One Health Approach in molecular caracterisation of gram negative bacilli resistant to carbapenem and colistine isolated from fish in South-Kivu, Eastern of DRC

1. Overview

The emergence of carbapenem- and colistin-resistant Gram-negative bacilli poses a major threat to public health worldwide. As part of the aquatic ecosystem and the human food chain, fish can serve as reservoirs and vectors for antimicrobial-resistant bacteria, which could promote the spread of resistance genes among humans, animals and the environment. The One Health approach integrates human, animal and environmental health perspectives to comprehensively understand the molecular mechanisms underlying the resistance of these bacteria isolated from fish in South Kivu. This is essential for developing targeted interventions to curb the spread of resistance in this region and beyond. The project is supported by researchhub. Molecular biology and microbiology analyses will be carried out at the Department of Medical Biology of the Higher Institute of Medical Techniques in Bukavu (ISTM-Bukavu), which is equipped with modern facilities for bacterial culture, antimicrobial susceptibility testing and molecular characterisation (qPCR, agarose gel electrophoresis). Ethical approval has been granted by the ethical comittee of ISTM-Bukavu, ensuring compliance with guidelines on environmental sampling and biosafety.

Estimated Timeline
Initial sample collection and bacterial isolation: Months 1–3
Antimicrobial susceptibility testing: Months 3–4
Molecular characterisation (DNA extraction, PCR, gene detection): Months 4–7
Data interpretation and analysis: Months 7–9
Report writing and publication: Months 10-12.

Data Sharing Commitment

All data generated from this study, including antimicrobial susceptibility profiles, molecular characterization results, will be made publicly available through appropriate and shared with relevant stakeholders, including local health authorities and the scientific community, ensuring transparency and fostering collaborative efforts to combat antimicrobial resistance.

2. Introduction

Antibiotic resistance remains a source of concern for the scientific community, as it has become a public health issue and a recurring obstacle in the treatment and control of infectious diseases worldwide. In our country, the Democratic Republic of Congo, information on the epidemiology of carbapenem and colistin resistance is scarce. We are focusing on the One Health concept and using molecular techniques and a metagenomic approach to better identify them. Resistant species employ various mechanisms of carbapenemase production and colistin resistance, making sensitivity testing and other biochemical tests for the detection of carbapenem resistance unreliable.

Background

The One Health approach recognises the interconnectedness of human, animal and environmental health. Antimicrobial resistance (AMR) is a global health threat that transcends these domains. Gram-negative bacilli resistant to last-resort antibiotics such as carbapenem and colistin pose considerable challenges to treatment efficacy. Fish and aquatic environments can serve as reservoirs and vectors for the transmission of resistant bacteria, particularly in regions where antibiotic regulation and surveillance are limited, such as South Kivu in eastern DRC.

Key Litterature

Studies documenting the emergence and spread of carbapenem- and colistin-resistant Gram-negative bacilli in clinical, animal and environmental settings. Studies highlighting the role of aquatic animals and environments in harbouring and spreading resistant bacteria. Previous molecular characterisations have identified resistance genes and mechanisms in bacteria from food sources. Publications emphasising the importance of integrated surveillance within the One Health concept to combat antimicrobial resistance.

Research question

How common are gram-negative bacilli that are resistant to colistin and carbapenem that have been isolated from fish in South Kivu?
Which genes for molecular resistance are found in these bacteria?
What is the difference between these resistance profiles and those of the region's environmental and human isolates?
What potential routes exist for resistant bacteria to spread from fish to people and the environment?

Hypothesis

H1: Significant resistance to carbapenem and colistin is shown by gram-negative bacilli obtained from fish in South Kivu.

H2: The isolates' known carbapenemase and colistin resistance genes will be found by molecular analysis.

H3: Resistance genes discovered in fish isolates exhibit genetic resemblance to those in environmental and human isolates, indicating potential pathways of transmission.

H4: Multidrug-resistant bacteria that could affect the health of humans and animals are found in South Kivu's water environment.

