Session 4SAMPLING for surveillance and

monitoring of AMR in bacterial

pathogens from aquaculture

Aihua LiInstitute of Hydrobiology, Chinese

Academy of Sciences (IHB-CAS), China

liaihua@ihb.ac.cn

4.1 Purpose of AMR surveillance and monitoring

4.2 Overall purpose-based design for sampling

4.2.1 Prioritization of target aquatic animals and environment

4.2.2 Selecting antimicrobials for testing

4.2.3 Determining the timing/schedules for sample collection

4.2.4 Specimen numbering

4.2.5 Target bacterial species

4.2.6 Sample size

4.3 Sample collection from aquatic animals and environment

4.3.1 Sample collection from live/moribund animals (whole animal or its tissue)

4.3.2 Environmental sample (water samples, sediment)

4.3.3 Transportation of various types of samples

4.3.4 Biosecurity practices when collecting samples

4.4 Collection of sampling information

4.4.1 Clinical signs of sampled aquatic animals

4.4.2 Environmental parameters

4.4.3 Sample information recording

4.4.5 Other information

4.5 Preservation of bacterial isolates

Contents

⁃ The spread of resistant bacterial strains may greatly

reduce the available medical options including in

aquaculture itself.

⁃ We must determine the source and accumulation of

AMR in aquaculture settings, and monitor and analyze

the transfer of AMR among microbial communities,

environment and aquaculture products, in order to

better understand the impact on human and

environmental health and, to stop the situation from

getting worse

⁃ .

Why AMR surveillance and monitoring are needed

AM

R s

urv

eill

ance

and

monit

ori

ng

AMR profiles or patterns

The causes of the problem

The solutions to the problem

- FAO’s goal is to achieve food security for all and

make sure that people have regular access to enough

high-quality food to lead active, healthy lives.

- Control of bacterial antimicrobial resistance in

aquaculture is an important part of ensuring food

safety and food security.

⁃ This course is mainly based on “Regional AMR Monitoring and

Surveillance Guidelines Volume 3- Monitoring and Surveillance of AMR in Bacteria from

Aquaculture” (GL3).

⁃ Session 4 of this training course mainly introduces the

sampling method of fish specimen for AMR surveillance

corresponding to chapter 2 and 3 of GL3.

For molluscs： please refer to EU Reference Laboratory for Molluscs Diseases. Sample

processing in the context of mollusc mortality events (1st edition, 2020)

For crustaceans: please refer to Shields et al Collection techniques for the analyses of

pathogens in crustaceans Journal of Crustacean Biology 37 (6), 753-763, 2017

This guideline (GL3) places emphasis on linking AMR

surveillance with ultimately improving antimicrobial use in

aquatic species, as well as improving aquaculture production.

Recognizing the value of an internationally harmonized AST

data set, it was proposed by FAO-RAP to establish a network of

aquaculture AMR testing laboratories in Asia.

Regional Tools to Support Overarching Design of AMR, AMU and Residue Surveillance

Compiled by FAO-RAP

Mary Joy Gordoncillo,2021

Monitoring refers to a continuous, dynamic process of collecting data about

AMR in a given population or areas over a defined time period. Monitoring

usually concerns specific groups.

Surveillance refers to a systematic collection, analysis, interpretation and

timely dissemination of AMR data from defined populations or areas. These data

are then used to describe health hazard occurrence and to contribute to the

planning, implementation and evaluation of AMR mitigation action.

Surveillance system typically involves a number of data collection approaches,

and also incorporates data management, analysis and reporting system. Routine

reports (monitoring data), surveys and special studies, case investigations,

census are a part of surveillance.

These two concepts are often used together.

What is AMR surveillance and monitoring（AMRSM）

FAO promotes One Health in work on food

security, sustainable agriculture, food

safety, antimicrobial resistance (AMR),

nutrition, animal and plant health, fisheries, and

livelihoods. Ensuring a One Health approach is

essential for progress to anticipate, prevent,

detect and control diseases that spread between

animals and humans, tackle AMR, ensure food

safety, prevent environment-related human and

animal health threats, as well as combatting

many other challenges.

AMR surveillance and monitoring program generates

information on AMR patterns in aquaculture that can be

considered together with AMR patterns in humans.

Multi-sectoral review of the results will help to identify

potential links between AMR in humans and in aquatic

animals which can be investigated in more depth through

future surveillance and research.

This multi-sectoral approach will contribute to the design

of evidence-based policies and programs to mitigate AMR.

