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Focus - June 2022

Resident wild birds as potential disseminators of antimicrobial resistant bacteria

A research team at UCD (see panel) have studied the role of resident wild birds as potential disseminators of antimicrobial resistant bacteria in a large public amenity park in the greater Dublin area

Figure 1: An adult female (pen) mute swan with three cygnets, one of which is seen “disembarking” at the feeding trough. Photo: Theo de Waal.

Chloe Maloney

Undergraduate veterinary nursing student, School of Veterinary Medicine, University College Dublin.

Dr Sandra Aungier

MVM PhD MRCVS, Supervisor. Lecturer in Veterinary Nursing, School of Veterinary Medicine, University College Dublin. 

Dr Theo de Waal

BVSc PhD MRCVS, Associate Professor, School of Veterinary Medicine, University College Dublin. 

Shermin Shahriari

Undergraduate veterinary medicine student, School of Veterinary Medicine, University College Dublin.

Dr Mary Sekiya

PhD, Chief Technical Officer (Microbiology/Parasitology), School of Veterinary Medicine, University College Dublin.

Ms Amanda Lawlor

BAgrSc, Senior Technical Officer, School of Veterinary Medicine, University College Dublin.

Dr Sabine Harrison

PhD, Senior Technical Officer, School of Agriculture and Food Science, University College Dublin.

Dr Finola Leonard

MVB PhD MRCVS, Associate Professor, School of Veterinary Medicine, University College Dublin.

Antimicrobial resistance (AMR) is a significant and rapidly escalating concern globally due to its spread between humans, animals and the environment1. The occurrence of multi-drug resistant Escherichia coli (E.coli)bacteria in wild waterfowl including mute swans and mallard ducks has been documented with these hosts identified as reservoirs of avian pathogenic E. coli strains (APEC) and pathogenic human E. coli strain serotype H7:O1572. One study in Poland highlighted that the mute swan showed the highest number of E. coli strain isolates at 59.5 per cent closely followed by the mallard duck at 55.3 per cent3. The migratory lifestyle of these wild waterfowl is a contributing factor towards the spread of potentially zoonotic E. coli strains and multi-drug resistant (MDR) strains between various ecosystems across Europe and other countries. 

Thus, wild water birds may pose a potential risk to human and animal health by transmitting MDR E. coli strains to water bodies via contaminated faeces4.

Research on wild water birds acting as hosts for MDR E.coli has been widely documented in various countries worldwide. However, there is little data available on wild water birds acting as hosts for MDR E.coli in local public amenities in Ireland. This research project identified one public water amenity in the greater Dublin area where the only water source for the water bodies was from surface water run-off in the immediate area. These water bodies were inhabited by wild waterfowl which were made up of mute swans (Figure 1), mallard ducks, seagulls and other seabirds. The water bodies were located along popular walking trails where dogs were allowed direct access. Therefore, the aim of this project was to determine if E.coli was present in these water bodies and whether the E. coli identified were resistant to certain veterinary and human antibiotics.

Figure 2: TBX agar plate showing blue Escherichia coli colony growth following culturing and incubation. Photo: Chloe Maloney.

Materials and Methods 

Collection, Filtration and Culturing

Water sampling was carried out weekly for four consecutive weeks from four water bodies in a Dublin public amenity area. Five 100 ml water samples were collected and pooled at each site. Samples of 100ml from each site were vacuum filtered using a sterile 0.45μm nitrocellulose filter membrane. Buffer Peptone Water (BPW) was added to rinse each membrane by vortexing. Samples (100μl) were cultured on i) TBX (Tryptone Bile X-glucuronide (Figure 2); ii) TBX with 1mg/ml cefotaxime; and iii) TBX with 1mg/ml ciprofloxacin. These plates were incubated for 18-24 hours at 37°C. Potential E. coli isolates were sub-cultured onto MacConkey agar (Figure 3) and then checked by antibiotic disc diffusion on Mueller Hinton agar5.

Figure 3: Escherichia coli colonies were sub-cultured onto MacConkey agar to ensure pure colonies were obtained for subsequent carrying out of an antibiotic disc diffusion test. Photo: Chloe Maloney.

Antibiotic Disc Diffusion Test 

Antibiotic resistance trends were determined for seven antibiotics commonly used in veterinary and human medicine (Figure 4)5.

Figure 4: Antibiotic susceptibility patterns of Escherichia coli isolated from the public amenity water bodies. Photo: Chloe Maloney.

The European Committee of Antimicrobial Susceptibility Testing (EUCAST) Zone of Diffusion chart was used to classify each isolate as resistant (R), intermediate (INT), or sensitive (S) dependent on the zone of inhibition (Table 1 overleaf).

MALDI-TOF Mass Spectrometer

The MALDI-TOF Mass Spectrometer (bioMerieux Vitek MS) facility in the UCD School of Agriculture and Food Science was used to identify E. coli and other coliform isolates (Figure 5)6.

Figure 5: MALDI-TOF Mass Spectrometer used for bacterial identification. Photo: Chloe Maloney.

