Download Salmonella

Salmonella Infection and
Antimicrobial Resistance
Cheng-Hsun Chiu, MD, PhD
Department of Pediatrics
Chang Gung Children’s Hospital
Chang Gung University College of Medicine
Taoyuan, Taiwan
Salmonella Overview
• Member of the family Enterobacteriaceae.
• Gram-negative, non-spore forming bacilli.
• Taxonomically, medically important Salmonella
belong to a single species, Salmonella enterica, that is
comprised of many serogroups and > 2500 serotypes.
• Human infections caused by only a few serotypes.
e.g. S. enterica serotypes Typhimurium, Choleraesuis, and
Enteritidis
2
Nomenclature
• 1999, World Health Organization
Kauffmann-White scheme
(O - somatic antigens, H - flagellar antigens, Vi – capsular antigens)
two species, seven subspecies
Salmonella enterica (2502 serotypes)
S. enterica subsp. enterica ( or subspecies I)
S. enterica subsp. salmae (or subspecies II)
S. enterica subsp. arizonae (or subspecies IIIa)
S. enterica subsp. disrizonae (or subspecies IIIb)
S. enterica subsp. houtenae (or subspecies IV)
S. enterica subsp. indica (or subspecies VI)
Salmonella bongori (21 serotypes)
S. bongori subspecies V
3
Nomenclature
• 2000, Centers for Disease Control and Prevention
(accepted by the American Society for Microbiology)
Genus (italic)
Salmonella
Species (italic)
enterica
(subspecies I, II, IIIa, IIIb, IV, VI)
bongori
Serotype
(subspecies V)
(capitalized, not italic)
1. The first time a serotype is mentioned in the text; the name
should be preceded by the word "serotype"or "ser.“
2. Serotypes are named in subspecies I and designated by
antigenic formulae in other subspecies.
3. Members of subspecies II to IV, and VI, and S. bongori retain
their names if named before 1966.
4
Nomenclature
• Salmonella enterica (subspecies enterica)
serotype Choleraesuis
• Salmonella Choleraesuis
• S. Choleraesuis

Somatic O antigen
serogroups A, B, C1, C2, D, and E
5
Typhoid fever
Typhoid fever, a bacterial disease caused by Salmonella enterica
serovar Typhi (S. Typhi), is a major cause of morbidity and
mortality worldwide, with an estimated 21 million new infections
and 0.2 million deaths each year.
(Crump et al., 2004)
There are four stages in the classical typhoid fever, each lasting
approximately one week.
• First: temperature rise slowly, malaise, headache, cough,
leukopenia and Widal test negative
• Second: high fever (40℃), rose spots, hepatoslenomegaly,
diarrhea, blood culture and Widal test positive
• Third: intestinal hemorrhage and perforation, encephalitis,
dehydration, cholecystitis, endocarditis, osteitis
• Final: defervescence and/or being carriers.
(Disease info typhoid fever, CDC)
6
S. Typhi infection
1st infection
2nd infection
(Denise M et al., 2004, Nat. Rev. Microbiology)
7
Non-typhoid Salmonella
infection
• Most persons infected with Salmonella bacteria develop
diarrhea, fever, and abdominal cramps 12 to 72 hours
after infection. The illness usually lasts 4 to 7 days, and
most persons recover without treatment.
• However, in some persons, the diarrhea may be so severe
that the patient needs to be hospitalized.
(Disease info typhoid fever, CDC)
• The pathology of S. Typhimurium-infected C57BL/6 mice
was characterized by recruitment of neutrophils to the site
of inflammation, submucosal oedema and crypt
destruction.
(Grassl et al., 2010)
8
Epidemiology of typhoid fever
Mastroeni P and Maskell D. Salmonella Infection.
Cambridge Press. 2006
9
Complications of typhoid fever
Mastroeni P and Maskell D. Salmonella Infection. Cambridge Press. 2006
10
Antimicrobial resistance of S. Typhi
(I)
Mastroeni P and Maskell D.
Salmonella Infection.
