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Sunday, June 6, 2010


See First: Salmonellosis a Biological Weapon of Mass Destruction

A ProMED-mail post

ProMED-mail is a program of the
International Society for Infectious Diseases

Date: Thu 3 Jun 2010
Source: Eurosurveillance [edited]

Infections with _Salmonella enterica_ account for the 2nd largest
burden of bacterial gastrointestinal disease in the European Union
(EU) (1). The majority of _Salmonella_ infections result in mild,
self-limited illness and may not require treatment with
antimicrobials. Nevertheless, treatment with an appropriate
antimicrobial can be life-saving in immunocompromised patients and in
invasive disease, such as Salmonella bacteraemia and meningitis.

Serotyping according to the Kauffmann-White scheme is a widely used
method for the initial characterisation of Salmonella isolates and is
based on the antigenic variability of the somatic (O) and flagellar
(H) antigens present in the cell wall of the organism (2). Despite
identification of more than 2500 different serovars [serotypes -
Mod.LL], the majority of cases of human infection are caused by a
limited number of serovars. Most serovars are biphasic and express 2
distinct flagellar antigens encoded by fliC (phase-1 flagellin) and
fljB (phase-2 flagellin). However, some serovars fail to express
either the phase-1 or phase-2 flagellar antigen, therefore are
classed as monophasic.

_S. enterica_ serovar 4,[5],12:i:- is considered a monophasic variant
of serovar Typhimurium (4,[5],12:i:1,2) due to antigenic and
genotypic similarities between the 2 serovars (3,4). Serovar
Typhimurium is the 2nd most common serovar associated with human
cases of salmonellosis in the EU (1). In contrast, isolates of
serovar 4,[5],12:i:- were rarely identified before the mid-1990s but
are now among the top 10 most common serovars isolated from humans in
several countries (3-8). According to Enter-net data, this serovar
was the 4th most common serovar in confirmed cases of human
salmonellosis in the EU in 2006 (1). Cases of infection with serovar
4,[5],12:i:- have reportedly been severe, with a 70 percent
hospitalization rate during an outbreak in New York City in 1998 (9),
although a much lower rate of 21 percent was observed during an
outbreak in Luxembourg in 2006 (6). Infections have also been
particularly associated with cases of septicemia in Thailand and
Brazil (7,10). Overall, cases of infection have been linked to a
number of sources, including poultry and cattle, but particularly
pigs and pork products (4,6,10-13). Serovar 4,[5],12:i:- was among
the top 10 most common serovars isolated from both pigs and pig meat
in the EU in 2006 (1).

A marked increase in prevalence of _S. enterica_ serovar 4,[5],12:i:-
with resistance to ampicillin, streptomycin, sulphonamides and
tetracyclines (R-type ASSuT) has been noted both in food-borne
infections and in pigs/pig meat in several European countries over
the last 10 years (6,8,14,15). In the baseline study from fattening
pigs (Commission Decision 2006/668/EC), Spanish strains of _S.
enterica_ serovar 4,[5],12:i:- represented 14.3 percent of the
isolates, 52.5 percent of which were of R-type ASSuT (VISAVET
Salmonella database, unpublished data). In England and Wales cases of
serovar 4,[5],12:i:- infection have risen from 47 in 2005 to 151 in
2009 (a 321 percent increase) against a backdrop of an overall
decrease in the number of salmonellosis cases, with R-type ASSuT
accounting for approximately 30 percent of these strains (Health
Protection Agency (HPA) Salmonella database, unpublished data). In
France isolations of serovar 4,[5],12:i:- increased from 99 to 410
between 2005 and 2008 to become the 3rd most common serovar isolated
from humans, with 62 percent of strains in 2007 being of R-type ASSuT
(16). In Italy cases of serovar 4,[5],12:i:- infection have risen
from 59 in 2003 to 641 in 2009, with 75 percent of monophasic strains
isolated in 2009 belonging to R-type ASSuT (with or without
additional resistances) (Istituto Superiore di Sanita Salmonella
database, unpublished data). A recent study described emergence of a
clonal group of serovar Typhimurium and 4,[5],12:i:- R-type ASSuT
strains in Italy, Denmark and the United Kingdom (UK) (17).
Resistance genes blaTEM-1, strA-strB, sul2 and tet(B) encoding
resistance to ampicillin, streptomycin, sulphonamides and
tetracyclines were localised on the bacterial chromosome. On the
basis of resistance gene content and the lack of class 1 integrons,
these observations have suggested the existence of a new resistance
island that differs from the Salmonella Genomic Island-1 (17).

