NATIONAL SALMONELLA, SHIGELLA & LISTERIA REFERENCE
LABORATORY OF IRELAND
(HUMAN HEALTH)
ANNUAL REPORT FOR 2013
NATIONAL SALMONELLA, SHIGELLA & LISTERIA REFERENCE LABORATORY
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Introduction
The National Salmonella, Shigella & Listeria Reference Laboratory was established in 2000 with
support from the Department of Health and Children to provide reference services related to human
health. It is a public service laboratory which currently operates with 1.8 WTE scientific staff
representing a reduction from 3 staff working in the service until 2011. The service operates from the
Department of Bacteriology at the Clinical Science Institute, NUIGalway. The NSSLRL website is at
http://www.nuigalway.ie/salmonella_lab/. The reference laboratory uses a number of phenotypic
methods (serotyping, antibiotic-resistance testing and phage typing) and molecular methods (pulsefield gel electrophoresis (PFGE) and multi-locus variable number tandem repeat analysis (MLVA) to
precisely characterise Salmonella isolates. This process can be considered as genetic fingerprinting of
Salmonella. The goal of this genetic fingerprinting is to assist relevant agencies in protecting public
health by identifying and interrupting chains of transmission of Salmonella infection. In addition the
NSSLRL provides typing services for Shigella species and Listeria monocytogenes.
One of the interesting features in recent years is an apparent trend towards decline in the proportion of
human S. Typhimurium isolates accounted for by DT104 and DT104b. Theis apparent trend has not
been tested for statistical significance
One of the major challenges that NSSLRL will need to address in the near future is the transformation
of molecular typing methods. It appears increasingly likely that whole genome sequencing is rapidly
being accepted as a key method in molecular typing of pathogens. In 2013, NSSLRL continued to
explore ways in which it can develop and apply these methods within a very resource constrained
environment.
The Laboratory is committed to providing a high quality and timely service and has achieved
accreditation to the ISO15189 standard from the Irish National Accreditation Board (INAB).
The
continued success of the laboratory is entirely dependent on the support of the staff in the laboratories
that submit isolates for typing. My colleagues and I appreciate that the preparation, packing and
dispatch of isolates is a significant burden and would like to thank you for your support over the years.
I would also like to acknowledge the support of all those agencies with whom we work closely to ensure
that the service we provide works as information for action. In particular I would like to thank
Galway University Hospitals, NUI Galway, the Food Safety Authority of Ireland, the Health Protection
Surveillance Centre and colleagues in Public Health Departments and Environmental Health
2
Departments throughout the country and to acknowledge the work of colleagues in the National
Reference Laboratory Salmonella (Food, Feed and Animal Health)1.
If you have any comments or questions arising from the report please feel free to contact me at the
email address given below.
Martin Cormican
Director National Salmonella, Shigella & Listeria Reference Laboratory
martin.cormican@hse.ie
1.
The Annual Report of National Reference Laboratory Salmonella (Food, Feed and Animal
Health is available at
http://www.agriculture.gov.ie/media/migration/animalhealthwelfare/labservice/nrl/NRLSalmo
nellaAnnualReport2011.pdf
3
Salmonella
In 2013, 825 isolates were submitted to the National Salmonella, Shigella & Listeria Reference Laboratory.
When non-Salmonella, QC, contaminants and duplicate isolates were removed a total of 657 Salmonella
isolates were typed. This represents an 8% decrease in the number of isolates received in 2012.
There were 345 human clinical isolates, including 319 faecal isolates, 18 from blood (including 7 S.Typhi, 2
S.Paratyphi A and 1 S.Paratyphi B), 2 other invasive isolates, and 6 urine isolates. S.Typhimurium (n = 76)
and its monophasic variant 4,[5],12:i:- (n = 53) and S.Enteritidis (n = 49) predominated (Table 2). There was
marked seasonal variation with the highest number of isolates occurring in months May to October. This
coincides with the warmer months of the year and with the peak season for foreign travel (Fig.1) and may be
related in part to one or both of these factors.
Table 1: Number of Salmonella isolates received in NSSLRL
Year
Human
Non-human
2013
345
312
2012
319
391
2011
320
381
2010
364
559
2009
364
368
2008
447
815
2007
457
653
2006
430
308
2005
357
494
2004
420
650
2003
486
634
2002
394
540
2001
508
574
2000
636
214
In some cases more than one isolate was received from a patient.
For example we may have received an
invasive isolate (e.g. from a blood culture) and an isolate from faeces from the same patient. Where invasive
and faecal isolates come from the same patient, only the invasive isolate is recorded to avoid duplication. The
average turnaround time for human isolates was 7 days (range 2-18 days). The number of human Salmonella
isolates received is now just over half that observed when the laboratory was established in 2000 (Table 1)
4
and has been sustained at that level for some years. This reduction in number of isolates reflects a true
reduction in the incidence of human salmonellosis and represents a significant public health achievement by
those agencies working in this area.
Table 2: Top 14 serotypes of Human Isolates (inc typhoidal)
Serotype
Frequency
%
76
22.0
53
15.0
Enteritidis
49
14.2
Infantis
14
4.1
Dublin
12
3.5
Newport
11
3.2
Typhi
9
2.6
Agama
6
1.7
Stanley
5
1.4
Anatum
5
1.4
Montevideo
5
1.4
Virchow
5
1.4
Kentucky
4
1.2
Braenderup
4
1.2
Others
87
25.2
Total
345
100
Typhimurium
Monophasic Typhimurium
1
1
The antigenic formula 4,[5],12:i:- is that of S. Typhimurium except that the phase 2 antigen is not expressed.