Surveillance and Monitoring is widely acknowledged as critical

components of the response to antimicrobial resistance (AMR)

and are one of the five strategic priorities of the Global Action

Plan on AMR.

Research activity is not surveillance and monitoring, but can be

used as a part of AMR surveillance program, and the data can be

aggregated with data from the surveillance and monitoring

program if both are carried out using standard AST protocol.

National AMR surveillance framework

- Government commitment to support the surveillance program for AMR at

country level and development of policies and plans

- Develop a national laboratory-based surveillance system at country level

- Establish coordinating body with responsibility to systematically collect

and analyze data and share data.

- Allocate at least one reference laboratory for bacterial identification and

susceptibility testing with competence of phenotypic and genotypic

determination of presence of AMR

- Set up a network for data collection

- Emphasis on laboratory quality systems

Prerequisites for implementing an AMRSM program

Sharing the results with the stakeholders

Data analysis and conclusion

Laboratory methodology

Sample collection

Sampling design

National surveillance framework

Objectives setting

General procedure of AMR surveillance and monitoring

4.1 Purpose of AMR surveillance and monitoring

Objectives and usefulness of AMR

surveillance and monitoring

Evaluate the status of AMR level in aquatic pathogenic bacteria

populations isolated from different fish species or different

rearing areas.

Collect information on AMR trends in relevant microorganisms,

aquatic animal species, and areas.

Provide information for professionals prescribing the use of

antimicrobials in aquatic animals, predict clinical outcomes of

infections caused by AMR bacteria

Explore the epidemiology of AMR, describe the spread of

resistant bacterial strains and resistance genes, and identify their

affecting factors such as season and water environment.

Explore the potential relationship between AMR in aquatic animal bacteria and

antimicrobial use (AMU);

Provide clues for the identification of AMR source in aquaculture settings.

Conduct risk analyses as relevant to aquatic animal and human health;

Provide a basis for policy recommendations for animal and human health;

Detect the emergence of novel AMR mechanisms.

Evaluate interventions measures- such as use of probiotics and vaccines.

Obtain isolates for a national bacterial culture collection for future investigation.

Provide direction for the development of new antimicrobials and alternatives for

aquaculture.

Provide data for education on current and emerging hazards

Help establishing new or improve existing CLSI ECVs or EUCAST ECOFFs.

- between different target aquatic animals

- between different pathogenic bacteria regardless of the sources.

- between pathogenic bacteria and commensal counterparts.

- Bacterial strains isolated from different production system,

such as ponds vs RAS, ponds vs rice paddy, etc.

- between farms with and without the use of antimicrobials.

- Changes in AMR levels after discontinuation of antibiotic

treatment

Comparative analysis of AMR levels under various

settings can yield very instructive results and findings

The key reasons for surveillance of AMR described by Hunter and

Reeves ( 2002) are to determine:

The size of the AMR problem in aquaculture

Whether, how and why resistance is increasing

Whether previously unknown types of resistance are

emerging

Whether a particular type of resistance is spreading

Whether a particular type of resistance is associated with a

particular outbreak.

AMR investigations can and should clarify the

relationship between various factors and AMR levels

to facilitate the development of relevant measures to

slow the development and spread of AMR in

aquaculture.

These factors include: water type (freshwater, brackish water or

marine culture), geographical location , season, feed, stocking

density, fish value, farming system, aquatic animal species,

monoculture or polyculture, bacterial species, and types and

intensity of antimicrobial use, relevant legislations, and so on.

AMR surveillance can identify the causes leading to more severe AMR

situation by analyzing the relationship between AMR and factors.

Types of surveillance Description

Passive surveillance

The collection, analysis and dissemination of AMR

information obtained “passively” or collected for

other purposes.

Active surveillance

The active collection of AMR data following a

structured surveillance design; this can be either

active surveillance of a random sample or of a

targeted sample

Combination

Passive collection as baseline with one or more

additional active surveillance methods; includes

detection of cases as these occur, and active case

finding in representative or targeted samples

Determining the type of surveillance to be carried out

Active surveillance and monitoring are the core part of a national AMR surveillance

programmes. Passive surveillance and monitoring may offer additional information.

4.2 Overall purpose-based design for sampling

Components of AMRSM program

1. Aquatic animal species

2. Antimicrobials

3. Bacteria: species and pathogenicity (pathogenic or commensal, or

both), isolation method, identification method

4. Sampling timing and schedule

5. Sample size and information

6. AST methodology: K-B disk diffusion; 96-well microdilution plate

7. QC system

8. AMR data analysis and reporting

- Having a well-designed AMR surveillance plan provided clarity and

helped in their implementation.

