Abstract
Background: Many infectious diseases emerge when pathogens from animal reservoirs spill-over into human populations. It has been observed that a significant proportion, approximately 60%, of all documented infectious disease agents that affect humans are of zoonotic origin. Therefore, the interface between wildlife, livestock, and humans is a critical for the emergence and spread of infectious diseases. These interfaces represent complex, multi-host environments where pathogens can cross the species barriers and evolve in new ways. Rats are widely acknowledged as prominent reservoirs of infectious diseases, surpassing all other terrestrial animal species in this context. Moreover, a few studies have indicated that rats can carry bacteria that are resistant to antimicrobial agents. These observations suggest that rats may act as reservoirs and distributors of antimicrobial-resistant bacteria and their genetic determinants to both animals and humans. Furthermore, there is evidence that the antimicrobial resistance repertoire in rats may potentially reflect their ecological habitats. However, to date, only a few fragmented studies have been conducted to characterize and understand antimicrobial resistance in rats from ecologically diverse regions. Furthermore, anthropogenic activities, such as changing the environmental landscape (farming, urbanisation etc.) have greatly increased the interactions between wildlife, domestic animals and humans. The intensification of direct contacts between humans and animals accelerates the evolution and increases the risk of transmission of zoonotic diseases and antimicrobial resistance (AMR) between different species, with great implications for animal and public health. Peridomestic rats are considered the ideal sentinel peridomestic wildlife species to explore the ecology of AMR and zoonoses and to improve our understanding on disease dynamics at the wildlife–livestock–human interface, in line with the One Health principle.Objectives: The specific objectives of this PhD project were: (i) to estimate the prevalence and to characterize phenotypically and genotypically specific types of clinically relevant antimicrobial resistant bacteria in peridomestic rats in Hong Kong Special Administrative Region (SAR), (ii) to investigate how the AMR repertoire in rats relates to their specific habitats they live in, and (iii) to characterize zoonotic pathogens that might be present in these rats across diverse habitats of Hong Kong SAR, including potential factors associated with the presence of these zoonoses.
Materials and Methods: From October 2020 to August 2021, live rats from 16 locations covering nine districts in all three regions of Hong Kong SAR, including ten urban areas, two swine farms, two poultry farms, and two horse-riding schools were captured. The rat species were characterized using polymerase chain reaction (PCR) and Sanger sequencing. Antimicrobial-resistant microorganisms were isolated from the rats' caecal content and nasopharynx. Four types of selective agars were used to identify potential antimicrobial-resistant bacteria including extended-spectrum β-lactamases (ESBL) producing Enterobacteriaceae, colistin-resistant Enterobacteriaceae (CoRE), carbapenem-resistant Enterobacteriaceae (CRE), and methicillin-resistant Staphylococcus aureus (MRSA). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to determine the bacterial species. The antimicrobial susceptibility test (AST) was performed using Kirby-Bauer disk diffusion test and broth microdilution test, targeting different specific types of AMR. Whole genome sequencing (WGS) was carried out on ESBL-producing E. coli (ESBL-EC) and MRSA isolates. To assess the relationship between specific types of AMR phenotypes and potential variables, such as species, sex, weight, developmental stage, regions, types of area, and trapping location characteristics, a logistic regression model was developed. Moreover, different PCR methods were employed to detect and characterise other pathogens including Leptospira spp. and Capillaria hepatica.