These isolates are generally referred to as monophasic S. Typhimurium and are so called in this report.
Figure 1
Seasonal Variation in numbers of Salmonella isolates 1
5
60
50
40
30
20
Total
Ent
Typ/4,5.12:i
FT
10
0
1.
The line “FT (Foreign Travel)” describes the number of cases of salmonellosis for which an association
with recent foreign travel was reported to NSSLRL.
Reporting of recent foreign travel is likely to be
incomplete.
It is important to note that there is always an interval gap between the time of onset of symptoms and date of
isolate receipt in the NSSLRL. This includes time taken for patient to access doctor, taking and transporting
the sample to the primary laboratory, isolation of Salmonella, and referral to NSSLRL. Fig 1 refers to date
of receipt in the NSSLRL.
Salmonellosis non-typhoidal
S.Typhimurium and its monophasic variant.
S.Typhimurium 4,[5],12;i:2 and its monophasic variant (4,[5],12;i:-) together accounted for 37.4% of all cases
of Salmonella. Phage type DT193 was the most common (30.2%) phage type among human isolates in 2013.
Phage types Untypable* accounted for 14%, DT104b 8.5%, DT104 6.2% and DT120/DT120 low accounted
for 5.4% of the total number of S.Typhimurium/4,[5],12:i:- isolates. The proportion of S. Typhimurium
isolates accounted for by phage type DT104b at 8.5% was somewhat higher than in the previous 2 years
(2012, 4.9% and 2011 3.5%) but remains below that observed in in 2010 and years prior to that [2010 (11%),
2009 (12%), 2008 (20%), 2007 (12%)]. The proportion of isolates accounted for by phage type DT104 at
6.2% has also decreased in recent years [2012 (11.4%), 2011 (23.5%), 2010 (19.7%), 2009 (20.3%), 2008
(20.1%), 2007 (18.4%)].
* Does not react with typing phages.
6
S.Enteritidis
S.Enteritidis accounted for 14.2% of all cases of Salmonella. The predominant phage types were PT14b
(22.4%), PT1 (16.3%), PT8 (16.3%) and PT4 (10.2%).
Salmonellosis Typhi and Paratyphi
Nine isolates of S.Typhi, 3 isolates of S.Paratyphi A and 2 S.Paratyphi B isolate were received. A history of
recent travel was recorded for 8 of the S.Typhi isolates; 7 to the Indian subcontinent and 1 to Nigeria. Two of
the S.Paratyphi A isolates were associated with recent travel, one to Pakistan and one to Indonesia. Both
S.Paratyphi B isolates had a history of travel to Bolivia.
Antimicrobial resistance
Fifty-six percent of isolates (n = 193) were susceptible to all antimicrobial agents tested. Thirty percent of
isolates (n = 103) were multi-drug resistant (three or more different classes of antibiotics). Of the 30% of
isolates that were multi-drug resistant, 41% (n = 42) had the profile of resistance to ampicillin, streptomycin,
sulphonamide and tetracycline (ASSuT) and were mainly monophasic S. Typhimurium. Overall the pattern
is similar to that observed in previous years.
Five extended spectrum beta-lactamase (ESBL) producing isolates were detected. ESBL producing isolates
included a patient with travel history to India (S. Stanley), two patients with travel to Thailand (Typhimurium
and monophasic Typhimurium- both with ciprofloxacin resistance), a patient with travel history to Morocco
(Typhimurium) and 1 patient with travel to Cuba (S.Muenchen). Two AmpC producing S. Typhimurium
(phage types DT135 and DT104b) isolates were received from patients with no histories of foreign travel.
Five isolates of Salmonella resistant to ciprofloxacin were detected, all with travel history to the Far East.
These included a S.Typhi from a patient who visited India and a S.Kentucky from a male with travel history
to Nepal. Resistance to ciprofloxacin (based on widely used interpretive criteria up to and including 2013) is
rare among Salmonella but a ciprofloxacin-resistant S. Kentucky clonal group has arisen and spread from
North Africa in the last decade. Two ESBL producing (S.Typhimurium and monophasic S.Typhimurium)
isolates with resistance to ciprofloxacin were received from patients with travel history to Thailand. One
patient with travel to China had a ciprofloxacin resistant monophasic Typhimurium.
In addition 50 isolates resistant to nalidixic acid and with reduced susceptibility to ciprofloxacin (minimum
inhibitory concentration of > 0.06 mg/l) were detected, compared with 39 in 2012. It is worth noting that the
EUCAST interpretive criteria for resistance to ciprofloxacin for Salmonella changed on January 1st 2014 and
isolates with ciprofloxacin MIC of > 0.06 mg/l are now reported as resistant. By current EUCAST criteria
therefore 15.9% (n = 55) of Salmonella isolates from 2013 would be considered ciprofloxacin-resistant.
7
Ciprofloxacin
Figure 2. Ciprofloxacin MIC Distribution of Salmonella from humans typed in NSSLRL in 2013 as
performed using TREK Sensititre.
Travel related infection
A history of recent foreign travel was recorded in 38 % (n = 132) of human cases of infection (Table 3).