- Developing a scientific and well-thought-out surveillance program is

half the battle.

Basis for prioritization Target population to be selected

Economic value The aquatic species which has the highest

economic value in the country

Aquaculture production The aquatic species that are most produced in

the country.

Antimicrobial sales or use data The aquatic species for which the most

antimicrobials have been sold or used.

As aligned with other national

AMR surveillance initiatives

The aquatic species that will be most relevant to

the overarching national AMR surveillance plan

of the country

Basis for selection of target aquatic species population

4.2.1 Selection of target aquatic animals

Commensal bacteria from healthy aquatic

animals?

Pathogens from diseased aquatic animals?

Bacteria from the aquaculture environment?

(This is the concern of FAO GL3 and this training course )

(This is the concern of FAO GL4)

- For aquaculture clinical purpose, pathogenic bacteria should be

sampled and investigated.

- For One Health related surveillance program, samples should only

be collected from healthy food animals. In this case, sampling sick

animals should be avoided as these may not represent the status of

resistance in bacteria carried by healthy animals that enter the food

chain.

Actually, it depends on the objective of surveillance

- Areas with high aquaculture production

- Farms by randomly selected

- Sentinel farms

- Farms known to predispose to the target disease

- Areas easy access to the laboratories

- Voluntary participation

- Areas known to high intensity of antimicrobial use or vice versa

Selection of farms or sampling areas

The AMR status of any single region does not represent an AMR

profile of the country. So we should take samples from as many farms

or regions as possible to get the national AMR picture.

4.2.2 Selecting antimicrobials for AMR testing

- Extensively used antimicrobials in therapy of aquaculture

animals in the country/ Antimicrobial use data

- Human critically important antimicrobials

- Antibiotics with the highest content in the environment if the

data is available

- Antimicrobials prohibited in aquaculture, such as

furazolidone in many country.

- Representatives of different classes of antimicrobials

- The common antibiotics in the international AMR

surveillance frameworks for comparison purpose.

The availability of protocol-specific QC values. Currently CLSI protocol-specific QC values have been established for tests carried out using CAMHB (MIC) or MHA (disc) with incubation at 28℃ (and 22℃) for 10 agents.

ampicillin AMP gentamicin GEN

enrofloxacin ENR ormetoprim-sulfadimethoxine ORS

erythromycin ERY oxolinic acid OXO

florfenicol FLO oxytetracycline OXY

flumequine FLU trimethoprim-sulfamethoxazole TRS

If the reproducibility of the test protocol can be guaranteed, other antibiotics, even

without QC values, can also be included if they are considered mandatory for

testing, because the results can be used at least for comparison and trend analysis.

4.2.3 The timing/schedules for sample collection

- Pre-planning as to when to commence the sample collection, and how

regular the surveillance activities will be, is essential. A repeatable

population-based sampling plan will facilitate comparison of results over

time.

- Frequency should be based on the incidence and seasonality of bacteria or

diseases under surveillance.

- Ideally, surveillance should be continuous with sample collection separated

in different months around the year. However, in field reality, this will

depend on a variety of factors which need to be carefully considered to

enhance implementation feasibility, future sustainability, as well as the

quality of information to be later generated.

4.2.4 Specimen numbering

- A uniform sample coding system must be in place for an AMR

surveillance program especially those involving multi-labs.

- Each laboratory or each surveillance program has its own sample

coding scheme

The following is an example of sample ID

Laboratory ID + presumptive target bacteria code + serial numberFor the specimen label, it should contain：

Sampling Location + Sampling Date+ Animal species +/- Sample type (kidney, liver, etc.)

- Write the specimen label information on both the cap and the

wall of the container.

- It is recommended that specimen type code, lab ID, location

ID, etc. be set up according to WHONET's rules as much as

possible.

- All specimens coming into the laboratory are required to be

labeled throughout all phases.

- Inappropriate specimen identification can lead to adverse

outcomes such as unnecessary or delayed treatments and even

cause confusion or mistakes.

– Bacteria that are native to the aquatic environment in the

ecosystem (freshwater, marine, brackish water);

– Bacterial pathogen relevant to the aquacultured species in the

culture system in the country;

4.2.5 Determination of target bacterial species

Animal bacterial pathogens relevant to the countries‘ priorities

should be considered as the major target bacteria.