Cambridge Press. 2006
11
(II)
Mastroeni P and Maskell D.
Salmonella Infection.
Cambridge Press. 2006
12
Typhoid fever in Taiwan
Lee CJ, et al. JMII 2013;46:469-73.
13
First isolation of ciprofloxacinresistant S. Typhi in Taiwan
Lee CJ, et al. JMII 2013;46:469-73.
14
First isolation of ciprofloxacinresistant S. Typhi in Taiwan
Lee CJ, et al. JMII 2013;46:469-73.
15
Foodborne diseases
The public health perspective
FOODNET surveillance, CDC
16
Non-typhoidal Salmonella not just
cause self-limited diarrhea!
• Bacteremia/sepsis: associated with fever,
chill, and toxicity in ~5% of patients overall,
and more frequently in infants.
• Extraintestinal metastatic infections: septic
arthritis, osteomyelitis, meningitis most
common, patients with sickle cell disease
and immunodeficiency are at a higher risk
for such focal infections.
17
Extra-intestinal infections caused
by non-typhoid Salmonella
• Non-typhoid Salmonella entrocolitis is
complicated by bacteremia (sometimes transient)
in approximately 5% of patients overall, and more
frequently in infants.
• Interestingly, among the >2,500 serotypes of nontyphoid Salmonella, a few, such as S. Dublin and S.
Choleraesuis, may rapidly invade to the blood
stream with little or no intestinal involvement.
18
FoodNet, CDC
19
BMC Infect Dis 2011
20
Most common serotypes of non-typhoid Salmonella isolates in 2005:
Typhimurium (19%), Enteritidis (18%), Newport (10%), Heidelberg
(6%), and Javiana (5%) (MMWR April 14, 2006).
US CDC
21
Table 1. Distribution of Salmonella serotypes with >50 cases identified
during 1996-2006 and proportions of specified outcomes.
Outcome, %
Serotype
Total, no. (%)
Hospitalization
Invasive disease
Death
24736
22.8
6.7
0.5
Typhimurium
10894 (44.0)
24.2
5.7
0.6
Enteritidis
7572 (30.6)
20.6
6.7a
0.5
Newport
4779 (19.3)
21.9
1.4a
0.3
Stanely
311 (1.3)
17
4.2
0
Anatum
307 (1.2)
21.2
2.6a
1
Derby
267 (1.1)
19.9
3
0.8
Schwarzengrund
240 (1.0)
25.4
15.4a
0.4
Panama
211 (0.9)
25.1
18a
0.5
Dublin
100 (0.4)
67
64a
3
Choleraesuis
55 (0.2)
60
56.4a
1.8
All
a Statistically
significant difference, compared with S. Typhimurium.
Jones et al., JID 2008
22
23
Pediatrics 2006
24
Mycotic aneurysm
25
Risk factors:
1. Old age
2. Serogroup C
(S. Choleraesuis)
infection
Hsu et al. CID
2003;36:829
26
Antimicrobial therapy for non-typhoid
salmonellosis
• Drugs of choice: ampicillin, chloramphenicol,
trimethoprim-sulfamethoxazole, 3rd generation
cephalosporins, fluoroquinolones.*
• Antimicrobial resistance in Salmonella is now a
serious, global problem!
*FDA approved the use of fluoroquinolones in children
with complicated UTI. More indications may be
expected in the near future.
27
Dissemination of antimicrobial
resistance in Salmonella
• Spread of resistant clones
1. Fluoroquinolone resistance
2. MDR (ACSSuT) phenotype
• Lateral gene transfer
28
Mechanisms of fluoroquinolone
resistance
• Mutations in the quinolone resistance-determining
region (QRDR) of the DNA gyrase genes
1. gyrA
codon 83 : serine  phenylalanine, tyrosine, or alanine
codon 87 : aspartic acid  glycine, asparagine, or tyrosine
2. gyrB
codon 463 : tyrosine  serine
3. parC
codon 80 : serine  isoleucine or arginine
recently been reported in S. Typhimurium and S. Choleraesuis
Su LH, et al. CID 2004;39:546
Chiu CH, et al. CMR 2004;17:311
29
30
31
Euro Surveillance 2003 Feb.