Ireland was noted as the country of infection in 43.2 % (n = 149) of cases while 18.5 % (n = 64) had no
country of infection recorded. Spain, Thailand, India and Pakistan were the most commonly recorded travel
destinations. S. Enteritidis accounted for a high proportion of isolates associated with travel to Spain (38.5%)
and all isolates from Turkey (100%) while S. Stanley was strongly associated with travel to Asia (80% (4/5)
Stanley isolates were from Asia). Thirty-two (65 %) S.Enteritidis isolates were associated with foreign travel
compared to 21.7 % for S. Typhimurium and its monophasic variant combined. Although NSSLRL does not
have access to data on the number of Irish people who travel to each country it is likely that the number of
cases associated with each country is at least in part accounted for by the popularity of the country as a
destination.
8
Table 3: Foreign travel history for Salmonella isolates
Continent
Country
Number
Spain
26
Turkey
4
Poland
4
Portugal
4
France
3
Italy
2
Germany
1
Romania
1
UK
1
Lithuania
1
Belarus
1
Spain/Germany
1
Spain/Italy
1
Nigeria
7
Morocco
4
Sudan
1
Tanzania
1
Ghana
1
Egypt
1
Angola/Oman
1
Europe (n = 50)
Africa
(n = 16)
Australasia (n = 53)
Thailand
14
India
11
Pakistan
11
Indonesia
3
China
2
The Philippines
2
Bangladesh
1
Vietnam
1
Cambodia
1
Nepal
1
Lebanon
1
9
Bahrain
1
Indonesia/Hong Kong
1
Thailand/Cambodia
1
Thailand/U.A.E.
1
U.A.E.
1
Americas (n = 12)
Foreign travel
Mexico
3
Bolivia
2
Cuba
2
South America
1
Puerto Rico
1
Brazil/Argentina
1
Jamaica
1
Trinidad
1
Country unknown (n = 1)
* Country not stated
Clusters
Twenty-one clusters of cases involving 64 isolates were identified in 2013. S. Typhimurium/monophasic S.
Typhimurium was involved in 10 clusters (29 isolates) while S. Enteritidis was implicated in 1 cluster (2
isolates).
Six of the clusters (15 patients) were family outbreaks, i.e. all patients affected were from one family.
The outbreak of S. Typhimurium phage type DT8 that began in August 2009 continued through 2010 and
2011. This phage type is associated with ducks and several of the cases were linked epidemiologically with
ducks or consumption of duck eggs. However the number of DT8 isolates from human cases typed in the
NSSLRL decreased from 28 in 2010 to 9 in 2011, just 2 in 2012 and 3 in 2013. This suggests that the control
procedures put in place are working, leading to reduced burden of human infection from this source.
The NSSLRL liaises with the European Centre for Disease Control (ECDC) in the investigation of outbreaks
that may have an international dimension. PFGE images and analysis from Salmonella and Listeria isolates
are also uploaded to a centralised ECDC database where they can be compared with isolates from other
countries to check for matches.
10
Evidence of Links between S. Typhimurium in Humans, Food and Animals.
Multi locus variable number tandem repeat analysis (MLVA) is a relatively new technology that allows for
very fine discrimination between isolates that appear very closely related by other methods including PFGE.
This precise discrimination has proved a useful technique in showing persuasive evidence of association
between human S.Typhimurium clusters and individual cases and recent food and animal sources.
Table 4
illustrates evidence of association between human infection and food /food animal types.
The value of using standardised and readily comparable methods for “fingerprinting” human, food and animal
isolates of Salmonella not just within Ireland but across Europe and the world is illustrated by this body of
work. The laboratory analysis is most valuable when combined with relevant epidemiological data to support
links with particular sources of infection.
Table 4 MLVA profiles of human and food/animal isolates
Isolate no.
Source
Phage type
Resistance
MLVA profile
Typhimurium
(n = 5)
Human
Untypable
none
2-15-16-11-212
(n = 13)
Chicken
Untypable
not tested
2-15-16-11-212
(n = 2)
Bovine
Untypable
none
2-15-16-11-212
(n = 2)
Human
Untypable
none
2-15-16-NA-212
(n = 1)
Bovine
Untypable
none
2-15-16-NA-212
(n = 4)
Human
Untypable
none
2-15-17-11-212
(n = 1)
Chicken
Untypable
not tested
2-15-17-11-212
(n = 1)
Human
DT104
ACSSuT
3-13-24-13-311
(n = 2)
Bovine
DT104
ACSSuT
3-13-24-13-311
(n = 3)
Human
DT104b
ACSSuT
3-14-15-23-311
(n = 1)
Swine
DT104b
not tested
3-14-15-23-311
(n = 3)
Human