In addition, the following bacteria may also be considered as target

bacteria for AMR surveillance.

– Human pathogens like Salmonella, if there has been an

established link between the aquaculture system and outbreaks

of fish poisoning;

– Zoonotic bacteria , such as Streptococcus agalactiae (GBS)

– Commensal bacteria

E. coli may be sampled from animals, animal feed, and then

environment. These bacteria are commonly used in surveillance

and monitoring programs as indicators, providing information on

the potential reservoir of antimicrobial resistance genes, which

may be transferred to pathogenic bacteria of both animal and

human pathogenic bacteria.

Pathogen Host of pathogen

Aeromonas hydrophilaCatfish, carp, trout, eel, sturgeon, tilapia and bass, etc.

Aeromonas salmonicida Salmon, trout, carp and catfish

Other Aeromonas species Carp, catfish, eel, sturgeon, tilapia, etc.

Edwardsiella ictaluri Catfish, yellow catfish

Edwardsiella piscicida Turbot, flounder, carp, catfish, eel and tilapia

Flavobacterium columnare Carp, mandarin fish trout, tilapia , catfish and salmon

Flavobacterium psychrophilum Trout

Citrobacter spp. Carp, sturgeon, crab, crayfish, softshell turtle,

Acinetobacter spp. sturgeons

Photobacterium spp. Sturgeon, sea bream, yellow catfish, sea bass, snakehead

Pseudomonas spp. Carp, catfish, eel, salmon

Vibrio spp. Most of the marine fish species, crayfish

Yersinia ruckeri Trout and salmon

List of common pathogenic Gram-negative bacteria in aquaculture

Pathogen Host of pathogen

Lactococcus garvieae flounder, soft-shell turtle, crayfish

Nocardia sp. Snakehead, large yellow croakers, seriola, largemouth

bass, Trachinotus ovatus

Streptococcus agalactiae Tilapia, Grouper

Streptococcus iniae Tilapia, sea bream, flounder, hybrid striped bass

Streptococcus dysgalactiae Sturgeon

Weissella sp. Trout

Mycobacterium spp. sturgeon

List of pathogenic Gram-positive bacteria reported in aquaculture

According to my statistics from the literatures, there are at least

14 classes and 74 genera of bacteria with pathogenicity to

aquatic animals naturally (unpublished data).

Basis for prioritization Target pathogen to be selected

1. Prevalence of disease

The bacterial pathogen causing the most commonly

encountered infection of the target aquatic species farmed in

the area.

2. Outbreak frequencyThe bacterial pathogen causing the most significant disease

epizootics in the major aquatic species farmed in the area

3. Economic impact

The bacterial pathogen causing the biggest economic burden to

the target population, such as mass mortality or export

rejection.

5. Antimicrobial use impactThe bacterial pathogen causing infections that result in the

most antimicrobial use.

6. Current laboratory capacities

The bacterial pathogen that the designated laboratory can

isolate, identify and perform antimicrobial susceptibility

testing, and if QC data is available.

7. As aligned with the international

AMR surveillance program

The bacterial pathogen included in many other programs on

aquatic animal pathogens for international comparison

Basis for selection of target bacterial

pathogens(the countries‘ priorities )

For marine fish

For freshwater fish

For tilapia and marine fish

Recommended by FAO GL3

2.1.2 肠道内容物菌群的组成（属的水平）

• This figure is showing that genus Aeromonas is one of the main normal

groups in the intestine flora of healthy grass carp, crucian carp and bighead

carp.

• Of course, it may not be the case in other species of fish

Aeromonas

- Additionally, as reported, among all the detected bacterial

taxa, Aeromonas was associated with the highest number of

ARGs (Stalder et al., 2019).

- Aeromonas may play an important role in the dissemination

of ARGs and resistance plasmids in aquatic environment. In a

study, 73% and 100% of tested aeromonads could act as a

recipients and donors of DNA, respectively.

- Therefore, Aeromonas spp. isolated from healthy fish

intestine and the aquaculture environment may be a good

indicator bacteria for AMR surveillance.

If there is no diseased fish to be taken at a given sampling time point, and

what samples should be taken?

In the case, I personally recommend collecting the intestinal contents of

healthy fish for isolation of target bacteria if necessary. Because when fish

ingest antibiotics, all bacteria in the intestine will have the opportunity to

come into contact with the antibiotics and be subjected to its action, so the

AMR levels of the commensal bacteria in the intestine will also reflect the

AMR status of pathogenic bacteria in that culture system.