32
The appearance of S. Typhimurium DT104 strains with
combined MDR and ciprofloxacin resistance - ACSSuTCip
Euro Surveillance 1997 Nov.
33
S. Typhimurium definitive type 104
(DT104) in the US
• DT104 emerged as the most common multidrugresistant strain of Salmonella in the US in mid-1990s.
• In 1998, 28% of S. Typhimurium isolates were
ACSSuT; of these isolates, 86% were DT104.
• Overall 7% of human Salmonella infections in the US
in 1998 were caused by DT104.
Rabatsky-Ehr T. EID 2004;10:795.
34
int
thdF
0
Predicted ORFs and
function in SGI1
S009
DR-L (146bp)
xis
rep
S004
S013
X
trhG
10
S014
X
S025
floR
tet(G)
tetR
30
orf6
40
S010
trhG
10
S019
aadA2
X
tnpR
IRi
30
blaPSE-1
groEL/int1
20
qacED1 sulD1
int1
orf1
orf2
S023
S022
S020
qacED1
sul1
orf5
X
40
S044
tnpA
IRt
IS6100
X
S026
S008
S021
S015
S018
S016
S024
S007
S017
trhH
X
20
S006
S005
IRt
X
DR-R (42596 bp)
Complete Nucleotide Sequence of a 43-Kilobase Genomic Island Associated with the
Multidrug Resistance Region of Salmonella enterica Serovar Typhimurium DT104
Boyd et al. 2001 J Bacteriol. 183:5725
35
Generation of variants of the SGI1 complex integron
by gene replacement
IRt
IRi
IRt
SGI1
intI1
aadA2
sul1∆
floR
tetR
tet(G)
orf1
orf2
qacE∆1
IRi
blaPSE-1
groEL/intI1
sul1
orf6 IS6100
qacE∆1
orf5
IRt
IRt
SGI1-F
intI1
dfrA1
orfC
sul1∆
floR
tetR
tet(G)
orf1
orf2
qacE∆1
blaPSE-1
groEL/intI1
sul1
orf6 IS6100
qacE∆1
orf5
IRt
IRi
IRt
SGI1-H
intI1
sul1∆
aadA7
aac(3)-Id
floR
tetR
tet(G)
orf1
orf2
qacE∆1
blaPSE-1
groEL/intI1
sul1
orf6 IS6100
qacE∆1
orf5
IRt
IRi
IRt
SGI1-I
intI1
aadA2
sul1∆
floR
tetR
tet(G)
orf1
orf2
qacE∆1
dfrA1
orfC
groEL/intI1
sul1
qacE∆1
IRi
orf6 IS6100
orf5
IRt
IRt
SGI1-J
intI1
dfrA1
orfC
sul1∆
floR
tetR
tet(G)
orf1
orf2
qacE∆1
sul1
groEL/intI1
orf6 IS6100
orf5
IRt
IRi
IRt
SGI1-L
intI1
dfrA15
sul1∆
qacE∆1
floR
tetR
tet(G)
orf1
orf2
blaPSE-1
groEL/intI1
sul1
qacE∆1
orf6 IS6100
orf5
36
Distribution of SGI1 and its variants
• S. Typhimurium phage types
– DT104, DT1, DT12, DT120, U302
• Other serotypes
–
–
–
–
–
Agona (poultry, Belgium)
Meleagridis (animal, USA)
Albany (fish, Thialand)
Newport (human, France)
Paratyphi B dT+ (tropical fish, Singapore, humans,
Canada, UK, France)
– Derby (human, Taiwan)
37
Is SGI1 a mobile element?
• Liquid and solid mating experiments not
successful using DT104 as a donor.
• Conclusions: stably inserted in genome and not
easily transferable, but so why SGI1 is seen in
other serotypes of Salmonella?
38
Mobility of SGI1?