Results and Discussion

Microbiology culture results displayed in Table 2 showed E. coli growth on 11/20 (55%) culture plates across the four-week study period. Of these 11 plates, 46 E. coli colonies were isolated from the four water bodies analysed. Tap water culture samples, used as controls, showed no growth of E. coli. Escherichia coli are almost exclusively faecal in origin, and the presence of E. coli is suggestive of water faecal contamination from resident wildlife including mallard ducks and swans. From the literature, the incidence rate of E. coli strain isolates in mute swans is 59.5 per cent, closely followed by the mallard duck at 55.3 per cent3. Escherichia coli growth was only seen on “TBX only” culture plates, with no E. coli growth seen on “TBX + Ciprofloxacin” and “TBX + Cefotaxime” culture plates. All three TBX plates, across the four weeks, displayed some coliform growth in the form of white or cream colonies of various sizes. 

In terms of antimicrobial resistance, the E. coli detected had higher levels of resistance to certain antibiotics as shown in Table 3. Ninety-six per cent of E. coli isolates were resistant to cephalexin. Cephalexin is a first generation cephalosporin commonly used in canines. It is known to be clinically resistant to E. coli6. Escherichia coli resistance to cefpodoxime, a third generation cephalosporin, was lower at 35 per cent. 

Tetracycline is widely known in veterinary medicine for its broad-spectrum activity against bacterial infections7. In the present study, 85 per cent of E. coli isolates were resistant to tetracycline. In Poland, a study on wild birds which included mute swans and mallard ducks, found that 50 per cent of E. coli isolates were resistant to tetracycline2. 

This study found that only 15 per cent of E. coli isolates were resistant to enrofloxacin and sulfamethoxazole/trimethoprim. Fluoroquinolones are categorised as “highest priority critically important antimicrobials” (HPCIA) in both human and veterinary medicine8 with enrofloxacin (Baytril) being widely used in both companion and food producing animals to treat gram-negative and gram-positive infections9. Enrofloxacin is partially metabolised to ciprofloxacin in animals, and high levels of ciprofloxacin resistance in multi-drug resistant E. coli isolates have been found in the mallard duck in Poland at 46.8 per cent2. Therefore, the low levels of resistance in the Irish water bodies studied towards enrofloxacin and no resistance to ciprofloxacin is an interesting finding. 

Table 1. EUCAST Zone of Diffusion chart, displaying the disc contents in µg and diameter breakpoints measured in mm, was used to determine the resistance and susceptiblity levels of the Escherichia coli isolates towards the antimicrobials5.

Table 2. Culture demographics from the four-week culture period.

Sulfamethoxazole/Trimethoprim is a human preparation based on a combination of the antimicrobial sulfamethoxazole and the antibiotic trimethoprim, whereas trimethoprim and sulfadiazine is the veterinary preparation. Both combinations can be used in veterinary medicine10. 

The low resistance levels of the E. coli isolates towards the human preparation of sulfamethoxazole/trimethoprim is promising as it is used to treat a range of gram-negative and gram-positive bacterial infections in companion and domestic farm animals10.

The E. coli isolates displayed relatively high resistance levels at 30 per cent towards amoxicillin/clavulanic acid and gentamicin. Amoxicillin-clavulanic acid (Co-Amoxiclav, Synulox) is a commonly used veterinary and human antibiotic that has a broad spectrum of activity against gram-negative and gram-positive species. However, it is not indicated for gram negative bacteria including E. coli, as it is known to be clinically resistant11. 

It has been documented that there is growing resistance towards this beta-lactamase inhibitor combination. It has also been suggested that its use should be reserved alongside the critically important antimicrobials including the third and fourth generation cephalosporins and fluoroquinolones to slow the growing resistance levels12, 13.

The findings of this study highlight the need for monitoring of water quality of all water bodies to which the public have direct access. Currently, there is a European Council Directive (76/160/EEC) which only applies to sites where bathing is not prohibited, and the sites are frequented by a large number of bathers14. Under the Directive, local authorities are responsible for sampling of waters at the bathing sites in their areas. Analysis of microbiological parameters such as total coliforms and faecal coliforms are carried out. Ideally, this Directive should be amended to include shallow water bodies with public access. Dogs can still drink/bathe in these waters and the public can still access them to paddle in, if they wished.

Table 3. Antibiotic resistance levels of Escherichia coli isolates.

The MADLI-TOF Mass Spectrometer identified a number of other coliforms in the water bodies that were obtained from the culture plates (Table 4). While the MALDI-TOF database is very extensive, the E. coli strains and serotypes could not be identified due to their protein profile not being in the database. However, the E. coli bacteria were confirmed as members of the E. coli family which further confirmed that E. coli is present in the water bodies. Similarly, 31.25 per cent of isolates could not be identified as there were no matching profiles in the MALDI-TOF database.

Table 4. Coliforms identified using the MADLI-TOF Mass Spectrometer.