For AMR surveillance and monitoring in aquaculture, we

should sample pathogenic bacteria as much as possible.

- The sample size should be large enough to allow detection or

determine prevalence of, or trends in, existing and emerging

antimicrobial resistance phenotypes.

- The sample should avoid bias and be representative of the animal

population whilst taking into account the expected prevalence of

the bacteria in the sample type, the expected prevalence of the

resistance phenotype and the desired level of precision and

confidence.

- Samples from which bacteria were not isolated cannot be used in

the calculation of prevalence of the resistance phenotype.

4.2.6 Sample size

- Several statistical methods can be employed to calculate the number of

isolates needed for testing

- When setting the sample size, it is necessary to consider the following factors

• the desired precision for estimates of the prevalence of resistance and the

magnitude of change in resistance to be detected over a specified period of

time

• the initial or expected prevalence of resistance and the size of the

population to be monitored

• the desired level of statistical significance and power to detect a change

when it occurs

• Bacterial isolates from different fish in the same pond may have different

resistance pattern.

Table 4: Number of isolates required to estimate prevalence of resistance to a specific

antimicrobial in a particular bacterial species with a 95% confidence level, for two levels

of precision (5% and 10%). (Extracted from OIE Terrestrial Animal Health Code).

- Although this table is designed for surveys of bacterial

resistance in healthy food animals, I believe it is equally

applicable to surveys of pathogenic bacterial resistance.

This is because we know that only a sufficient number of

bacterial strains, whether pathogenic or commensal or

environmental, can reflect the overall level of resistance

in a fish farm or an region.

- Larger sample numbers are generally required for screening purposes

than for diagnosis of mortalities, or other abnormalities.

- When there is a fixed total number of samples, the most precise

estimates of AMR prevalence are obtained by maximizing the number

of farms of origin from which animals are sampled and testing a

single isolate of each target bacteria per aquatic animal. (Yamamoto et

al., 2014; Persoons et al., 2011).

- The number of samples collected and the frequency of sampling

should match the laboratory’s capacity to process samples in addition

to their routine workload.

My personal suggestion and practice:

- Take one isolate of bacterium per bacterial species per fish.

- Take no more than five fish from each pond

- Take no more than three ponds per species of fish per farm

- Take no more than five isolates from environment (water or

mud) per pond

- Maximize the diversity of strains' sources

- Another principle is to avoid two or more strains originating

from the same clone. This principle is used to design all

aspects of the sampling process.

An AMR surveillance project plans to collect a total of 400

strains of pathogenic bacteria. If only 4 laboratories are

involved, then each laboratory needs to select 2 farms and

collect 5 times throughout the year, each time selecting 2 ponds

from each farm as sample ponds, then each pond needs to

collect at least 5 diseased fish each time.

400/4/2/5/2=5

Let's take an example:

Collection of fish samplesA total of 288 fish with an average of 6 fish per farm were collected from 40 purposively selected fish farms from the following districts….. The farms included 33 earthen ponds, 5 cages and 2 tanks. It also included 57 fish collected from 8 wild water sites that are majorly landing sites around Lake Victoria(Table 1). The water temperatures ranged between 24.3-28℃ with an average of 25℃ for all sites where temperature measurements were taken. The collected fish were immediately transported in cool boxes or buckets containing their source waters, to the Microbiology Laboratory…

4.3 Collection sample from aquatic

animals and environment

Sampling strategiesThe success of a surveillance system is dependent on a

reliable process for sample and data collection. Sampling

should be conducted on a statistical basis. The sampling

strategy should ensure:

- the sample is representative of the population of

interest and meets the objectives of the surveillance;

- Sufficient sample size.

- the robustness of the sampling method. Any time a

sample is suspected to be contaminated, it must be discarded

before isolation is performed.

- Design a sampling plan for each sampling

- Prepare a sample collection form to record sample

information.

- Prepare SOP for sample collection and transport

- Purchase consumables for sample collection

- Prepare sample collection kits

- Train sample collection staff

- Trial sample collection in each surveillance area

- Prepare a sampling timetable

Preparations for sample collection

- Complete a sample collection form for each sample to

capture descriptive information that will help correctly

interpret the AMR results.