S. enterica
E. coli
thdF
TTCTGTATTGGTAAGTAA
3’end thdF (attB)
+
TTCTGTATTGGGAAGTAA
SGI1 site-specific sequence (attP)
SGI1 : Site-specific
integrative element
Circular
SGI1
SGI Integrase
yidY (S. enterica)
thdF
tnaL (E. coli)
DR-L
SGI1 site-specific sequence
new 3’ end of thdF
Integrated SGI1
DR-R
former 3’end of thdF
39
Doublet et al. Mol. Microbiol. (2005) 55:1911-1924
Mobility of SGI1
S. enterica
E. coli
thdF
TTCTGTATTGGTAAGTAA
3’end thdF (attB)
+
TTCTGTATTGGGAAGTAA
SGI1 :
Mobilizable
site-specific
integrative
element
Circular
SGI1
SGI1 site-specific sequence (attP)
Plasmid IncC
R55
(and others?)
SGI Integrase
yidY (S. enterica)
thdF
tnaL (E. coli)
DR-L
SGI1 site-specific sequence
new 3’ end of thdF
Integrated SGI1
DR-R
former 3’end of thdF
Doublet et al. Mol. Microbiol. (2005) 55:1911-1924
40
The mobilizable SGI1 crossed the
border of species:
IRi
SGI1 in Proteus miribilis 18306
intI1
aadA2
IRt
IRt
SGI1
DT104
sul1∆
floR
tetR
tet(G)
orf1
orf2
qacE∆1
blaPSE-1
groEL/intI1
sul1
qacE∆1
orf6 IS6100
orf5
IRt
IRi
IRt
SGI1-L
P. miribilis
intI1
dfrA15
sul1∆
qacE∆1
floR
tetR
tet(G)
orf1
orf2
blaPSE-1
groEL/intI1
sul1
orf6 IS6100
qacE∆1
• First report of SGI1 in a bacterium other than
Salmonella.
• AbR gene cluster identified corresponded to that of
the SGI1-L complex reported previously in S.
Newport.
Ahmed et al. 2007. JAC
41
Dissemination of antimicrobial
resistance in Salmonella
• Clonal spread
• Lateral gene transfer
- cephalosporin resistance
42
Average 1.5% in all
serogroups/serotypes
Su LH et al. Increasing ceftriaxone resistance in nontyphoid Salmonella in a university hospital in Taiwan.
J Antimicrob Chemother 2005; 55:846
43
44
Su LH et al. JAC 2005
CTX-M-3
plasmids
Liu SY et al. IJAA
2007
45
Inter-species
spread of
CTX-M-3
ESBL
46
Plasmid-mediated CTX-M-3 in E. coli and Kp. Liu SY. IJAA 2007
Ceftriaxone-R
Enterobacteriaceae
*10 genera
*17 species
*140 isolates
(different genotypes)
Plasmid-mediated
AmpC lactamase(CMY-2)
*9 genera
*10 species
*34 isolates
47
Su et al. J Antimicrob Chemother 2006; 57: 424.
The plasmids containing
either CTX-M-3 ESBL or
CMY-2 AmpC genes are
conjugative and have a
broad host range!
Through plasmid transfer, the
situation of worldwide spread
of the ceftriaxone resistance
among Salmonella and other
Enterobacteriaceae is an
“epidemic of plasmids.”
48
Winokur et al. Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates
expressing a plasmid-mediated CMY-2 AmpC -lactamase. AAC 2000;44:2777
Dunne et al. Emergence of domestically acquired ceftriaxone-resistant Salmonella infections
associated with AmpC -lactamase. JAMA 2000;284:3151
49
Prevalence MDR-AmpC
S. Newport
JID 2003;188:1707
The multidrug-resistant S. Newport contained the chromosomal
SGI1 (like DT104) as well as the plasmid-mediated AmpC (CMY-2)
β-lactamase gene; so they expressed the “MDR-AmpC” phenotype.
