Unveiling Rare Pathogens and Antibiotic Resistance in Tanzanian Cholera Outbreak Waters

Abstract

The emergence of antibiotic resistance is a global health concern. Therefore, understanding the mechanisms of its spread is crucial for implementing evidence-based strategies to tackle resistance in the context of the One Health approach. In developing countries where sanitation systems and access to clean and safe water are still major challenges, contamination may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the environment. This contamination can increase the risk of exposure and community transmission of ARGs and infectious pathogens. However, there is a paucity of information on the mechanisms of bacteriophage-mediated spread of ARGs and patterns through the environment. Here, we deploy Droplet Digital PCR (ddPCR) and metagenomics approaches to analyze the abundance of ARGs and bacterial pathogens disseminated through clean and wastewater systems. We detected a relatively less-studied and rare human zoonotic pathogen, Vibrio metschnikovii, known to spread through fecal--oral contamination, similarly to V. cholerae. Several antibiotic resistance genes were identified in both bacterial and bacteriophage fractions from water sources. Using metagenomics, we detected several resistance genes related to tetracyclines and beta-lactams in all the samples. Environmental samples from outlet wastewater had a high diversity of ARGs and contained high levels of blaOXA-48. Other identified resistance profiles included tetA, tetM, and blaCTX-M9. Specifically, we demonstrated that blaCTX-M1 is enriched in the bacteriophage fraction from wastewater. In general, however, the bacterial community has a significantly higher abundance of resistance genes compared to the bacteriophage population. In conclusion, the study highlights the need to implement environmental monitoring of clean and wastewater to inform the risk of infectious disease outbreaks and the spread of antibiotic resistance in the context of One Health.

Keywords: ESBL; One Health; antibiotic resistance; bacteriophage; cholera; microbiota; transduction; water; zoonotic pathogen.

Figure 1

Schematic representation of sampling areas in Tanzania. Water sources from Tanga city and the nearby village Pongwe (orange dots) were collected, according to the defined geographical locations (Table 1).

Figure 2

Microbial and fungal composition and diversity in water samples. Water samples were isolated from two general areas in Tanzania (P = Pongwe; T = Tanga City) from a recent cholera outbreak region (cc), city center (c), taps (t), hospitals (h) or medical ward taps (mw). The samples were divided into a bacterial and phage fraction, and the microbial composition determined using amplicon sequencing of conserved regions. Relative abundance of microbes is given in (a,d) for bacteria and fungi, respectively. Alpha (b,e) and beta (c,f) diversity of bacteria and fungi, respectively, are also given.

Figure 3

Water samples from different sources were collected and 16S amplicon sequencing performed on the purified bacterial DNA. Prevalence of several common GI-associated pathogens as well as opportunistic pathogens (a,b) are denoted based on number of reads in the amplicon data (total reads 37,291–140,684 depending on sample). (c) Log₁₀ numbers of gene copies for different resistance genes from both bacterial and bacteriophage fractions.