- It is important to ensure that a unique sample

identification numbering system is put in place covering

all surveillance laboratories so that every sample,

regardless of its origin, has a unique sample ID. Ensure that

the ID number written on the form matches the ID number

on the sample tube.

- Each sampling requires a detailed plan, and bring all necessary

materials and tools with you. Especially it is very important to

make the plan and the schedule known to everyone involved in

the sampling and subsequent processing and analysis.

- It is likely to be most practical to collect samples on the first two

days of the working week so isolates can be grown and identified

by the end of the week and laboratory staff will not be required

to work weekends.

Making plan for each sampling

- Quality of sample affects quality of result

- Moribund fish are the preferred sample. Collection of a sick fish

found at the surface or water's edge is best. Collecting fish with a

seine or hook is not recommended for sampling pathogenic

bacteria because most fish collected this way will be healthy.

- Do not collect dead fish, excluding freshly dead fish

- Use aseptic procedures for collecting samples

- Ensure all container with samples are put into the cooler

- Ensure that the field data sheet has been completed, including

photos, site map screenshot (from smart phone) and GPS readings.

Precautions when sampling

- Prepare your work area and get it organized. If an on-site laboratory is not

available, set aside an area indoors that can be used for sample collection.

Make sure you have good light and minimal air flow.

- Avoid sampling outdoors, particularly in summer.

- Disinfect the work area before and after sample collection.

- Dispose of waste, particularly carcasses and blood after sampling

carefully.

- Keep the time between collection and sampling as short as practicable

- Two or three people working on sample collection is ideal

- Dissection equipment: scalpel, forceps, scissors, alcohol burner, paper

towel, cutting board, loops, slides, syringe, tube, etc.

Preparing a work area for sample collection

- Examine fish carefully and record any obvious abnormalities

- Measure the length and weigh of each fish.

- Aseptically open the fish.

- Record any internal gross abnormalities

- Sample sites: Internal-kidney, liver, brain, eye; External -skin

lesions, gills

- If tissues or organs are collected in situ, place each piece of

tissue in a tube of sterile. Do not pool tissues from several fish

in one tube.

- Label the samples clearly correctly.

- Label plates on the agar side, not the lid

Procedure for taking sample from a fish

※ Small fish: live fish or iced fish

※ Large fish

※ Dying fish or fresh dead fish

※ Pond water or sediment

Type of specimen

4.3.1 Collection sample from live/moribund animals

(whole animal or its tissue)

1. Place the small live fish collected in a strong plastic bag with a

minimum amount of water (not more than one-third full). Fill the

bag with pure oxygen, tie it securely. Place it in another plastic bag

if possible and seal it. Place the double bags in a plastic box with

insulation. During summer months, pour crushed ice into a separate

plastic bag and place it in the box next to the fish bag.

2. If there is no oxygen supply at the collection site, small fish

specimens collected can also be packed directly into bags and

placed in insulation boxes filled with crushed ice.

※Small live fish specimen collection and shipping

※collection and shipping of large fish specimen

- If the sample fish are large in size and in number and lack

sufficient living transport conditions, in this case, a simple

laboratory need to be set up at the sample collection site to

collect the tissues and organs (such as viscera, gill,

gastrointestinal contents) of the sampled fish under aseptic

conditions into separate sterile tubes and bring them back to

the laboratory for bacterial isolation.

※Dying fish or fresh dead fish

- If the sample fish is fresh dead fish, or sick fish but cannot

tolerate hours of transport, they can be directly packed

without water into plastic bags and placed in an insulated

box containing enough ice and brought back to the

laboratory for bacterial isolation as soon as possible.

- If the number of target bacteria in the pool water is

expected to be relatively abundant, a small amount of

pond water can taken directly into a sterile tube, and

brought back on ice to the laboratory for bacterial

isolation.

4.3.2 Environmental sample collection (water

samples, sediment)

- If the abundance of target bacteria in the pool water is expected to be

relatively low, at least 100 ml of pond water needs to be collected with

a water collector and brought back on ice to the laboratory for bacterial

isolation. It is necessary to flush the water collector with the pond water

twice to avoid contamination of the water sample by the water collector.

The collected water samples were centrifuged to collect the bacterial

cells, and then cultured by streaking or spreading on the appropriate

agar media. Recording the water parameters such as temperature and

pH and so on.

- For the collection of pond mud, according to the actual

conditions of the site, a sample of about 10g can be

collected into a sterile container by using various

possible aseptic means, and then brought back on ice to

the laboratory for bacterial isolation.