50
JID 2003;188:1707
51
52
90
14
80
12
70
60
10
50
8
40
6
30
Chiu et al. (2002) New Engl J Med
2001-4
2001-2
2000-4
2000-2
1999-4
0
1999-2
0
1998-4
10
1998-2
2
1997-4
20
1997-2
4
Resistant Isolates (%)
16
1996-4
No. of Total Isolates
Fluoroquinolone resistance in S. Choleraesuis has
emerged in Taiwan since 1999, but they remained
suscpetible to ceftriaxone!
53
54
A ciprofloxacin-resistant S. Choleraesuis strain SC-B67
acquired the plasmid-mediated AmpC gene, thus expressing
resistance to both ciprofloxacin and ceftriaxone
Strain
Resistance genes
identified
Mutations in DNA
gyrase and
topoisomerase Ⅳ genes
Plasmids
(kb)
Antibiotic resistance
phenotypes
ATCC8392
None
None
None
T
SCB43
blaTEM-1, sulI,
dfr, aadA2
gyrA, parC
90, 50
ACSSuTGmKTpCip
blaTEM-1, sulI,
dfr, aadA2, blaCMY-2
gyrA, parC
120, 50
ACSSuTKTpCroCip
SC-B67
PCR was used to identify the genes and sequencing to detect the mutation.
A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfonamide; T, tetracycline; Gm, gentamicin; K,
kanamycin; Tp, trimethoprim; Cro, ceftriaxone; Cip, ciprofloxacin.
Chiu CH, et al. Lancet 2004;363:1285
55
Ye J, et al.
Plasmid
2011
56
Ye J, et al. Plasmid 2011
57
Figure 1. Secular trends in (A) annual numbers (bars) and proportions to total nontyphoid
Salmonella isolates (line) and (B) rates of resistance to ciprofloxacin and ceftriaxone of S.
Choleraesuis isolates and (C) ceftriaxone resistance among major serogroups of nontyphoid
Salmonella in CGMH, 1999-2009.
10
30
5
40
Ciprofloxacin
Ceftriaxone
20
9
Serogroup C
Serogroup D
6
3
0
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
0
Serogroup B
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
0
60
12
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
0
80
Ceftriaxone resistance (%)
60
100
Resistance (%)
15
% Choleraesuis/Salmonella
90
No. of isolates
(C)
(B)
(A)
Year
Year
Year
58
A: Kaohsiung B: Linko
Su LH, et al. EID
April 2014
*Approval and importation of vaccine for swine. †Promotion of the Certified Agricultural
Standards quality food certification system, monitoring of sale of antimicrobial drugs for animal
use, inspection of chemical residues in swine farms and pork market, launch of educational
programs about safe use of drugs in animals; inspection of illegal slaughtering and sale of farmed
animals dying of unidentified disease; and establishment of Taiwan Agriculture and Food
Traceability system. ‡Initiation of death insurance program for pigs in 2 representative centralsouthern counties. §Extension of death insurance program to another 8 neighboring counties.
¶Full implementation of death insurance program throughout all Taiwan counties. #Establishment
59
of Taiwan Good Agricultural Practice for pig husbandry.
(A)
Kb
1
2
3
4
5
6
7
8
9
10
(B)
Kb
1 2 3 4 5 6 7 8 9 10 11 12 131415 16
Kb
-1135
582485388-
-452.7
9050-
-336.5
291-
-244.4
194145.5-
(C)
-138.9
97-
-78.2
48.5-
-33.3
60
Figure 3. Minimum-spanning-tree analysis of the pMLST results among the 8 blaCMY-2harboring IncI1 plasmids (pSC-B134, pSC-B136, pSB5, pSB28, pSB105, pSB151, pSB193,
pSD166) derived in the present study and other published IncI1 plasmids that also harbored
blaCMY-2 (25-27). Detailed allele variants are shown in Table 3. (ST, sequence type; CC,
clonal complex)
ST54
(pSB28, pSB193)
ST19
ST4
CC-12
ST52
(pSC-B136)
ST53
(pSB5)
ST51
(pSC-B134)
ST20
ST21
CC-5
ST18
CC-2/CC-12
ST12
CC-12
ST2
CC-2
ST56
(pSB105,pSD166)
ST55
(pSB151)
ST22
CC-12
ST23
CC-2
Single locus variant
Double locus variant
Triple locus variant
61
Clinical Impact of Antimicrobial Resistance
Decreased long-term survival
Helms et al. EID 2002;8:490
62
Clinical Impact of Antimicrobial Resistance (2)
• Fluoroquinolone susceptible
MDR-AmpC
 ciprofloxacin
• Fluoroquinolone resistant
AmpC producer (CMY-2)
cefepime? (may be safer to use carbapenem)
carbapenem
ESBL producer (CTX-M-3)
 carbapenem
 others? tigecycline
63
Carbapenem resistance in
Salmonella
• Extremely rare
• Two cases report in 2003 in AAC
- Imipenem resistance in a Salmonella strain due to
plasmid-mediated class A KPC in Atlanta, Georgia, US.