DT120/DT120 low ASu
3-15-8-14-311
(n = 1)
Swine
DT120 low
not tested
3-15-8-14-311
(n = 1)
Human
DT104b
ACSSuT
3-15-NA-11-311
11
(n = 1)
Swine
DT104b
not tested
3-15-NA-11-311
(n = 3)
Human
DT104b
ACSSuT
3-15-NA-12-311
(n = 1)
Human
DT104b
SSu
3-15-NA-12-311
(n = 1)
Swine
DT104b
not tested
3-15-NA-12-311
(n = 3)
Human
DT10var
none
3-17-NA-NA-311
(n = 1)
Human
RDNC
none
3-17-NA-NA-311
(n = 1)
Swine
RDNC
not tested
3-17-NA-NA-311
(n = 1)
Human
U302
ACSSuT
3-5-NA-14-311
(n = 1)
Swine
U302
not tested
3-5-NA-14-311
(n = 1)
Human
U310
none
4-12-8-8-211
(n = 4)
Bovine
U310
none
4-12-8-8-211
(n = 3)
Human
DT193
ASSuT
3-12-12-NA-211
(n = 4)
Bovine
DT193
ASSuT
3-12-12-NA-211
(n = 2)
Human
DT120
SSuT
3-12-9-NA-211
(n = 1)
Bovine
DT120
SSuT
3-12-9-NA-211
(n = 1)
Swine
DT120
not tested
3-12-9-NA-211
(n = 2)
Human
DT193
ASSuT
3-13-11-NA-211
(n = 1)
Bovine
DT193
ASSuT
3-13-11-NA-211
(n = 1)
Swine
DT193
not tested
3-13-11-NA-211
(n = 1)
Human
DT193
ASSuT
3-13-8-NA-211
(n = 2)
Swine
DT193
not tested
3-13-8-NA-211
(n = 1)
Human
DT193
ASSuT
3-13-9-NA-211
(n = 1)
Bovine
DT193
ASSuT
3-13-9-NA-211
(n = 1)
Swine
DT193
not tested
3-13-9-NA-211
(n = 1)
Human
DT193
ASSuT
3-14-10-NA-211
(n = 2)
Bovine
DT193
ASSuT
3-14-10-NA-211
4,[5],12:i:-
12
(n = 1)
Swine
DT193
not tested
3-14-10-NA-211
(n = 1)
Human
DT193
ASSuT
3-14-9-NA-211
(n = 1)
Human
DT193
ASSu
3-14-9-NA-211
(n = 6)
Bovine
DT193
ASSuT
3-14-9-NA-211
(n = 1)
Swine
DT193
not tested
3-14-9-NA-211
NT = Not tested
Some of the animal isolates have not been serotyped in the NSSLRL
Animal Contact
A history of animal contact was recorded for 103 patients with salmonellosis including contact with snakes,
lizards, turtles, birds, fish, horses, dogs and farm animals (Table 5). Dogs were the most common contact
animal (n = 61) while contact with cats was less common (n = 25). This is broadly similar to 2012.
In some cases typing of isolates from patients and individual animals they had contact with had the same
phenotype, e.g. S. Give isolate from a baby and a pet lizard.
Other strong links included single cases of S. subsp IIIa 44:z4,z23,z32:- and S. Muenchen and snake contact;
contact with lizards and a case of S. subsp IV 16:z4,z32:-.
Although public information on the risk (particularly to children) of contact with reptiles has been circulated
(http://www.fsai.ie/WorkArea/DownloadAsset.aspx?id=11207) it appears that this may not be reaching
relevant sections of the population or may not have resulted in modification of risk behaviour.
It may be
appropriate to consider if further steps to limit exposure of children to risk of salmonellosis from contact with
reptiles and other exotic pets is appropriate. In total 19 isolates of Salmonella (approx. 5.5 % of all human
cases) were associated with contact with exotic animals.
Among people that had a recorded history of living on farms or working with farm animals (n = 18), S.
Typhimurium/monophasic S. Typhimurium predominated (n = 13).
Many of the patients that had a history of animal contact also had other risk factors, e.g. recent history of
foreign travel, consumption of particular food products, etc. It is important to note that Salmonella is
primarily a foodborne disease and that contact with animals such as dogs and cats is very common in the
general population therefore contact with an animal species should not be taken to indicate that the animal is
the likely source of infection.
13
Table 5: Animal contact history or strains associated with animal contact
Source
Sub1.
Strain
Animal Contact
MS130312
Human
I
Muenchen
Snake
MS130779
Human
IIIa
44:z4,z23,z32:-
Snake
MS130085
Human
IV
16:z4,z32:-
Lizard
MS130439
Human
I
Oranienburg
Lizard, snakes, dog
MS130436
Human
I
Oslo
Bearded dragon, snakes
MS130714
Human
I
Give
Lizards, tropical fish, dogs
MS130241
Human
I
Braenderup
Turtles
MS130357
Human
I
Typhimurium U296
Turtle & Goldfish
MS130531
Human
I
Typhimurium DT104b
Goldfish
MS130367
Human
I
Braenderup
Fishtank, cat, dog
MS130420
Human
I
Braenderup
Fishtank- tropical fish, cat,
MS130286
Human
I
Typhimurium Untypable
Budgie
MS130479
Human
I
4,[5],12:i:- DT193
Canaries
MS130610 *
Human
I
Newport
Parrot
MS130495 *
Human
I
Senftenberg
Elephants
MS130509 *
Human
I
Stanley
Elephants, cats
MS130799 *
Human
I
Agona
Elephants, tigers
MS130398 *
Human
I
4,[5],12:i:- U302
Zoo animals, horse
MS130510 *
Human
I
Colindale
Zoo in Nigeria
NSSLRL no.
