence of physicochemical factors on bacterial communities in hospital wastewater from HJM. (A). PCA based on the months of the year and (B) PCA by season.