- Imipenem resistance in a Salmonella strain due to
porin loss plus CMY-4 AmpC-type -lactamase in
Tunisia.
• NDM-1 or other metallo--lactamase gene has not
been found in Salmonella by now.
• Carbapenem-resistance due to Omp deficiency plus
plasmid-mediated CMY-2 β-lactamase. (Su LH, et al. Clin
Microbiol Infect 2012;18:E91-4)
64
Clin Microbiol Infect 2012; 18:E91.
65
Liu CY et al. AAC 2008
66
Liu CY et al. AAC 2008
67
Tramsmission of non-typhoid
Salmonella to humans
Multiple vesicles
• Produce
• Eggs
• Poultry
• Other meat
• Direct contact with animals and their
environments
Plant-derived food and low-moisture
food-- growing importance!
68
43 states, DC,
Canada
contamination of
imported jalapeño and
serrano peppers grown
on a single Mexican
farm
contamination of one Georgia
producer's peanut butter
44 states,
Canada
NEJM March 5, 2009
69
Relative rates of laboratory-confirmed infections with Campylobacter, STEC* O157, Listeria, Salmonella, and Vibrio
compared with 1996–1998 rates, by year — FoodNet, USA, 1996–2009†
Vibro spp.
Salmonella
Listeria,
Campylobacter,
EHEC O157
* Shiga toxin-producing Escherichia coli.
† The position of each line indicates the relative change in the incidence of that pathogen compared with 1996--1998.
The absolute incidences of these infections cannot be determined from this graph. Data from 2009 are preliminary.
The figure above shows relative rates of laboratory-confirmed infections with Campylobacter, STEC* O157, Listeria, Salmonella, and
Vibrio compared with 1996-1998 rates, by year from the FoodNet, for the US during 1996-2009. In comparison with 1996-1998, rates
of infection in 2009 were lower for Shigella (55% decrease, CI = 37%-68%), Yersinia (53% decrease, CI = 41%-63%), STEC O157
(41% decrease, CI = 27%-52%), Campylobacter (30% decrease, CI = 24%-35%), Listeria (26% decrease, CI = 8%-40%), and
Salmonella (10% decrease CI = 3%-16%); rates were higher for Vibrio (85% increase, CI = 36%-150%). The incidence of infection
with Cryptosporidium did not change significantly. The incidence of Vibrio infection has been increasing since approximately 2001 and
the most marked decreases in Campylobacter, Listeria, and Salmonella infections occurred before 2004. The incidence of STEC O157
infection in 2009 was similar to that in 2004.
70
Foodborne disease in 2011:
the rest of the story
1.
2.
3.
Increasing non-O157
STEC
Previously un-recognized
vehicles for foodborne
infection, such as raw
produce like jalapeno
peppers in Salmonella
outbreaks
Improvement in
detection methods and
population-based
surveillance needed
Michael T. Osterholm. NEJM March 11 2011
71
Controlling the spread of the antimicrobial
resistance in non-typhoid Salmonella
• Restriction of the use of antimicrobial
agents both in food animals and humans.
• Reinforcement of infection-control
measures in clinical settings.
• Continued surveillance for antimicrobial
resistance trend in clinical as well as animal
isolates of Salmonella.
72
Thank you for your attention