Results: A total of 221 rats were trapped for this study, including brown rats (Rattus norvegicus, n = 144), Asian house rats (R. tanezumi, n = 67), Indochinese forest rats (R. andamanensis, n = 8), and South China white-bellied rats (Niviventer huang, n = 2). Phenotypic testing revealed 253 isolates belonging to four specific types of AMR. Among these, 70% were identified as ESBL-producing Enterobacteriaceae, 8% as CoRE, 5% as CRE, and 1% as MRSA. The statistical model showed that location characteristics and rat species were potential factors associated with ESBL-producing E. coli (ESBL-EC). The odds of carrying ESBL-EC were higher in R. norvegicus caught in livestock farms compared to other rat species and trapping locations. Extended-spectrum β-lactamases (ESBL) producing E. coli were detected only in R. norvegicus and R. tanezumi. The prevalence of ESBL-EC was estimated at 58.82% (130/221) (95% CI = 52.33 – 65.32%). The antimicrobial susceptibility test (AST) against 16 antimicrobial drugs covering 10 antimicrobial classes was performed on ESBL-producing E. coli (ESBL-EC) isolates from rats in city areas (N=61), chicken farms (N=36), pig farms (N=36), and a horse-riding school (N=1), respectively. The AST results revealed a significant proportion of 86% (113/134) multidrug resistant isolates, namely ESBL-EC in rats from city areas (72%, 44/61), chicken farms (92%, 33/36), and pig farms (100%, 36/36). No MDR ESBL-EC was observed in rats caught inside horse-riding school. Genotypic analyses by WGS revealed blaCTX-M-14 (40%), blaCTX-M-15 (14%), blaCTX-M-55 (23%), and blaCTX-M-65 (13%) as the predominant blaESBL genes. Three genes including blaCTX-M-14, blaCTX-M-15, and blaCTX-M-65 were common in rats from city areas, chicken farms, and pig farms, while blaCTX-M-55 was found to be shared only between rats in the city areas and chicken farms. Phylogenetic analyses revealed a total of 61 sequence types (STs), eight phylogroups, and 17 phylogenetic clusters. The sharing of STs was not high given that E. coli ST10 and ST155 were common to ESBL-EC from rats caught in city areas and chicken farms, and ST44 was found among ESBL-EC from rats trapped in city areas and pig farms. However, the diversity of STs across locations was high, especially in rats captured from city areas (33 STs), followed by rats from chicken farms (16 STs) and pig farms (8 STs). These phylogenetic results indicate a certain level of dissociation between city areas and farm areas. Methicillin-resistant Staphylococcus aureus (MRSA) was found in only two rats, and both isolates were community-associated (CA)-MRSA (MRSA ST30 SCCmec IVc Panton-Valentine leucocidin (PVL) - positive), which have previously been identified in the Hong Kong SAR population. The findings reflect a possible anthropogenic origin of these MRSA isolates.
Pathogenic Leptospira spp. were detected in seven R. norvegicus caught from both urban areas and farms using PCR analysis of kidney samples. The species detected were Leptospira interrogans and L. borgpetersenii, confirming the circulation of pathogenic Leptospira spp. in the rat population in Hong Kong. Capillaria hepatica was found in 81 rats (37%) collected from all location types. The statistical analysis revealed that R. norvegicus caught from urban areas of Kowloon Peninsula and Hong Kong Island were more likely to be infected with C. hepatica.
In conclusion, the findings of this study underscore the ability of peridomestic rats to acquire antimicrobial-resistant bacteria from their surrounding environment, potentially serving as reservoirs and vectors for the dissemination of such resistant bacteria to both humans and animals. The presence of a significant number of genes conferring resistance against various antimicrobial agents, observed in the ESBL-EC from rats indicates the existence of substantial selective pressures within the habitats occupied by peridomestic rats. The genetic differences between the populations of rodent ESBL-EC found in urban areas, poultry farms, and pig farms suggests a certain level of segregation between human and animal environments. Furthermore, this study provides evidence of the circulation of zoonotic pathogens within the peridomestic rat population of Hong Kong. The results of this study improve our understanding on the complex epidemiology and dynamics of AMR and zoonoses at the wildlife–livestock–human interface, in diverse habitats with anthropogenic influence. The knowledge gained from this PhD project can contribute to the development and implementation of tailored interventions to reduce the emergence and spread of AMR and zoonoses in the future and therefore safeguard animal and public health.
| Date of Award | 8 Aug 2025 |
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| Original language | English |
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| Supervisor | Dirk Udo PFEIFFER (Supervisor), Yrjö GRÖHN (External Co-Supervisor) & Ioannis MAGOURAS (External Co-Supervisor) |