In response to the rapid increase in the frequency of _S. enterica_
serovar 4,[5],12:i:-, R-type ASSuT strains, isolates from England and
Wales, Germany, France, Italy, Poland, Spain and the Netherlands were
compared using phage typing, resistance gene characterization,
pulsed-field gel electrophoresis (PFGE) and multilocus variable
number tandem repeat (MLVA) analysis to evaluate the possibility of
clonal spread of this emerging multidrug-resistant (MDR) strain.

Methods and Materials [can be found at original URL. - Mod. LL]

Some 122 serovar 4,[5],12:i:- isolates were sent to the HPA
Laboratory of Gastrointestinal Pathogens, of which 116 were confirmed
as serovar 4,[5],12:i:-. These comprised 41 from England and Wales
(20 from pigs and 21 from humans, including 3 from patients with a
history of recent travel to Thailand, Greece and an undisclosed
destination), 10 isolates from France (isolated from pig meat), 19
from Germany (12 from pigs, 6 from pig meat and one from a human), 23
from Italy (from humans), 5 from Poland (from humans), 8 from Spain
(from pigs) and 10 from the Netherlands (7 from human cases of
infection; 3 from pigs). The H:1,2 phase-2 flagellar antigen could be
serologically detected in the remaining 6 isolates.

Phage typing using the Typhimurium typing phages identified 16
different PTs (Table 1 - for tables, see original URL - Mod.LL). The
most commonly identified PTs were DT193 (51 isolates), DT120 (27
isolates) and RDNC (reacts but does not conform; 11 isolates). DT193
was the most common PT identified in England and Wales, France,
Germany, Spain and the Netherlands, while DT120 predominated in Italy
and Poland. All 116 isolates were PCR-positive for the
Typhimurium-specific fragment of the malic acid dehydrogenase gene
but only 4 isolates (one belonging to DT104, two to PT U302 and one
untypable) gave a product with primers targeting the 16S to 23S
spacer region specific to DT104 and the related PT U302 (19).

Overall, 94 of 116 isolates were PCR-negative for all variants of the
fljB gene coding for the phase-2 flagellar antigen, including 48 of
51 DT193 and 17 of 27 DT120 isolates. H:1,2-specific amplicons were
detected in the remaining 22 isolates.

84 isolates (72 percent) expressed resistance to ampicillin,
streptomycin, sulphonamides and tetracyclines (R-type ASSuT), with or
without additional resistance(s) (Table 2). Six isolates were fully
sensitive to all antimicrobials in the test panel. 83 of 92
ampicillin-resistant iolates carried blaTEM, 85 of 96
streptomycin-resistant isolates carried strA-strB, 88 of 99
sulphonamide-resistant isolates carried sul2 and 93 of 105
tetracycline-resistant isolates carried tet(B) (data not shown). Of
84 R-type ASSuT strains, 68 possessed blaTEM, strA-strB, sul2 and
tet(B) resistance genes. 82 percent of RDNC isolates, 80 percent of
DT193 and 74 percent of DT120 were of R-type ASSuT (with/without
additional resistance(s)), with resistance encoded by genes blaTEM,
strA-strB, sul2 and tet(B) in 78 percent, 75 percent and 56 percent
of isolates respectively. Isolates of R-type ASSuT were negative for
both class 1 and 2 integrase genes; these were found only in strains
expressing resistance to aminoglycosides and/or trimethoprim. Among
the remaining 16 R-type ASSuT strains from the present study that did
not carry blaTEM, strA-strB, sul2 and tet(B), 11 strains lacked only
one of tet(B), blaTEM-1 or sul2, one strain each lacked blaTEM-1 and
tet(B) or strA-strB and tet(B), one strain lacked blaTEM-1, strA-strB
and sul2 and one strain lacked all 4 genes. These strains belonged to
phage types DT120 (5 strains), DT193 (4 strains), RDNC (2 strains),
and one each belonged to phage types DT104, DT18 variant, U302, U311
and UT.

PFGE analysis and MLVA typing [can be seen at the original URL - Mod.LL]

Antimicrobial resistance is a serious public health problem limiting
the therapeutic options available to clinicians treating complicated
salmonellosis. In recent years there has been an overall decline in
the level of resistance in serovar Typhimurium in several European
countries as a result of a reduction in the number of isolates of
penta-resistant DT104 (14). To some extent this reduction has been
counteracted by an increase in prevalence of serovar 4,[5],12:i:-
isolates expressing resistance to ampicillin, streptomycin,
sulphonamides and tetracyclines (8,17).