- Sample collection should take place as close to

shipping time as possible, to reduce mortalities

during transportation. This is especially

important for moribund or diseased fish.

- Make sure all sample containers are closed

securely

- Keep samples chilled at all time

4.3.3 Transportation of various types of samples

- Freezing of samples should be avoided as it may kill

the bacteria or affect the carriage of plasmids.

- Samples should ideally be transported to the

laboratory on the day of collection. If not, they must

be stored in a refrigerator at 4 – 8℃ and transported

to the laboratory the next day.

- Sample collectors must apply good biosecurity practices

when collecting samples to avoid spreading disease from one

location to another. This is extremely important when

sampling from farms, both to ensure that pathogens are not

spread between farms and to avoid farmers associating a

disease outbreak that occurs by chance following sampling

with the presence of the samplers on their farm.

- Another aspect of biosecurity is the safety of the person

collecting the samples, this is because some fish pathogens

are zoonotic.

4.3.4 Biosecurity practices when collecting samples

• Streptococcus agalactiae (tilapia)

• Streptococcus iniae (tilapia)

• Edwardsiella tarda (eel, cichlids, ornamental fish)

• Vibrio vulnificus (eel)

• Photobact. damselae damselae (marine fish)

• Mycobacterium marinum (various warmwater fish, incl.

tilapia)

• Mycobacterium fortuitum (warmwater ornamental fish)

• Mycobacterium haemophilum (ornamental fish)

• Elisabethkingia meningoseptica （frog）

Zoonotic fish pathogenic bacteria from warmwater systems

include at least the following species

4.4 Collection of sampling information

4.4.1 Clinical signs of sampled aquatic animals

- Typical signs of diseased fish sampled should be recorded to

assist diagnosis.

- Particular attention should be paid to the presence of the

symptoms that the target disease should have. This helps to

avoid collection of pathogens that are not the target disease.

- Clinical signs include behavior in water, external signs, signs on

internal organs and mortality rate.

- Possible predisposing factors of the disease.

4.4.2 Environmental parameters

The following parameters are largely determined by the nature of the water

supply and are not significantly affected by most fish farm systems.

• pH

• temperature

• alkalinity

• salinity

• biocides

• suspended solids in inflowing water

• dissolved gases in inflowing water

• background nutrient levels

• metals

• hardness

The following water quality parameters may be

significantly affected by the aquaculture operation.

• dissolved oxygen

• ammonia

• nitrite

• biochemical oxygen demand (BOD)

• carbon dioxide

• suspended solids

• phosphorus

- All samples submitted for diagnosis and AST should

include as much supporting information as possible.

- All of these information will help identify the source of

AMR in the aquaculture system where the samples were

taken. These information are also very useful in analysis

of the AMR significance.

4.4.3 Sample information

Identification numberCOUNTRYState/ProvinceCityDistrictVillageLOCATIONAQUATIC SPECIES GROUPSPECIESECOLOGICAL HABITATWATER TYPEEPIDEMIOLOGICAL UNITFarm name/numberLocation typeGPS coordinatesBreedUtilizationMarket categoryRaising system Age

SPECIMEN TYPESPECIMEN DATESTATE OF HEALTH Transport conditions to the labSpecimen qualityCollected bySpecimen numberHistory of treatmentReasonLABORATORYORGANISMINCUBATION TEMPERATURESTORAGE NUMBERLocal organism codePrimary mediumEnrichmentSerotypeStorage location

The information marked in red is necessary

Relevant information Description

Farm number Code for the farm (if applicable)

Location type Where the specimen was collected

LocationName/code of location type (if

applicable)

GPS coordinatesLong/Lat where specimens were

collected

Aquatic species

group

Name/code of group of aquaculture

species

Species Species of the specimen

BreedBreed of aquatic spp from which

sample was taken

UtilizationPurpose for which the aquatic species

was raised

AgeAge of the animal source

(standardized)

Age category Newborn, young, adult

Market category Domestic, imported, for export, etc.

Relevant information Description

Collected by Name or identifier for specimen collector

Specimen type Specimen collected

Specimen number Code for the specimen

Specimen date Date sample was collected

State of health Status of health upon specimen collection

History of treatmentLists antimicrobials and regimen that the

sample was treated with;

Transport conditions to

the lab

Describes transport conditions for the

specimen

Specimen qualityDescribes the quality of sample upon lab

arrival

Reason Purpose for sample collection

Data on the specimenData on source of specimen GL3

4.4.5 Other information

The predisposing factors, such as water quality, handling,

transportation over crowding, nutritional deficiency and

non-lethal parasites play an important role in the

development and spreading of the bacterial diseases among

cultured fish. The identification of these predisposing factors

not only facilitates disease diagnosis, but also has

implications for the analysis of the development and spread

of AMR in the aquaculture system.

