International Journal of Food Microbiology 144 (2010) 310316

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Prevalence and characterization of antimicrobial resistance of foodborne Listeria

monocytogenes isolates in Hebei province of Northern China, 20052007
He Yan a, Sucharit Basu Neogi c, Ziyao Mo b, Wenying Guan d, Zhixin Shen d, Shuhong Zhang d, Lin Li a,
Shinji Yamasaki a,c, Lei Shi a,, Nanshan Zhong b
a
College of Light Industry and Food Sciences, South China University of Technology, 510640 Guangzhou, China
b
The State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, 510230 Guangzhou, China
c
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan
d
HeiBei Provincial Center for Disease Control and Prevention, 050051 Heibei, China

a r t i c l e i n f o a b s t r a c t

Article history: A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened
Received 12 May 2010 for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping,
Received in revised form 6 October 2010 antimicrobial susceptibility, pulsed-eld gel electrophoresis (PFGE), as well as PCR screening to identify
Accepted 17 October 2010
genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and
class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples
Keywords:
Listeria monocytogenes
representing various food products. The pathogen was mainly isolated from frozen food made of wheat
Prevalence our or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of
Antibiotic Resistance cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323)
Tetracycline Resistance Determinants of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to ve
serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance
was most frequently observed for ciprooxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%).
Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred
among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than ve antimicrobials.
Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one
harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as
scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The
widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes,
and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate
that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.
2010 Elsevier B.V. All rights reserved.

1. Introduction survive under adverse environmental conditions such as low tempera-

Listeria monocytogenes is a Gram-positive, facultative intracellular,
foodborne pathogen that can cause listeriosis in humans and other
animals, e.g., cattle, sheep, poultry, birds, etc. Invasive listeriosis is more
frequently associated with immunocompromised individuals, elderly
(aged 65 years and older), pregnant women, unborn infants and
neonates, whereas non-invasive infections can also cause illness among
healthy persons (Hof, 2004b). The opportunistic L. monocytogenes
bacterium is found widely distributed in the natural environment,
thus can easily contaminate various raw and processed foods, including
milk and dairy products, meat, fermented sausages, fresh vegetables as
well as seafood and sh products (Yucel et al., 2005). The bacterium can
Corresponding author. Tel.: + 86 20 87113848; fax: + 86 20 87112734.
E-mail address: leishi88@hotmail.com (L. Shi).
ture and pH, high concentrations of salt or bile, oxidative stress, carbon
starvation, etc. (Razavilar and Genigeorgis, 1998). The occurrence of
this bacterium is recognized as a major public health concern because
of its high case-fatality rate and severity of the disease manifested by
septicemia, meningitis, gastroenteritis, pneumonia, spontaneous abor-
tion, etc. (Vzquez-Boland et al., 2001). Over the past two decades
numerous foodborne listeriosis outbreaks have been reported all over the
world including the USA, Japan, New Zealand, Germany, England, France
and other European countries (Warriner and Namvar, 2009). In the USA,
during 19982002 approximately 43% of the annual deaths related to
food-borne pathogens were due to this pathogenic bacterium (Lynch
et al., 2006). Control of L. monocytogenes contamination represents a
signicant problem for the food industries, public health agencies, and
government bodies (Ryan et al., 2009).
China is one of the major producers and consumers of animal
source foods. In this country, the per capita consumption of meats and