One of the 1st reports of serovar 4,[5],12:i:- in Europe was of an
isolate grown in the late 1980s from a chicken carcass in Portugal
(26). This serovar emerged in Spain in strains from humans and pork
or pork products during 1997, and subsequently became the 4th most
common Salmonella serovar identified from 1998 to 2000 (11). All
isolates belonged to phage type U302. These isolates were classed as
monophasic variants of serovar Typhimurium due to presence of an
IS2000 fragment located in a Typhimurium-specific location within the
fliB-fliA intergenic region and amplification of a Typhimurium DT104-
and U302-specific region (3). All 116 monophasic isolates in this
study harboured the Typhimurium-specific fragment of the malic acid
dehydrogenase gene, suggesting that these strains are monophasic
variants of serovar Typhimurium. However, the majority (97 percent)
were negative for the DT104- and U302-specific region, suggesting
that these monophasic isolates may not be related to the serovar
4,[5],12:i:- strain(s) that emerged in Spain. This was confirmed by
phage typing, which identified DT193 as the most common PT, followed
by DT120, thereby adding to the diversity of phage types of serovar
4,[5],12:i:- linked to serovar Typhimurium. DT193 and DT120 have
consistently fallen within the top 5 phage types of serovar
Typhimurium from cases of human infection in England and Wales in
recent years (HPA Salmonella database, unpublished data). It is
plausible that at least some of this increase may be attributed to
the emergence of serovar 4,[5],12:i:- DT193 and DT120 strains.
Putative Typhimurium isolates sent from primary diagnostic
laboratories to the HPA Salmonella Reference Unit are only
phage-typed and not routinely subjected to further serological
examination. This may result in misclassification as serovar
Typhimurium and under-reporting of this serovar in England and Wales,
and in other countries where phage typing is used in lieu of full
serotyping to identify strains as serovar Typhimurium. Serovar
4,[5],12:i: DT193 strains have previously been isolated from human
cases of infection and/or pigs in Luxembourg and Spain (6,13), while
monophasic DT120 strains were identified in Italy (8).

The Spanish PT U302 serovar 4,[5],12:i:- strains were PCR-negative
for H:1,2 (11), as were the majority (81 percent) of monophasic
isolates in this study. Previous published work has shown that the
lack of phase-2 flagellar expression may be due to different
mutations (including point mutations) and partial or complete
deletions in fljB and adjacent genes (4,27). Monophasic strains in
which the phase-2 flagellar antigen is not detected serologically but
can be detected by PCR may contain deletions in a part of fljB that
leave the H:1,2-specific PCR primer binding sites intact, or they may
represent "serotype inconsistent" strains (27). These are serovar
Typhimurium strains in which serological detection of the phase-2
flagellar antigen may be inconsistent. This may be due to problems
with flagellar phase reversal, which is a time-consuming and
technically demanding procedure that may result in misclassification
of Typhimurium strains as serovar 4,[5],12:i:-. Alternatively, the
invertible promoter controlling expression of fljB and fliC may have
become locked in one position allowing only expression of fliC in
these strains (4). The range of mechanisms that can result in
non-expression of the phase-2 flagellar antigen make definitive
identification of serovar 4,[5],12:i:- problematic. It is possible
that molecular serotyping could be used as a basis to define such
strains as serovar 4,[5],12:i:- or Typhimurium, but as yet such
methods lack standardisation, are not in place in most countries and
may not be suitable for laboratories other than reference facilities.
Given that there may be discrepancy in detection of the phase-2
flagellar antigen between classical and molecular serotyping, an
international agreement both on the definition of monophasic strains
and on detection methodology is required. Without reaching such a
consensus the true incidence of such Typhimurium-like strains is
difficult to assess; only the harmonisation and the sharing of
methods will allow accurate comparison of reported data.