Exotic animals
dog
Farm or Food animals (including horses)
MS130180
Human
I
Typhimurium DT8
Cattle, dog
MS130364
Human
I
4,[5],12:i:- DT193
Cattle, sheep, slurry
MS130429
Human
I
Typhimurium Untypable
Cattle, chicken factory
MS130492
Human
I
Typhimurium DT135
Cattle
MS130497
Human
I
4,[5],12:i:- DT193
Cattle, dog, cat
MS130637
Human
I
Agama
Cattle farm
MS130793
Human
I
Agama
Cattle farm, sheep, dog, cat
MS130796
Human
I
Typhimurium Untypable
Cattle, dog
MS130294
Human
I
Typhimurium Untypable
Sheep, dog
MS130080
Human
I
Typhimurium DT15A
Pony (sick), dogs
14
MS130264
Human
I
Typhimurium DT10 var
Horses, dogs, cats
MS130273
Human
I
Typhimurium DT193
Hens, goats, horses, dogs,
MS130545
Human
I
Anatum
Horses
MS130770 *
Human
I
Enteritidis PT21 var
Farm animals
MS130802
Human
I
Dublin
Farm animals
MS130022
Human
I
Typhimurium Untypable
Hens
MS130305
Human
I
4,[5],12:i:- DT193
Hens, ducks
MS130486 *
Human
I
Typhimurium DT193
Chickens, turkeys, cat
MS130002
Human
I
Gaminara
Dog
MS130011
Human
I
4,[5],12:i:- DT193
Dog
MS130012
Human
IIIb
53:z10:z35
Dog
MS130079 *
Human
I
Newport
Dog
MS130083
Human
I
Kasenyi
Dog, cat
MS130167 *
Human
I
Enteritidis PT1
Dog
MS130170
Human
I
4,[5],12:i:- DT193
Dog, cat
MS130176
Human
I
Typhimurium DT135
Dog (sick), cat
MS130225
Human
I
Enteritidis PT8
Dog
MS130228
Human
I
4,[5],12:i:- DT107
Dog- puppy
MS130265
Human
I
Typhimurium DT104b
Dog
MS130270
Human
I
Typhimurium DT104b
Dogs
MS130276
Human
I
4,[5],12:i:- DT120
Dog
MS130285
Human
I
Indiana
Dogs x 2
MS130366
Human
I
Typhimurium Untypable
Dogs
MS130395
Human
I
Typhimurium Untypable
Dog, bird
MS130399
Human
I
Telelkebir
Dog
MS130416
Human
I
4,[5],12:i:- DT193
Dog
MS130419
Human
I
Typhimurium DT135
Dog
MS130422
Human
IIIb
48:z:-
Dog
MS130444
Human
I
Typhimurium DT8
Dogs x 2
MS130483
Human
I
Typhimurium DT120 low
Dog
MS130489
Human
I
Typhimurium DT193
Dog
MS130496
Human
I
Typhimurium DT10
Dog
MS130502
Human
I
Montevideo
Dog
MS130507
Human
I
4,[5],12:i:- Untypable
Dogs, cats
MS130517
Human
I
4,[5],12:i:- DT193
Dogs, cats
MS130520 *
Human
I
Typhimurium DT136
Dog
petfood
Domestic pets
15
MS130523
Human
I
Typhimurium DT69
Dog
MS130533
Human
I
Enteritidis PT6
Dog
MS130552 *
Human
I
Enteritidis PT3
Dog
MS130574
Human
I
Stanley
Dogs
MS130577
Human
I
Enteritidis PT3
Dog
MS130584 *
Human
I
Enteritidis PT14b
Dog- puppy
MS130587
Human
I
Newport
Dog, cat
MS130602
Human
I
Typhimurium Untypable
Dog
MS130612
Human
IIIb
65:z10:e,n,x,z15
Pigeons, dogs
MS130613 *
Human
I
Enteritidis PT2
Dog
MS130635
Human
I
Typhimurium U302
Dog, rabbit
MS130665
Human
I
4,[5],12:i:- DT193
Dogs, oysters
MS130712
Human
I
Typhimurium DT104b
Dog
MS130734
Human
I
4,[5],12:i:- DT193
Dogs, pet rabbit
MS130742
Human
I
Typhimurium DT104
Dogs
MS130748
Human
I
Enteritidis PT24 var
Dogs
MS130763
Human
I
Typhimurium DT104
Dog
MS130780
Human
I
Enteritidis PT4
Dog
MS130788
Human
I
Enteritidis PT21
Dog
MS130801*
Human
I
Coeln
Dog
MS130817
Human
I
Typhimurium U287 var
Dog
MS130132
Human
I
4,[5],12:i:- DT193
Cats
MS130289
Human
IIIa
18:z4,z23:-
Cats
MS130303 *
Human
I
Stanley
Cat
MS130490
Human
I
Enteritidis PT14b
Cat
MS130566 *
Human
I
Montevideo
Cats
MS130572
Human
I
Typhimurium DT104b
Cats
MS130591
Human
I
4,[5],12:i:- DT193
Cats
MS130593 *
Human
I
Thompson
Cat
MS130699
Human
I
Dublin
Cat
MS130754
Human
I
Infantis
Cat
MS130755
Human
I
Dublin
Cat
MS130771
Human
I
Dublin
Cats
MS130666
Human
I
Typhimurium DT193
Pet Rabbit
MS130743
Human
I
Agama
Pets
MS130789
Human
I
Kottbus
Pet food
16
1. Sub = Subspecies. There are over 2500 Salmonella serotypes of which approximately 60% belong to
subspecies I. These account for about 99% of human infections. Subspecies I is present in both warm and
cold blooded animals while the other Salmonella subspecies are generally associated with cold-blooded
animals.
* Patient had history of recent foreign travel
Non-Human isolates
In 2013, 311 isolates of Salmonella of non-human origin were submitted to the NSSLRL. This represents a
decrease of 20 % in the number of non-human isolates received in 2012. The majority of isolates were from
swine (n = 138), bovine (n = 84) and poultry (n = 56) sources. S. Typhimurium/monophasic S. Typhimurium
(n = 56) were the most prevalent serovars (Table 6).