Study Importance and Impact Statement:

This research is important because it addresses antibiotic resistance at the nexus of human, animal, and environmental health in a vulnerable area by utilizing the One Health concept. It closes knowledge gaps about the reservoirs and transmission dynamics of important resistance genes outside of clinical settings by molecularly profiling resistant bacteria from fish. The results could improve integrated surveillance systems, direct antibiotic usage and environmental management strategies, and inform risk assessments. Notwithstanding the results, this study contributes to our knowledge of the ecology of AMR, aids international efforts to control resistance, and safeguards public health by spotting possible dangers before they become serious.

By offering vital information on the frequency and mechanisms of antibiotic resistance in Gram-negative bacilli in the Sud-Kivu region, our study has the potential to greatly improve public health. Targeted therapies that lower infection rates can be informed by the identification of certain resistant strains and their genetic drivers. Better health outcomes, reduced morbidity and mortality from resistant infections, and fewer medical expenses for treating antibiotic-resistant infection-related consequences could result from this. Our project's results will improve healthcare systems by offering evidence-based suggestions for antibiotic stewardship. We may encourage a culture of accountability and well-informed decision-making in clinical settings by educating healthcare professionals about antibiotic resistance and ethical prescribing practices.

3. Methods

In order to isolate gram-negative bacteria that are resistant to antibiotics, this study will gather fish samples from freshwater habitats and markets in South Kivu, Democratic Republic of the Congo. Antimicrobial susceptibility testing, genetic analysis of resistance genes in using genes such as blaKPC, blaNDM, blaGES,blaIMI for Carbapenem resistant and mcr1,mcr2,mcr3,mcr4 for colistine resistants genes, and microbiological identification will all be performed on the samples. Power analysis justifies a sample size of 300. To investigate relationships between resistance patterns and environmental factors, data will be evaluated using regression models and descriptive statistics. Data will be safely stored and made available to the public when ethical approval was secured. A pilot study guided the study design and validated the method's viability.

Participants/Sample

Target Sample:

Gram-negative bacilli isolated from fish from a variety of aquatic habitats in South Kivu, Democratic Republic of the Congo (DRC), will be the main focus of this investigation. Included will be fish species that are often consumed in the area as well as those that reflect various ecological niches.

Materials and Procedures

Inclusion criteria

Fish samples gathered from South Kivu's fish markets and freshwater sources from aquaculture met the inclusion criteria.
samples that, according to preliminary microbiological screening, show the presence of gram-negative bacteria.
Antimicrobial-resistant isolates will be verifying by first susceptibility testing.

Exclusion criteria

All samples that are outside aquatic environments are excluded.

Planned Analyses

Recruitment Strategy:

Local fishermen and merchants will be enlisted to assist with sample collection, which will be carried out through planned field trips to markets and fishing locations. We'll use random stratified sampling to capture market and ecological heterogeneity.

Justification for Sample Size:

In order to identify significant correlations between resistance genes and environmental factors with 80% power at α = 0.05, a minimum of 300 fish samples will be targeted based on power analysis. Based on regional data, this estimate takes into account an anticipated 30% prevalence of antibiotic resistance.

Supplies and Methods

Fish specimens will be aseptically collected, and their exterior surfaces will be swabbed as part of the sample collection and processing process. Under sterile conditions, samples of internal organs (intestines, gills) will be taken. Samples will be sent to the lab for microbiological investigation via cold chain.

Microbiological Identification:

Selective media, such as MacConkey agar, will be used to isolate gram-negative bacilli. The identity of the bacteria will be verified by biochemical assays (API 20E), colony morphology, and Gram staining.

Antimicrobial Susceptibility Testing (AST):

In accordance with CLSI and EUCAST standards, AST will be carried out using the disk diffusion method. Commonly used antibiotics that have been found from Lake Kivu fish will be examined.
Molecular Characterization:

Commercial kits will be used to extract DNA from isolates. We will do Polymerase Chain Reaction (PCR) and gel electrophoresis agarose experiments that target and confirm important resistance genes, such as blaCTX-M, blaTEM;…etc, and mcr. Sequencing will be done to confirm positive amplicons.
Settings for Data Collection:

Standardized forms will be used to document every procedure. Biosafety level 2 conditions will be used for laboratory work.