In contrast to the monophasic variants isolated in Thailand and
Spain, which commonly expressed additional resistance to gentamicin
and trimethoprim-sulphamethoxazole and/or chloramphenicol (10,11) and
to serovar 4,[5],12:i:- strains isolated in Brazil and New York City,
which were infrequently MDR (7,9), the countries participating in
this study observed an increase in isolates of serovar 4,[5],12:i:-
with resistance to ampicillin, streptomycin, sulphonamides and
tetracyclines only. Characterisation of the resistance genes
responsible for this phenotype identified blaTEM, strA-strB, sul2 and
tet(B) in 81% of isolates. Such genes have also been identified in
isolates of Typhimurium DT193 R-type ASSuT obtained during 2005 in
England and Wales from raw beef and a human case of infection,
although the majority of strains tested harboured tet(A) rather than
tet(B) (unpublished data). Analysis of a 10 kb chromosomal region of
a Typhimurium DT193 revealed the presence of an
strB-strA-sul2-repC-repA region derived from plasmid RSF1010 located
upstream of blaTEM-1 and downstream of a class 1 integron [28]. The
resistance genes encoding the tetra-resistant phenotype in isolates
of serovars Typhimurium and 4,[5],12:i:- from Italy, Denmark and the
UK were also identified as blaTEM-1, strA-strB, sul2 and tet(B), but
all isolates were negative for class 1 integrons (17). Transfer
experiments were unsuccessful and probes specific for these genes
bound to a 750 kb I-CeuI digest fragment, suggesting a chromosomal
location and existence of a new resistance island. As in the present
study, strains with other R-types than ASSuT, but with related PFGE
profiles and harbouring one or more of blaTEM-1, strA-strB, sul2 and
tet(B) were identified. This suggests that rearrangements or
deletions may occur within the resistance island leading to partial
resistance patterns [17]. In contrast, resistance to ampicillin,
streptomycin, sulphonamides and tetracyclines was mediated by
plasmid-borne blaTEM-1 and tet(A), and a class 1 integron harboring
aadA2 and sul1 in the Spanish serovar 4,[5],12:i:- U302 isolates (29).

[Discussion on PFGE and MLVA typing can be found at the original URL. - Mod.LL]

The data presented here suggest that a serovar 4,[5],12:i:- DT193
R-type ASSuT clone with PFGE profile STYMXB.0131 has emerged from
serovar Typhimurium and spread within several European countries,
with pigs as a likely reservoir of infection. Isolates of serovar
4,[5],12:i:- DT120 R-type ASSuT with closely related PFGE profiles
were identified in humans and pigs from 5 of the participating
countries. The diversity of PFGE and MLVA profiles within serovar
4,[5],12:i:- DT193 and DT120 R-type ASSuT isolates, and the
differences between these isolates and those previously described in
Spain (30), suggests that serovar 4,[5],12:i:- is likely to represent
several clones or strains that have emerged independently from
serovar Typhimurium. Recent genotypic studies have shown that in
addition to the Spanish 4,[5],12:i- clone, other 4,[5],12:i:-
lineages exist (27).

In the 1st 10 months of 2009, DT193 and DT120 accounted for 18
percent and 11 percent of Typhimurium isolates in England and Wales,
respectively. In contrast, DT104 accounted for only 7 percent of
Typhimurium isolates (HPA Salmonella database, unpublished data).
Serovar 4,[5],12:i:- has already caused substantial outbreaks in
several countries, with reports of severe infections and also deaths
(6,7,9,10). In order to prevent a global epidemic of these newly
emerging clones or strains, as occurred with Typhimurium DT104,
appropriate intervention strategies need to be put in place as soon
as possible, particularly in pig husbandry throughout the EU.

[References can be found at original URL. - Mod.LL]

[Authors: Hopkins KL, Kirchner M, Guerra B, et al]

Communicated by:

[2] France (dried sausage)
Date: Sat 5 Jun 2010
Source: eFoot Alert [edited]

An outbreak of salmonellosis due to salmonellosis (serotype 4,12:i:-)
was traced late in May 2010 to contaminated dried sausages produced
by the French sausage company Salaisons du Lignon, based in
Saint-Maurice de Lignon (Haute Loire).

The outbreak, which was still under investigation as of 28 May 2010,
has sickened at least 88 people, 18 severely enough to require
hospital treatment, in 49 departements throughout France. Illnesses
were reported between 15 Mar and 9 May 2010.

This particular Salmonella Typhimurium-like serotype 1st appeared in
Europe in the mid 1990s; it now has become the 2nd most common type
of salmonellosis reported by European Union member countries. Cases
of salmonellosis associated with this type tend to be more severe
than average, and result in a higher rate of hospitalization than is

Based on patient interviews, investigators from France's Institut de
Veille Sanitaire traced the outbreak to a single production lot of
Lou Montagnard brand La Pause Auvergnate style dried sausages
(saucisses seches droites nature La Pause Auvergnate). Salaisons du
Lignon recalled the implicated production lot (Lot No. 040020900)
distributed both in France and in Belgium on 27 May 2010.

Yesterday (4 Jun 2010), without fanfare or explanation, Salaisons du
Lignon expanded its recall in Belgium, but not yet in France, to
encompass all product expiration dates up to and including 24 Aug
2010. There have been no published reports of salmonellosis cases in
Belgium that are tied to the French outbreak.

