Dangerous Dublin: The Virulent Salmonella

1
Contact Details Salmonella Reference
Laboratory UCHG 091-544628 e.mail
colette.ohare_at_nuigalway.ie
Dangerous Dublin The Virulent Salmonella Colette
O Hare1, Conor Burke2, Suzanne Gray3, Niall
Delappe1, Ger Doran1, Grainne McHale2, Dr. Gerard
Solan2 and Prof. Martin Cormican3 1 Salmonella
Reference Laboratory, Medical Microbiology, UCHG.
2 Medical Microbiology, Mayo General Hospital. 3
Medical Microbiology, UCHG

• RESULTS
• Of the S. Dublin human isolates (n34) 29 were
  isolated from non-fecal samples blood (n10),
  urine (n1), CSF (n1) and pleural fluid (n1).
  
• 82 of non-human S. Dublin isolates were from
  bovine origin (incl milk/cheese)
• 35 of S. Dublin isolates were from children
  under 5 years of age (n12) and 35 from adults
  over 60 years of age (n12). Table 1
• 12 (n7) of isolates had resistance to one
  antibiotic.
• No C8 esterase activity, as indicated by clear
  colony colour growth was obtained from 36
  non-duplicate isolates of S. Dublin on ASAP agar
• 10/14 S. Dublin isolates showed presence of the
  80kb serovar specific plasmid (ssp). Figure 1
• Six banding patterns (A-A5) were observed (one
  to two band differences) for 28 human S. Dublin
  isolates using XbaI endonuclease. 78.5 were
• pattern A. Figure 2

ABSTRACT Salmonella Dublin is a particularly
virulent serovar of Salmonella enterica. It is
frequently recovered from cattle, but can be
transferred to humans via meat and dairy
products. In early 2003 Salmonella enterica was
isolated from CSF and blood samples from a 1
month-old baby in a General Hospital. The
isolates were sent to the Salmonella Reference
Laboratory. Serotyping was performed by standard
methods and susceptibility to 15 antimicrobial
agents tested by the NCCLS disc diffusion method.
These isolates and other Salmonella Dublin
isolates were subcultured to ASAP agar (Aes
Laboratoire Salmonella agar) and were typed by
Pulsed Field Gel Electrophoresis (PFGE) using the
Pulse-Net protocol. Isolates were examined for
plasmids by the methods of Birhboim and Doly.
Serotyping confirmed the isolates as Salmonella
Dublin (9,12g, p). Isolates were susceptible
to all 15 antimicrobial agents tested. All
Salmonella Dublin isolates failed to produce the
expected pink/purple colonies on ASAP agar. On
Pulsed Field Gel Electrophoresis (PFGE) the two
isolates from the infant were indistinguishable
from each other and indistinguishable from an
isolate submitted from the Eastern region in
2001. A 80 kb plasmid, known to be associated
with virulence in Salmonella Dublin was detected
in most isolates. Salmonella Dublin was
confirmed as the cause of blood stream infection
and meningitis in the infant (isolate number
170/03). ASAP agar does not reliably detect
Salmonella Dublin. PFGE allows discrimination of
subtypes within the serovar Salmonella Dublin
although the difference are relatively minor (1
to 2 band differences) in most cases.
INTRODUCTION Salmonella enterica is a major
human pathogen. More than 2400 serovars are
recognised (1). Human infection with most animal
serovars is associated with acute self-limiting
diarrhoea, however in the very young, the aged
and other vulnerable individuals, life
threatening invasive infection may occur. At
present Salmonella enterica serovar Enteritidis
and Salmonella enterica serovar Typhimurium are
most common in Western Europe (2) and accounted
for the majority of isolates in Ireland from
1996-2002 (3, 4). Salmonella Dublin is
particularly associated with invasive disease and
is frequently isolated from blood of infected
patients. Its antigenic components include the
Vi (virulence) antigen, which is also commonly
found in Salmonella enterica serovar Typhi. From
2000-2003, 56 isolates of S. Dublin were received
in the Reference Laboratory from humans and
food/animals. This study was proposed to
investigate both phenotypic and genotypic typing
methods for S. Dublin
A

• MATERIALS AND METHODS
• 34 human and 22 non-human Salmonella Dublin
  isolates submitted from 2000-date
• Susceptibility testing against 15 antibiotics
  (Oxoid) using the NCCLS disc diffusion method (5)
• C8 esterase activity on ASAP agar (AES
  Laboratoire) determined by colony colour from
• growth aerobically at 37oC (6)
• Plasmid isolation using the Birhboim and Doly
  method (7)
• PFGE using PULSENET protocol developed in
  CDC, Atlanta, USA

A1
A2
A3
A4
A5
Figure 2. PFGE dendogram analysis of clinical S.
Dublin isolates

• CONCLUSIONS
• S. Dublin cause high percentage of invasive
  disease
• High numbers of S. Dublin isolated from
  patients at extremes of age
• Low levels of single antimicrobial
  resistance.
• No multiple antimicrobial resistance carraige
• Plasmid analysis poor discriminatory tool for
  genotyping
• PFGE good discrimination for genotyping

Figure 1. Plasmid analysis of S. Dublin isolates
(lanes 2-5, 9-11). Control plasmid size markers
(Lanes 6-8 62Mdal, 72Mdal, 166Mdal).
Supercoiled marker (lanes 1, 12)
Table 1.
REFERENCES (1) Popoff MY, Le Minor L. (1997)
Antigenic formulae of the Salmonella serovars. 7e
rev ed (2) EnterNet. (2002) Enter-net Quarterly
Salmonella reports. (3) Cormican M, Butler C,
Morris D, Corbett-Feeney G, Flynn J. (1998)
Antibiotic resistance amongst Salmonella enterica
species isolated in the Republic of Ireland. J.
Antimicrob. Chemother. 42(1)116-118. (4) OHare
C, Doran G, Delappe N, Morris D, Buckley V,
Corbett-Feeney G, Cormican M. Antimicrobial
resistance and phage types of human and non-human
Salmonella enterica isolates in Ireland from
1998-2002. Submitted to CDPH (5) NCCLS. (2002)
Performance standards for antimicrobial
susceptibility tests, sixth edition In CDSC,
NI (6) Gray S, Clancy J, O Hare C, Doran G,
Cormican M. (2003) Failure to detect Salmonella
enterica serovar Dublin on Aes Laboratoire
Salmonella agar plate. J. Clin. Microbiol. 41
(8) 4003 (7) Birhboim H. C, Doly J. (1979) A
rapid extraction procedure for screening
recombinant DNA. Nucleic Acids Res. 1513-1523

ACKNOWLEDGMENTS We would like the thank Dr.
Gabriel Fox, Mayo General Hospital for his case
history