Table 6 Top 7 serotypes among non-human isolates
Serotype
Frequency
%*
Typhimurium
32
10.3
Monophas Typhimurium 24
Not serotyped
1
7.7
226
72.4
Dublin
8
2.6
Tennessee
5
1.6
Montevideo
3
1
S.subsp II 42:c:5
2
0.6
Others
11
3.8
Total
311
100
* Approximate figures
1
These are Typhimurium/monophasic S.Typhimurium (n = 223) and Enteritidis (n = 3) isolates received from
CVRL for phage typing only.
17
Salmonella serotypes and correlation with Human Infection
S.Typhimurium
S.Typhimurium and its monophasic variant 4,[5],12;i:- accounted for 89% of all non-human isolates and were
isolated from a variety of sources predominantly swine (n = 137) but also including poultry (n = 52) and
bovine (n = 70) sources. Phage types DT193 (n = 74), DT120/DT120 low (n = 24), DT104b (n = 15), U302
(n = 7), DT104 (n = 6) and Untypable (n = 3) were the most common phage types from swine. Phage types
DT104 (n = 17), DT193 (n = 18), Untypable (n = 12), U310 (n = 7) and DT104b (n = 5) were the most
common phage type among bovine isolates. Phage types Untypable (n = 31) and DT104 (n = 13) were the
most common phage types among poultry isolates. As illustrated in Table 4 (above) there is evidence from
molecular typing of a significant link between isolates from pigs and pig products and also with cattle and
human infection.
Salmonella Enteritidis.
Three S. Enteritidis isolates were received from non-human sources in the NSSRL in 2013, 2 from poultry
and 1 from avian source. One of these was from poultry imported from Asia. It is of interest that S. Enteritidis
is rarely detected from poultry or poultry products in Ireland but that S. Enteritidis is the first or second most
common serovar isolated from human infections each year. In this context it is worth noting that a history of
travel outside of Ireland is reported in relation to 65 % of S. Enteritidis cases compared with only 21.7 % of S.
Typhimurium/monophasic S. Typhimurium cases in 2013.
Sources of Isolates
Swine
A total of 3 serovars accounted for the 138 isolates received from swine with S.Typhimurium and its
monophasic variant 4,5,12:i:- (n = 137) accounting for the majority of isolates. Phage type DT193 was by far
the most frequent phage type (n = 74), while DT120/DT120 low (n = 24), DT104b (n = 15), U302 (n = 7) and
DT104 (n = 6) were also common in the swine isolates tested. These phage types were also typed from
human cases in 2013; DT193 (n = 39), DT120/DT120 low (n = 7), DT104b (n = 11), U302 (n = 3) and
DT104 (n = 8). MLVA typing provided further evidence of links between Salmonella in swine and human
infection (see Table 4).
Poultry
A total of 56 isolates comprising 3 serovars were received from poultry sources. As with swine S.
Typhimurium and its monophasic variant 4,[5],12:i:- (n = 55) were the most common serovars received. S.
Typhimurium isolates were from a variety of poultry sources including duck and chicken. Three isolates of S.
Enteritidis were received, a PT6a isolate from imported chicken from Asia, a PT6a isolate from poultry
(source not stated) and a single isolate of PT8 from an avian culture.
18
Isolates of all of these serovars were associated with human infections (S. Typhimurium/ monophasic
Typhimurium (n = 129), S. Enteritidis (n = 49), and S. Java (n = 2) in 2013).
Bovine
A total of 84 isolates were received from bovine sources and again S. Typhimurium and its monophasic
variant 4,[5],12:i:- accounted for the majority of isolates (n = 71) with DT104 (n = 17), DT193 (n = 18) and
Untypable (n = 12) being the most common phage types. These phage types were also received from human
cases of salmonellosis in 2013. MLVA typing provided further evidence of links between Salmonella in cattle
and human infection (see Table 4). There were also 7 S. Dublin isolates (and 12 human cases).
Figure 3
Fall in Salmonella Typhimurium DT104/DT104b isolates among isolates from swine
300
250
200
150
100
Total
DT104
DT104b
DT193
50
0
The fall in the number of isolates of S. Typhimurium DT104/DT104b in humans appears to correspond in
time with a decline in the proportion of S. Typhimurium from pigs accounted for by these phage types. This
may support the view that pigs/pig products are an important source of domestically acquired S.
Typhimurium in Ireland.
Antimicrobial Resistance among non-Human isolates
Antimicrobial susceptibility testing was performed on 84 of the 311 non-human isolates (227 were referred
for phage typing only). Of the isolates tested 54% (n = 45) were susceptible to all antimicrobial agents tested
while 44% (n = 37) were multi-drug resistant (three or more different classes of antibiotics). Seventeen
percent of isolates tested had the profile of resistance to ampicillin, chloramphenicol, streptomycin,
sulphonamide and tetracycline (ACSSuT) and most of these typed as S. Typhimurium DT104 or DT104b.
None of the isolates were resistant to ciprofloxacin; however one isolate exhibited resistance to nalidixic acid
and reduced susceptibility to ciprofloxacin. As discussed earlier in this report this isolate would now be
considered ciprofloxacin resistant by current EUCAST criteria.