Conclusion 

  1. Microbiological analysis of the water samples concluded that E. coli is present in these four public amenity water bodies. The areas surrounding these water bodies are popular trails where dogs are regularly taken for walks. If water is ingested, there is the potential for the health of both the owners and their pets to be affected. This interplay between humans, animals and the environment highlights the relevance of regular microbiological monitoring of our public water amenities. 
  2. The MALDI-TOF Mass Spectrometer did not identify the specific strain of E. coli. However, it is still potentially a zoonotic pathogen and diligence must be taken if dogs are given unrestricted access by their owners to these water bodies. 
  3. This study focused on E. coli, but it is likely that there are various other pathogens in these waters that may have zoonotic potential and owners should take precaution around open stagnant water bodies to protect their own health as well as their pets.
  4. Water body sites with public access should not be assumed to be safe. The public should be alerted to this fact by the placing of local authority public health warning notices in each vicinity.
  5. A strong trend of resistance towards tetracycline and cephalexin was observed. The high levels of E. coli resistance to some of the antibiotics tested in this study further supports the escalating trends of resistance towards commonly used antibiotics in human and veterinary medicine. This study highlights that the resistance levels to certain antibiotics are low. However, it is probable that these resistance levels will continue to increase if antibiotics are not used wisely. This in turn will have serious implications for the future of One Health. 

    Acknowledgements

    The study was funded by the Summer Research Project Fund (School of Veterinary Medicine, UCD)

References
  1. Prestinaci F, Pezzotti P, Pantosti A. Antimicrobial resistance: a global multifaceted phenomenon. Pathogens and global health 2015, 109(7):309-18
  2. Nowaczek A, Dec M, Stępień-Pyśniak D et al. Antibiotic resistance and virulence profiles of Escherichia coli strains isolated from wild birds in Poland. Pathogens (Basel) 2021, 10(8):1059-72
  3. Kuczkowski M, Krawiec M, Voslamber B et al. Virulence Genes and the Antimicrobial Susceptibility of Escherichia coli, Isolated from Wild Waterbirds, in the Netherlands and Poland. Vector borne and zoonotic diseases 2016, 16(8):528-36
  4. Merkeviciene L, Klimiene I, Siugzdiniene R et al. Prevalence and molecular characteristics of multi-resistant Escherichia coli in wild birds. Acta Veterinaria Brno 2018, 87(1):9-17
  5. CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed. CLSI standard M02. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute;. 2018
  6. Papich MG. Cephalexin. Papich Handbook of Veterinary Drugs (Fifth Edition). St. Louis (MO). W.B. Saunders; 2021. p. 165-6
  7. Grossman TH. Tetracycline antibiotics and resistance. Cold Spring Harbor perspectives in medicine 2016, 6(4):a025387-a
  8. Römer A, Scherz G, Reupke S et al. Effects of intramuscularly administered enrofloxacin on the susceptibility of commensal intestinal Escherichia coli in pigs (sus scrofa domestica). BMC veterinary research 2017, 13(1):378-88
  9. Trouchon T, Lefebvre S. A Review of Enrofloxacin for Veterinary Use. Open journal of veterinary medicine 2016, 6(2):40-58
  10. Papich MG. Trimethoprim and Sulfamethoxazole. Papich Handbook of Veterinary Drugs (Fifth Edition). St. Louis (MO). W.B. Saunders; 2021. p. 945-7
  11. Papich MG. Amoxicillin and Clavulanate Potassium. Papich Handbook of Veterinary Drugs (Fifth Edition). St. Louis (MO). W.B. Saunders; 2021. p. 45-7
  12. Burch DGS, Sperling D. Amoxicillin—current use in swine medicine. Journal of veterinary pharmacology and therapeutics 2018, 41(3):356-68
  13. Huttner A, Bielicki J, Clements MN et al. Oral amoxicillin and amoxicillin–clavulanic acid: properties, indications and usage. Clinical microbiology and infection 2020, 26(7):871-9
  14. Environmental Protection Agency. The Quality of Bathing Water in Ireland (2002)(Internet). Ireland: Environmental Protection Agency; 2003 [cited 2022 April 6th]. Available from: https://www.epa.ie/publications/monitoring--assessment/freshwater--marine/EPA_quality_bathing_water_2002.pdf
Readers questions and answers

1. How many Genera of PCV virus have been identified to date?

A. 3

B. 2

C. 4

D. 1

2. Mutations in the PCV virus are most commonly seen in which gene which encodes for the PCV2 protein coat?

A. Open Reading Frame 2 (ORF2) gene

B. Open Reading Frame 1 (ORF1 ) gene

C. Open Reading Frame 3 (ORF3) gene

D. E2 Ubiquitin Ligase

3. What percentage of PCV2 field strains are recombinant viruses?

A. 10%

B. 22%

C. 40%

D. 33%

4. Subclinical disease is the most common form of PCV2 infection.  True or False?

A. True

B. False

5. Which genotype of PCV2 form the basis of the vaccines produced before 2021?

A. a

B. d

C. g

D. h

Answers: 1C; 2A; 3D; 4A; 5A.