Salmonella enterica Bredeney: Third commonest cause of human infection in Ireland in 1999

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Salmonella enterica Bredeney Third commonest
cause of human infection in Ireland in 1999

• C. A. O Hare, M. Cormican. G. Corbett-Feeney, S.
  Fanning and J. Moore.

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Abstract
Salmonella enterica is an important human
pathogen. In 1999 three serotypes predominated
amongst strains submitted for typing to the
Interim National Salmonella Reference Laboratory.
As in Europe generally, S. Typhimurium (42) and
S. Enteritidis (33) predominate. Salmonella
Bredeney is not a common human pathogen in most
countries. However isolates of this serotype
accounted for 8 and 17 of the total number of
Salmonella enterica isolates received in 1998 and
1999 respectively. We have performed a detailed
analysis of a collection of 112 isolates of S.
Bredeney from humans (n52), animals (n40) and
food (n20) sources from throughout the island of
Ireland. Antimicrobial susceptibility testing
was performed by the disk diffusion method of the
National Committee for Clinical Laboratory
Standards (NCCLS). Activity of ten antibiotics
were assessed (ampicillin 10mg, chloramphenicol
30mg, sulphonamides 300mg, streptomycin 10mg,
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Abstract
tetracycline 30mg, trimethoprim 5mg, nalidixic
acid 30mg, kanamycin 30mg, ciprofloxacin 5mg and
nitrofurantion 300mg (Oxoid). Pulsed field gel
electrophoresis was performed with digestion of
whole chromosomal DNA with XbaI, and DNA
amplification fingerprinting (DAF) were performed
on all isolates. Resistance to sulphonamide and
trimethoprim (SuTm) was observed in one human
isolate (1), 20 animal isolates (18) and 3 food
(3) isolates. Eight distinct PFGE patterns (3
or more band differences) were observed with a
single pattern accounting for 86 of strains.
Nine distinct patterns were observed by DAF.
Groupings identified by DAF were in close
agreement with those identified by PFGE.
Resistance to SuTm was associated with PGFE type
I and DAF types I and II. These results indicate
that most isolates of S. Bredeney from human,
food and animal sources are closely related. It
is interesting however that antimicrobial
resistance was observed much more frequently in
isolates from animal than from human sources.
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Introduction

• Salmonella enterica an important human and
  animal pathogen
• In humans it is most commonly associated with
  self-limiting gastroenteritis- Infection may be
  invasive and life-threatening in vulnerable
  groups
• Serotypes most often associated with human
  infection in Ireland
• S. Typhimurium (42 isolates, 1999)
• S. Enteritidis (33 isolates, 1999)
• S. Bredeney (12 isolates, 1999)
• In Ireland, S. Bredeney isolate numbers rose
  from less than 2 in 1997 to 12 in 1999. No
  corresponding increase was reported elsewhere in
  Europe.
• S. Bredeney in USA is a rare cause of food
  poisoning. Less than 0.1 of isolates to CDC
  annually.

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Introduction

• Only three major outbreaks reported since S.
  Bredeney was
• typed by Kauffmann in 1937.
• 1983 Brazil (ref)
• 1995 Austrailia (ref)
• 1998 Alabama, USA (ref)
• Italian study (ref) found that S. Bredeney
  isolates had a higher
• MIC to mercury based disinfectants than other
  serovars
• Objective of study. To investigate the degree
  of diversity of
• S. Bredeney strains from Ireland using molecular
  and non-molecular
• methods.

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Materials and Methods

• Bacterial strains 112 isolates of S. Bredeney
  from Ireland and Northern Ireland. Sources
  included human, animal and food
• Antibiotic sensitivity testing Disk diffusion
  methods of the NCCLS (1).
• Pulse field gel electrophoresis Plugs cut
  with XbaI. 1.3 agarose gel ran for 17h at 160V
  with a ramp switch of 5-50s at 8oC.
• Plasmid DNA prepared by the method of Kado and
  Lui (2). Plasmid DNA ran on 0.7 gel at 70V for
  2h.
• DNA amplification fingerprinting 10-mer primer
  P1254 used to PCR. samples ran on 1 agarose gel
  at 100V for 90min.

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Table 1 Origin of Salmonella Bredeney isolates
Isolate source Number of isolates (ntotal
no) antibiotic resistant Human
(n52) 1 Poultry1 (n27) 16 Porcine
(n1) 1 Bovine2 (n9) 3 Deer
(n3) 2 Food3 (n20) 4
23/25 isolates had resistance to sulphonamides
and trimethoprim (SuTm) 1one isolate tet only, 1,
2one amp only, 3 one ASSuTTm
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Figure 1 DNA Amplification fingerprinting (DAF)
patterns of Salmonella Bredeney isolates
Lanes M molecular weight markers
0.56-21.2kb 1. S. Typhimurium DT104 control 7.
B039 DAF II 13. B061 DAF V 2. B003 DAF
I 8. B070 DAF II 14. B072 DAF V 3. B037
DAF I 9. B053 DAF III 15. B058 DAF V 4.
B062 DAF I 10. B056 DAF III 16. B052 DAF V 5.
B024 DAF VII 11. B054 DAF IV 17. B023 DAF
VI 6. B001 DAF II 12. B067 DAF III 18. B071
DAF IX
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Figure 2 PFGE types I to VIII of Irish
Salmonella Bredeney isolates
1 2 3 4 5 6 7 8 9 10 11
Lanes 1. Concatamer l ladder 2. PFGE type I 3.
PFGE type I 4. PFGE type II 5. PFGE type III 6.
PFGE type I 7. PFGE type IV 8. PFGE type V 9.
PFGE type VI 10.PFGE type VII 11.Concatamer l
ladder
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Table 2 Distribution (n) of PFGE types among
sources of Salmonella Bredeney
PFGE type Human Poultry Bovine Porcine
Deer Food I 50 23 4 1
1 18 II 1 0 0
0 0 0 III 1
0 0 0 0 0 IV 0
2 4 0 0 0 V 0
0 0 0 1 0 VI
0 0 0 0 0
1 VII 0 0 0 0 0
1 none 0 2 1
0 1 0
Samples gave the appearance of
smearing/degradation on PFGE analysis. Repeated
in triplicate.
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Figure 3 Plasmid analysis of S. Bredeney
Lanes 1. Clinical isolate 2. Clinical
isolate 3. Clinical isolate 4. Clinical
isolate 5. Poultry isolate (SuTm) 6. Poultry
isolate 7. E.coli 36R891 marker 8. Clinical
isolate 9. Food isolate (SuTm) 10 Food
isolate 11.Clinical isolate Resistance to
Sulphonamides and tetracycline
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Results

• Seven different PFGE types observed (I to VII)
• 1 PFGE type accounted for most isolates (n97)
  and
• predominates in humans, animals and food
• Nine DAF patterns observed (I to IX). Very
  good agreement between
• PFGE and DAF patterns.
• Antimicrobial resistance much more common in
  poultry.
• Isolates resistant to ampicillin only or
  ASSuTTm, were assigned to PFGE types IV, VII or
  were untypable by PFGE (DAF V)

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Results

• Small plasmids of similar size (gt20kb) were
  observed in eight out
• of ten isolates of PFGE Type I.
• Observed plasmid carriage was not always
  associated with antibiotic
• resistance.

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Bibliography
1. National Committee for Clinical Laboratory
Standards (NCCLS) (1997). Performance standards
for antimicrobial susceptibility tests, sixth
edition approved standard. M2-A6. Villanova,
PA NCCLS. MARTIN - TO BE DONE TOMORROW