0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.10.015
 H. Yan et al. / International Journal of Food Microbiology 144 (2010) 310316
seafood has increased signicantly over the past decades. Conse-
quently, there is an increased potential for exposure to foodborne
pathogens. During 2000, the National Surveillance Network for Food
Contamination and Foodborne Diseases System was launched which
recognized L. monocytogenes as a serious risk to food safety and public
heath in China. However, there is a paucity of data regarding the
prevalence, types of serovars, antimicrobial susceptibility, and PFGE
proles of L. monocytogenes from food samples in China.
L. monocytogenes is usually susceptible to antibiotics active against
Gram-positive bacteria except for the cephalosporins, uoroquinolones
and fosfomycin (Charpentier and Courvalin, 1999a). Currently, ampi-
cillin plus gentamicin is standard therapy for listerosis while trimeth-
oprim-sulfamethoxazole, vancomycin and erythromycin are also used
to treat bacteremia and listeriosis in pregnant women (Charpentier
and Courvalin, 1999a; Hof, 2004b). However, an increasing number of
strains are becoming resistant to most of the known antibiotics
including those used for treating listeriosis (Charpentier and Courvalin,
1999a; Lyon et al., 2008). The levels of resistance are varied and
inuenced by antimicrobial use to humans and animals, as well as the
geographical differences. Therefore, it is necessary to monitor the
antibiotic resistance patterns of L. monocytogenes in food and environ-
mental sources from different geographic areas.
Diverse subtyping methods such as serotyping (Thvenot et al.,
2006), phage typing (Shen et al., 2006), molecular methods like
ribotyping (Bruce, 1996) and pulsed-eld gel electrophoresis (PFGE)
(Fugett et al., 2007), etc. have been used in discriminating L.
monocytogenes strains of different origin. So far this bacterial pathogen
has been differentiated into 13 serotypes which can be grouped into
genetic lineage I (serotypes 1/2b, 3b, 3c, and 4b) from human clinical
listeriosis, lineage II (serotypes 1/2a, 1/2c, and 3a) from food isolates
and animal clinical cases, and less common Lineage III (serotypes 4a
and 4c, as well as some 4b) from animal and environmental specimens
(Nadon et al., 2001). Among molecular typing tools, PFGE is recognized
as the gold standard method because of its high discriminatory power
and reproducibility. Additionally, the creation of PulseNet (http://
www.cdc.gov/pulsenet) allows the comparison of PFGE patterns of
L. monocytogenes associated with food-borne listeriosis outbreaks.
In the present study, a 3-year-long surveillance on the occurrence
of L. monocytogenes in different food items was conducted from nine
different cities of Hebei province in northern part of China. Our overall
aim was to detect the occurrence of this pathogen in food samples,
determine their serotype and genetic relatedness, and evaluate
susceptibility of L. monocytogenes isolates to 18 antibiotics currently
used in veterinary and human therapy.
2. Materials and methods
2.1. Collection of food samples and isolation of Listeria monocytogenes
A total of 2177 retail food samples, comprised of raw meats
(n = 733), cooked meats (n = 302), quick-frozen food made of wheat
our and rice products (n = 252), non-fermented bean products
(n = 204), seafood (n = 343), and vegetables (n = 323), were obtained
from nine surveillance points (including open-air markets and large
supermarkets) in Hebei province, located in the northern part of
China during 2005 to 2007 as part of the national active food-borne
pathogens surveillance system. The samples were randomly collected
from the markets from May to October of each year.
Strain isolation and identication were performed as described
previously (Chao et al., 2007; Norton et al., 2001).
2.2. Serotyping
After conrmation as L. monocytogenes species, the isolates were
serotyped using commercially available antisera for subtyping this
311
pathogen (Denka, Seiken, Tokyo, Japan), according to the manufac-
turer's instructions.
2.3. Antimicrobial susceptibility testing
All the L: monocytogenes isolates were tested for their resistance
to 18 antimicrobial agents currently used in veterinary and human
therapy by the broth microdilution method (Zhang et al., 2007).
Ampicillin, ampicillin-sulbactam, penicillin, cephalothin, cefotaxime,
imipenem, ciprooxacin, moxioxacin, levooxacin, tetracycline,
doxycycline, erythromycin, gentamicin, vancomycin, rifampin, chlor-
amphenicol, streptomycin, trimethoprim-sulfamethoxazole obtained
from Sigma were included in the susceptibility study. Strains were
inoculated in BHI broth using 96-well microtiter plates and growth
was recorded after 18 to 24 h of incubation at 37 C by measuring
OD at 630 nm using a VICTOR3 multilabel counter (PerkinElmer). Each
of the experiments was done in triplicate. Clinical and Laboratory
Standards Institute (CLSI) breakpoint guidelines for L. monocytogenes
and other gram-positive bacteria were used for data analysis (Clinical
and Laboratory Standards Institute, CLSI, 2006a,b). For moxioxacin,
as there is no universal guideline, the isolates were categorized as
susceptible, intermediate, or resistant according to breakpoints: 1 g/
ml MIC N 2 g/ml, as described previously (Grayo et al., 2008.).
Staphylococcus aureus ATCC 29213T, Escherichia coli ATCC 25922T,