Communicated by:

[This discussion is taken from Salmonellosis, frozen poultry pie -
USA (multistate)(04): recall 20071013.3355:

As readers will note from past outbreaks of salmonellosis, the
specific organism involved is usually referred to as _Salmonella
enterica_ followed by its serotype (or serovar) name such as _S.
enterica_ serotype Tennessee. Serotype Tennessee was associated with
the well-publicized USA-wide peanut butter-associated outbreak
affecting more than 600 people.

As discussed by Ana Paccagnella (supervisor of the Enteric
Bacteriology Section at the British Columbia Centre for Disease
_Salmonella_ as a genus has 2 species, _enterica_ and _bongori_. _S.
enterica_ is divided into 6 subspecies: _enterica_, _salamae_,
_arizonae_, _diarizonae_, _houtenae_ and _indica_. _S. enterica_
subspecies _enterica_ (or subspecies I) strains are the ones usually
isolated from humans or warm-blooded animals and represent a majority
of the salmonellas that are clinically important.

The designation _Salmonella_ I 4,[5],12:i:- means:
- the "I" reflects subspecies _enterica_;
- the "4, [5], 12" are the O (or somatic) antigens associated with
the organism; It should be noted that serotype Typhimurium carries
somatic antigens 4, 5 and 12 (the Group B antigens). Paccagnella
pointed out to me in an email that an O antigen underscored or placed
in [ ] can refer to whether the antigen is related to phage
conversion or not.
- the nomenclature after the colon, in this case i:-, reflect the
flagellar or H antigens. It is these H antigens that define the
serotype identity within an individual group of salmonellae.

Almost all of these organisms are biphasic in regard to the H
antigens, that is, they can switch from 1 flagellar antigen to
another. As an example, _S._ Typhimurium is I 4, 5, 12:i:1,2. The
outbreak organism here, however, is monophasic. Echeita and
colleagues (1) have found that this monophasic strain appears to be
such due to the absence of the _flj_B gene which is involved in the H
antigen switching mechanism. According to Paccagnella, around 1903
Smith and Reagh reported on the different behavior of the flagellar
and somatic antigens of salmonella strains. Their work was mostly
ignored until Weil and Felix, working on _Proteus_ cultures, noted 2
forms, the swarming form called the H form (_mit Hauch_, in English:
with breath) and the non-swarming form, called the O form (_ohne
Hauch_, in English: without breath). The H form contained both O and
H antigens (correctly termed the OH form). These parallels were
transposed to the salmonella antigens.

If O antiserum is added to a culture motility is preserved, whereas
if H antiserum is added the culture does not move. Therefore, H
antigen was involved with swarming in the agar plate (breathing might
have been used to imply the ability to move). (Salmonellae, of
course, do not swarm on certain agar plates like _Proteus_ bacteria

Several other foodborne outbreaks related to 4, 5, 12:i:- salmonellae
have been reported including one in New York City reported in 2002
(2). Additional information about this monophasic organism, which may
be resistant to multiple antimicrobial agents, can be found in
references 3-5.

1. Echeita MA, Herrera S, Usera M. Atypical, fljB-negative
_Salmonella enterica_ subsp. _enterica_ strain of serovar 4,5,12:i:-
appears to be a monophasic variant of serovar Typhimurium. J Clin
Microbiol 2001; 39: 2981-3.
2. Agasan A, Kornblum J, Williams G, et al. Profile of _Salmonella
enterica_ subsp. _enterica_ (subspecies I) serotype 4,5,12:i:-
strains causing food-borne infections in New York City. J Clin
Microbiol 2002; 40: 1924-9.
3. Guerra B, Laconcha I, Soto SM, et al. Molecular characterisation
of emergent multiresistant _Salmonella enterica_ serotype
[4,5,12:i:-] organisms causing human salmonellosis. FEMS Microbiol
Lett 2000; 190: 341-7.
4. de la Torre E, Zapata D, Tello M, et al. Several _Salmonella
enterica_ subsp. _enterica_ serotype 4,5,12:i:- phage types isolated
from swine samples originate from serotype Typhimurium DT U302. J
Clin Microbiol 2003; 41: 2395-400.
5. Mossong J, Marques P, Ragimbeaul C, et al. Outbreaks of monophasic
_Salmonella enterica_ serovar 4,[5],12:i:- in Luxembourg, 2006.
Eurosurveillance 2006; 12 (6).
. - Mod.LL]

[see also:
Salmonellosis, frozen poultry pie - USA (multistate)(09) 20071119.3748
Salmonellosis, frozen poultry pie - USA (multistate)(03): CDC 20071011.3337
Salmonellosis, human, pet turtles - USA (multistate): 2006-2007 20070709.2186]
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