Statistical Tests:

Gene distribution and prevalence of resistant isolates are summarized using descriptive statistics.
To evaluate relationships between resistance genes and fish species or sample locations, use chi-square or Fisher's exact tests.
Antimicrobial resistance predictions can be found using logistic regression models.
Specific Predictions for Hypotheses:
Significant correlations with environmental factors like water quality or human activities will be seen if resistance genes are common in fish from particular habitats.
Species-specific resistance patterns will be found if molecular profiles vary among fish species.

Ethics and Data Management

Data management and ethics
Ethical Approval:

The study protocol complies with both national and international ethical standards after being submitted to and approved by the Institutional Review Board of [Name of Institution] in the Democratic Republic of the Congo.
Measures for Confidentiality: Every sample identifier will be made anonymous. Only authorized personnel will have access to the safely stored data.
Data Sharing and Archiving:

After publication, sequence data will be placed in open repositories (like GenBank). To encourage openness and repeatability, de-identified datasets and analytic scripts will be made accessible upon reasonable request.
Optional Pilot Data
Twenty fish samples have been used in a preliminary pilot project that was carried out to confirm laboratory and sampling procedures. The feasibility of the full-scale investigation was supported by preliminary findings showing a 25% incidence of gram-negative bacteria that are resistant to antibiotics.

4. Pilot Data (Optional

5. Budget

· Personnel (salary, fringe benefits, tuition, or other direct compensation): 2, 930USD, This will cover the salary of the PI, accounting, lab technician and/or perdiem) for the 12 months of the project and the overhead and administration expenses that will support the entire operations of project. Laboratory space provided by the institution, access to equipment available in the laboratory, and associated information systems, support and administrative staff such as HR, general finance, accounting, IT for internet access, and legal, office space for project’s documents, publications, transportations.. Equipment: We request Micropipettes (2), , 4P200, 4P20, 4P10, 4P2 (200 USD), equipment’s transportation and taxes (100 USD), (500 gr Chrom Agar base (80USD), 2 NP Carba Test Kit (100 USD), Antibiotics discs for testing (80USD), 500 Eppendorf tubes (20 USD), Genes of resistance and virulence (300 USD), 500 fecal swabs and others bottle for collection (50USD).

6. References

Pan X, Zhao X, Song Y, et al.(2022) Molecular Characterization of WCK 5222 (Cefepime/Zidebactam)-Resistant Mutants Developed from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate. Gao B, éditeur. Microbiol Spectr. 10(1):e02678-21.

2. Bernabe KJ, Langendorf C, Ford N, et al. (2017) Antimicrobial resistance in West Africa: a systematic review and meta-analysis. International journal of antimicrobial agents 50 (50). Elsevier: 629-639.

3. Bourafa N, Chaalal W, Bakour S, et al.(2018) Molecular characterization of carbapenem-resistant Gram-negative bacilli clinical isolates in Algeria. Infect Drug Resist. Volume 11:735‑42.

4. Djahmi N, Dunyach-Remy C, Pantel A, et al. (2014) Epidemiology of Carbapenemase-Producing Enterobacteriaceae and Acinetobacter baumannii in Mediterranean Countries. BioMed Res Int. ;2014:1‑11.

5. Runcharoen C,Raven KE, Reuter S, et al. (2017) Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand. Genome medicine 9(1).Springer: 1-11.

6. Mahamat OO, Kempf M, Lounnas M; et al. (2022) Epidemiology and prevalence of extrended spectrum Beta-lactamase and carbapenemase-producing Enterobacteriaceae in humans, animals and environnement in Wet and Central Africa. International Journal of Antimicrobial Agents. Elsevier: 106203.

7. Osei Sekyere J and Reta MA (2020) Genomic and resistance epidemiology of Gram negative bacteria in Africa: a systematic review and phylogenomic analyses from an One Health perspective. Systems 5(6). Am Soc Microbiol: e00897-20

8. Osterblad M, Hakanen AJ and Jalava J (2014) Evaluation of the Carba NP test for carbapenemase detection. Antichemotherapy 58 (12). Am Soc Microbiol: 7553-7556.

Preprint

Preprint

One Health Approach in molecular caracterisation of gram negative bacilli resistant to carbapenem and colistine isolated from fish in South-Kivu, Eastern of DRC