19
Laboratory Contamination
False-positive Salmonella results due to laboratory cross-contamination are a serious problem for laboratories
and can be difficult to detect. Cross contamination in a laboratory can result in inappropriate diagnosis of
patient infection or in unfounded concerns regarding the safety of a food product. Detailed subtyping of
isolates by the NSSLRL helps in detection and confirmation of laboratory contamination incidents (Role of
Subtyping in Detecting Salmonella Cross Contamination in the Laboratory; BMC Microbiology: 9; 155).
There were 5 contamination incidents with human clinical samples where the contaminating isolates arose
from other patient’s samples while one laboratory had 2 food samples contaminated by the quality control
strain.
We would like to reiterate our request that all laboratories involved in testing Salmonella from any source use
Salmonella Nottingham NCTC 7382 as their positive control.
Listeria monocytogenes
Table 7: Number and serotypes of Listeria monocytogenes isolates from human sources received in
NSSLRL
Year
Total
4b
1/2a
1/2b
1/2c
2013
7
6
1
0
0
2012
8
5
1
2
0
2011
6
3
3
0
0
2010
4
2
1
0
1
2009
8
4
3
0
1
2008
14
9
4
0
1
2007
12
9
2
0
1
2006
1
0
1
0
0
2005
4
3
1
0
0
Listeria monocytogenes can be subdivided into 13 different serotypes based on their combinations of O and H
antigens. However serotypes 4b and the 1/2 group account for the vast majority of human infections.
20
Fig. 4
Pulse Field Gel Electrophoresis of L.monocytogenes isolates digested with ApaI
100
90
PFGE-ApaI
80
70
60
PFGE-ApaI
MQ130027
.Swine
.
.
1/2c
MQ130028
.Swine
.
.
1/2c
MQ130030
.Swine
.
food
.
1/2c
MQ130043
.Porcine
.
.
1/2c
MQ130014
.Cooked
.
crab
.
1/2c
MQ130037
.Blood
.
.
1/2a
MQ130034
.Shortbread
.
.
1/2a
MQ130059
.Bacon
.
Dinner
.
1/2a
MQ130060
.Bacon
.
Dinner
.
1/2a
MQ130061
.Bacon
.
Dinner
.
1/2a
MQ130001
.Meat
.
slicer
.
1/2a
MQ130031
.Swine
.
food
.
1/2a
MQ130035
.Shortbread
.
.
1/2a
MQ130036
.Shortbread
.
.
1/2a
MQ130013
.Cooked
.
turkey
.
Untypable
MQ130033
.Blood
.
.
4b
MQ130041
.Cooked
.
duck
.
4b
MQ130058
.Blood
.
.
4b
MQ130012
.Ham
.
& potato salad
.
4b
MQ130032/#1 .Blood
.
.
4b
MQ130026
.Blood
.
.
4b
MQ130029
.CSF
.
.
4b
MQ130042
.PM
. Pleural Swabs
.
4b
21
Fig. 5
Pulse Field Gel Electrophoresis of L.monocytogenes isolates digested with AscI
100
PFGE-AscI
90
80
70
PFGE-AscI
MQ130026 Blood
.
.
4b
MQ130029 CSF
.
.
4b
MQ130032 Blood
.
.
4b
MQ130042 PM
. Pleural Swabs
.
4b
MQ130033 Blood
.
.
4b
MQ130041 Cooked
.
duck
.
4b
MQ130012 Ham
.
& potato salad
.
4b
MQ130058 Blood
.
.
4b
The NSSLRL received 24 Listeria monocytogenes isolates in 2013. These included 7 human clinical isolates;
5 from blood cultures, 1 from CSF and 1 from placental swab (PM). Six of the isolates from humans typed as
serotype 4b and 1 typed as serotype 1/2a.
The majority of the food/animal/environmental isolates serotyped as 1/2a (n = 9), 5 typed as 1/2c and 2
isolates were serotype 4b.
The NSSLRL is working with colleagues in food and veterinary microbiology in Ireland and with colleagues
in Europe to build a library of typing data that may help to identify sources of human infection. A critical
limiting factor is the availability of human isolates for typing.
The total number of human clinical isolates in
Ireland per year is very small therefore it is critical that all such isolates are available for typing and we
appeal for all isolates to be forwarded for typing. Within the limited data available it is of interest that one
human case (MQ130033) is very similar to isolate to an isolated from cooked duck (MQ130041) by both
enzyme digest patterns although the NSSLRL is not aware that the food product is epidemiologically linked
with human infection.
22
Shigella species
Table 8: Number of Shigella isolates received in NSSLRL 2005-13
Year
Total
sonnei flexneri boydii
dysenteriae
2013
43
23
16
4
0
2012
20
12
6
2
0
2011
30
20
10
0
0
2010
39
17
18
3
0
2009
48
19
24
1
4
2008
43
22
16
5
0
2007
20
5
12
2
1
2006
20
7
12
0
1
2005
13
8
5
0
0
Shigella sonnei has only one serotype while the other Shigella species can be can be subdivided into a number
of different serotypes and subserotypes based on their lipopolysaccharide antigens.
Figure 6
Summary of Shigella isolates typed in NSSLRL from 2005-13
60 50 Total 40 S.sonnei 30 S.flexneri S.boydii 20 S.dysenteriae 10 0 2005 2006 2007 2008 2009 2010 2011 2012 2013 23
Fig. 7
Pulse Field Gel Electrophoresis image of S.sonnei digested with XbaI
100
PFGE-XbaI
95
90
85
PFGE-XbaI
MH130006
.
Africa
.
SSuTTm
MH130031/#1
.
.
ASSuTm
MH130033/#1
.
