Preplanned Studies: Antimicrobial Resistance Analysis and Whole-Genome Sequencing of Salmonella Isolates from Environmental Sewage — Guangzhou City, Guangdong Province, China, 2022–2023

Salmonella represents a prevalent foodborne pathogen globally. The emergent trend of MDR, exacerbated by the misuse and overuse of antibiotics, has compromised treatment effectiveness and led to therapeutic failures (2). It is, therefore, vital to examine the resistance patterns and the genetic basis of antimicrobial resistance in Salmonella, with a focus on MDR, to better manage and contain infections. Notably, the serovars S.1,4,[5],12:i:- and S. Rissen have gained recognition as emerging threats to human health in various countries (3–4). There remains, however, a substantial gap in the understanding of these serovars’ resistance profiles in China, particularly in Guangzhou. Our study aims to fill this crucial knowledge void. We conducted our research on Salmonella isolates obtained from environmental sewage, which offers distinctive insights. Conventional antimicrobial resistance surveillance primarily targets symptomatic clinical cases, thereby overlooking asymptomatic carriers and key environmental reservoirs including livestock, vegetables, and water bodies. Conversely, environmental sewage likely harbors Salmonella strains shed from multiple sources, offering a more comprehensive overview of the strains present. Additionally, we utilized SNP analysis to elucidate the genetic relationships among isolates, providing valuable data for tracing the origins of potential Salmonella outbreaks in the future.

In this study, we found that both S.1,4,[5],12:i:- and S. Rissen isolates exhibited high resistance levels to commonly prescribed clinical antibiotics, such as AMP, STR, CHL, SXT, AMS, as well as to TET, an antibiotic critically important in veterinary medicine. These findings underscore the need for judicious use of these antibiotics in both human medicine and animal farming to prevent treatment failures. However, antibiotics such as TIG, AMK and CZA have demonstrated high antimicrobial sensitivity and thus may offer effective treatment options. The extremely high prevalence of multidrug resistance observed in these isolates is alarming, with potential severe implications for both human health and life. A comprehensive strategy that integrates bacterial and fungal resistance surveillance, clinical prescription monitoring, and hospital infection control is essential to combat the spread of drug-resistant pathogens (5).

Drug efflux pumps are integral membrane proteins that actively expel antibiotics from the cell, representing a significant mechanism contributing to the MDR observed in Gram-negative bacteria (1). Our investigation identified six types of efflux pumps in both S.1,4,[5],12:i:- and S. Rissen isolates: the RND family, ABC superfamily, MFS, SMR family, MATE, and kdpDE. These systems are likely involved in the extensive antibiotic resistance demonstrated by S.1,4,[5],12:i:- and S. Rissen isolates. In addition, we discovered the mcr-3.1 subtype of the plasmid-mediated colistin resistance gene mcr-3, which has been infrequently reported in China (6). Literature suggests that mcr-3.1 is instrumental in propagating drug resistance via both plasmid transfer, or horizontal transmission, and chromosomal insertion, or vertical transmission (7). Ongoing surveillance of mcr-3.1 is vital to controlling its dissemination within China. Furthermore, our WGS analysis predicted genes responsible for resistance to fluoroquinolones, aminoglycosides, and tetracyclines, among others. Notably, there was a discernible correlation between the presence of these ARGs and the corresponding resistance phenotypes, underscoring the importance of ARGs in Salmonella resistance and the reduction in antibiotic efficacy.

Antibiotic-resistant bacteria (ARB), ARGs, and mobile genetic elements (MGEs), such as plasmids, are present in sewage and promote the horizontal transfer of ARGs among various microorganisms, leading to increased bacterial resistance (8). This study has identified numerous ARGs; consequently, their eradication from sewage is of paramount importance. Traditional disinfection methods, including chlorine, ozone, and ultraviolet light, exhibit minimal effectiveness in eliminating ARGs. While the combined photocatalytic oxidation-membrane bioreactor (MBR) process has proven effective at removing ARGs in laboratory studies, its application in real-world settings remains impractical (9). Therefore, there is an urgent need to develop innovative disinfection technologies suited for the efficient removal of ARGs from sewage.

The phylogenetic analysis indicates that Salmonella possesses the capability to disseminate various sources and geographical areas, including across international borders. The primary transmission vectors for Salmonella include contaminated food, water, and the international trade of animal feed (10). To mitigate the dissemination of Salmonella, it is imperative to enforce stringent food safety inspection protocols that encompass both food products and animal feed. Concurrently, the disinfection of environmental wastewater is of paramount importance.

This study is subject to some limitations due to the small sample size, which comprised only 35 S.1,4,[5],12:i:- isolates and 6 S. Rissen isolates. Consequently, these numbers may not sufficiently reflect the overall resistance features of these two Salmonella serotypes. In addition, owing to the limitations of second-generation genome sequencing methods for WGS, it’s impossible to obtain the location of resistance genes, such as whether it’s on the chromosomes or plasmids, which will limit further study on the resistance mechanism of Salmonella.

Nonetheless, the breadth of the sewage sample sources — from seven locations across eleven districts in Guangzhou city — lends some degree of representativeness to the findings. Additionally, the collection of sewage samples was carried out by trained professionals and the samples were transported to the laboratory for analysis within 48 hours, stored at 4 °C. The methodological procedures, including selective enrichment, isolation, morphological examination, biochemical testing, and serological typing, were methodically performed to ensure the isolation of Salmonella strains. The thoroughness of the experimental protocol supports the accuracy of the results.

No conflicts of interest.