• Low oxygen content.

• High turbidity of water.

• High ammonia in water.

• High temperature

• Change in pH of water.

• Over stocking.

• Injuries or damage to skin or scales.

• Toxic inorganic and organic substances，overuse of various

kinds of drugs.

• Rough handling and transportation of fishes.

• Malnutrition

• Nutritional deficiency especially vitamins.

• Spawning activity.

- Some common stressors predisposing factors in farmed aquatic

animals should also be recorded, such as

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AMR DATA ANALYSIS AND DISSEMINATION

Laboratory preparations

- Use internationally valid method for isolation and

identification of target bacterial species

- Establish an AMR data storage system, install

equipment and software, and prepare data entry form

- Conduct disease diagnostic training

- Perform disc diffusion and MIC assays as per CLSI or

other internationally validated guidelines

- Ensure laboratory quality control (QC) systems are in

place.

It is recommended that MIC values (μg/ml) and zone diameter

(mm) of each antibiotic/bacterial strain should be reported.

MIC50/MIC90/MIC range；AMR prevalence (Resistance rate)

• R/I/S(%) (if clinical breakpoints for fish bacteria are

available);

• WT/NWT(NWT%): based on ECV (CLSI) or ECOFF

(EUCAST), or COwt.

• AMR Trend curve

Resistance patterns/Resistance profiles

Multiple antibiotic resistance index (MAR index) /average MAR

index.

How are the results reported and displayed

One or more indictors above can be used to describe the AMR level.

Example 1

Sandrine Baron et al, 2017

Table 2. Number of resistant isolates (# R) and prevalence of resistance to

each antimicrobial with 95% confidence intervals (CI) examined for the

isolates obtained by passive and active surveillance of poultry S. Heidelberg.

Example 2

Mather et al, 2016

Antimicrobial resistance patterns of s. aureus isolates (n = 33)…. Example 3

Deyno et al, 2017

Multi-Drug Resistance Pattern of Bacterial Isolates

Example 4

Example 5

Trends of resistance to cephalosporins by specific organismsTrends of total antimicrobial resistance

by common antibiotics

Mhondoro et al, 2019

Average multiple antibiotic resistance index (MAR) at different sampling sites

Chitanand et al，2021

Example 6

Data generated from surveillance must be packaged and presented

as a report that can be shared with relevant stakeholders :

Application of data generated

• Fish farmers

• Aquatic animal health professionals

• Aquaculture extension officers

• Associations of aquacultures producers, exporters

• Academic institutions

• Pharmaceutical industry

• Public health sector

• Customs department

Having a suitable management strategy to respond to surveillance data is of utmost importance for the successful implementation of surveillance systems.

4.5 Preservation of bacterial isolates

If possible, isolates should be preserved at least until

reporting is completed. Preferably, appropriate isolates

should be permanently stored. Bacterial strain

collections, established by storage of all isolates from

certain years, will provide the possibility of conducting

retrospective studies.

Storage of bacterial strains

Session 5.3 of this course will give details on this issue

• Regional AMR Monitoring and Surveillance Guidelines Volume 3 edited by FAO-RAP

• A Protocol for Active AMR Surveillance in Poultry : Towards a One Health AMR

Surveillance System: protocol for active AMR surveillance in commercial broiler and

layer chicken populations for the Fleming Fund Grants Programme. Version 2

• Section 2 of "Asia Diagnostic Guide to Aquatic Animal Diseases" Edited by Melba G.

Bondad-Reantaso, et al. http://www.fao.org/3/y1679e/y1679e.pdf

• Section 6 of OIE- Aquatic animal health code (2019). https://www.oie.int/en/what-we-

do/standards/codes-and-manuals/aquatic-code-online-access/

• Guidance in Development of Aquaculture Component of a National Action Plan on

Antimicrobial Resistance.

• The FAO Action Plan on Antimicrobial Resistance 2016-2020.

http://www.fao.org/fsnforum/resources/fsn-resources/fao-action-plan-antimicrobial-

resistance-2016-2020

• Many other online literatures.

REFERENCES

THANKS FOR YOUR ATTENTION!