Ireland
.
SuTm
MH130035/#1
.
Ireland
.
SuTm
MH130036/#1
.
Ireland
.
SSuTm
MH130038/#1
.
Ireland
.
ASuTm
MH130023/#1
.
Yes-Unknown
.
SSuTTm
MH130068
.
Pakistan
.
SSuTTmNa
MH130049
.
Ethiopia
.
SSuTTm
MH130072
.
Ireland
.
STm
MH130004
.
Angola
.
ACSSuTTm
MH130024/#1
.
India
.
SSuTTmNaCp
MH130067/#1
.
Ireland
.
SSuTTmNaCp
MH130021/#1
.
Ireland
.
SSuTTmNaCp
MH130022/#1
.
India
.
SSuTTmNaCp
MH130032/#1
.
.
SSuTTmNaCp
MH130037/#1
.
India
.
SSuTTmNaCp
MH130045/#1
.
India
.
SSuTTmNaCp
MH130061
.
Germany
.
SSuTTmNaCp
MH130062
.
Ireland
.
SSuTTmNaCp
MH130063
.
Ireland
.
SSuTTmNaCp
MH130065/#1
.
Ireland
.
STTmNaCp
MH130047
.
India
.
ASSuTTmNaCpCtx
A total of 74 isolates were referred to the NSSLRL in 2013 for Shigella typing. When non-Shigella, QC and
duplicate isolates were removed a total of 43 Shigella isolates were typed. These included 23 S.sonnei, 16
S.flexneri and 4 S.boydii. The S.flexneri isolates were further divided into 2 S.flexneri 1b, 7 S.flexneri 2a, 1
S.flexneri 2b, 1 S.flexneri 3a, 1 S.flexneri 4a, 2 S.flexneri 6 and 2 S.flexneri X variants. This is the highest
number of Shigella species submitted since 2009. It is worth noting that laboratories that have changed to use
of direct molecular detection of pathogens in faces appear to have a striking increase in submission of
Shigella species for typing. This is most likely explained by the increased sensitivity of the method compared
to conventional culture. As many major laboratories have now changed to use of direct molecular detection it
seems likely that the increased in Shigella species submitted in 2013 is related, at least in part, to improved
ascertainment.
24
Eighty-four percent of isolates (n = 36) were multi-drug resistant (three or more different classes of
antibiotics). One ESBL producing Shigella sonnei was received in the NSSLRL in 2013 while 15 isolates
were resistant to ciprofloxacin (including the ESBL-producing isolate). The ciprofloxacin resistant isolates
included 12 Shigella sonnei, 5 of which were known to be associated with travel to India, and 3 S.flexneri
isolates, all with history of recent foreign travel, including, India (S.flexneri 6), Bangladesh (S.flexneri 2a),
and Estonia (S.flexneri 3a). The proportion of S. sonnei isolates resistant to ciprofloxacin is a real concern
given that this has increased significantly in recent years. As is clear from the PFGE image above, many of
the ciprofloxacin resistant isolates including some reported as associated with India, Germany and Ireland are
very closely related suggesting relatively recent dissemination of a ciprofloxacin-resistant clonal group. This
merits further study.
Twenty-four patients had a recorded history of recent foreign travel, including Europe (n = 3), Africa (n = 7)
and Australasia (n = 13). Fourteen patients had Ireland recorded as their country of infection while there were
no details for 5 patients.
Table 7: Foreign travel history for Shigella isolates
Continent
Africa
Country
Number
Nigeria
3
Africa
1
Angola
1
Ethiopia
1
Sudan
1
Estonia
1
Germany
1
Turkey
1
(n = 7)
Europe (n = 3)
Australasia (n = 13)
India
9
Pakistan
2
Bangladesh
1
U.A.E.
1
FT Yes-Unknown (n = 1)
25
There was 2 family outbreaks (n = 5) of Shigella flexneri 2a in 2013. There were also clusters of S.flexneri 1b
(n = 2) and S.sonnei ( n = 2).
26
NSSLRL Publications and Presentations 2013
Poster Presentations
PulseNet Multi-Locus VNTR Analysis MLVA Protocol for Salmonella enterica Serovar Enteritidis
complements Phage Typing. International Symposium Salmonella and Salmonellosis (I3S) Saint Malo,
France. May 2013.
PulseNet MLVA Protocol for Salmonella enterica Serovar Enteritidis applied to Salmonella enterica Serovar
Dublin. International Symposium Salmonella and Salmonellosis (I3S) Saint Malo, France. May 2013.
Source Attribution of Salmonella enterica in Ireland using the Microbial Subtyping Method. International
Symposium Salmonella and Salmonellosis (I3S) Saint Malo, France. May 2013.
Papers
Salmonella enterica biofilm formation and density in the Centers for Disease Control and Prevention’s
biofilm reactor model is related to serovar and substratum. Corcoran M, et al. J Food Prot.2013;76(4): 662-7.
Neutral Genomic Microevolution of a Recently Emerged Pathogen, Salmonella enterica Serovar Agona.
Zhou Z., et al. PLoS Genet. 2013:9(4):
International outbreak investigation of Salmonella Heidelberg associated with in-flight catering. Rebolledo J,
et al. Epidemiol. Infect. 2013: Jul 26:1-10.
Investigation and management of an outbreak of Salmonella Typhimurium DT8 associated with duck eggs,
Ireland 2009-11. Garvey P, et al. Eurosurveill. 2013:Apr 18:18(16)
27