and Enterococcus faecalis ATCC 29212T were used as control strains.
L. monocytogenes isolates representing intermediate resistance values
were considered as resistant to corresponding antimicrobial agent.
2.4. PCR, cloning and sequencing for detection of tetracycline resistance
determinants, Tn916 and class 1 integron
Chromosomal DNA from each of the L. monocytogenes isolates was
extracted by using Qiagen Dneasy Blood and Tissue Kit. The quantity
of the DNA was determined by using a UV-1800 spectrophotometer
(Shimadzu, Kyoto, Japan). In order to identify if tetracycline resistance
genes were present in a silent form in tetracycline-susceptible
isolates, all isolates were screened for tet(L), tet(M), tet(K), tet(S)
and tet(B) genes which are responsible for tetracycline resistance, by
PCR using primers and conditions as described previously (Wang
et al., 2006). The PCR products were cloned in pMD20-T using the
pMD20-T cloning kit (Takara Bio Inc., Shiga, Japan), then sequenced at
Invitrogen Biotechnology Co., Ltd (Guangzhou, China). The resulting
DNA sequence data were compared to data in the GenBank database
by using the BLAST algorithm (1) available at the National Center for
Biotechnology Information web site (www.ncbi.nlm.nih.gov). The tet
(M)-positive isolates were further investigated for the presence of
Tn916 transposon by PCR mapping as described earlier (Poyart et al.,
2000). The PCR products were also cloned using pMD20-T cloning kit
and then sequenced. Besides, the presence of class 1 integron which
can also play role in the transfer of tetracycline resistance was also
checked following previously described PCR method (Li et al., 2006).
2.5. Pulsed-eld gel electrophoresis (PFGE)
PFGE of all the isolates after digestion with ApaI was performed
according to the CDC PulseNet standardized procedure for typing
L. monocytogenes (Graves and Swaminathan, 2001) by using the CHEF
MAPPER apparatus (Bio-Rad Laboratories, Hercules, USA). The images
of PFGE patterns were captured with a Gel Doc system (Bio-Rad)
using the Quantity One software program (version 4.6.2; Bio-Rad).
PFGE patterns were compared using the BioNumerics software
package (Sint-Martens-Latem, Belgium, version 5.10). Cluster analysis
of the PFGE patterns was performed using the unweighted pair group
method using arithmetic averages (UPGMA) and the band-based Dice
correlation coefcient with a position tolerance of 1.0%. Isolates were
312 H. Yan et al. / International Journal of Food Microbiology 144 (2010) 310316
considered to have the same PFGE type when the numbers and
locations of the bands were indistinguishable.
3. Results
3.1. Prevalence of Listeria monocytogenes in retail food products
A total of 90 L. monocytogenes isolates were recovered, represent-
ing 4.13% of the total samples (n = 2177). The pathogen occurred
among all kind of food samples and by category 10.32% (26/252)
of quick-frozen food made of wheat our and rice products, 6.28%
(46/733) of raw meat samples, 3.31% (10/302) of cooked meat
products, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented
bean products and 0.62% (2/323) of vegetables samples (for further
details about isolation dates, types of foods, etc. see Fig. 2). The
majority of isolates were obtained from raw meat products, quick-
frozen food made of wheat our and rice products and cooked meat
samples (46, 26 and 10 isolates, respectively). Among raw meat
samples, the pathogen was detected among 8.94% (21/235) of raw
chicken meat, 10.81% (20/185) of raw pork, 2.55% (4/157) of raw
beef, and 0.64% (1/156) of raw mutton samples.
3.2. Serotyping
The L. monocytogenes isolates (n = 90) could be grouped into ve
different serotypes (1/2a, 1/2b, 1/2c, 3a, and 4b); the majority (44
isolates, 48.88%) were of serotype 1/2a, followed by serotype 1/2c (26
isolates, 28.89%) and serotype 1/2b (11 strains, 12.22%). Beyond these
three categories, eight isolates (8.88%) were grouped as serotype 4b, and
one isolate (1.11%) as serotype 3a (Fig. 2, Table 1). L. monocytogenes
belonging to serotype 1/2a and 1/2c could be isolated from raw and
cooked meat samples as well as quick frozen food made of our, wheat
or rice in all three consecutive years. The occurrences of other serotypes
were scattered, e.g., two of the eight isolates of serotype 4b were
detected from cooked meat products in 2005, ve were found in quick-
frozen food in 2006, and one were from raw chicken in 2007. If genetic
relatedness is considered, lineage II-related isolates (serotypes 1/2a, 1/
2c and 3a) were dominant (78.9%, 71/90), while the remaining isolates
(21.1%, 19/90) belonged to lineage I (serotypes 1/2b and 4b). No clear-
cut relationship between sampling site and serotype could be revealed;
at site 1 all the observed serotypes except 4b could be found, whereas
alternatively, serotype 1/2a could be isolated from 8 out of 9 cities
(Fig. 2).
3.3. Antimicrobial susceptibility proles
The results of susceptibility of L. monocytogenes isolates to 18
antibiotics by the microdilution method are shown in Fig. 1. The
majority of the isolates (77.8%, n = 70) was resistant to cefotaxime.
Table 1
Clusters, PFGE types, and serotypes of L. monocytogenes isolates recovered from selected foods in province HeiBei in China from 2005 to 2007.
Cluster No. of No. of Serotype(s)
PFGE types isolates (no. of isolates)
I 1 2 4b(2)
II 3 3 1/2b(2), 1/2a(1)
III 5 7 1/2b(3), 1/2a(2), 4b(2)
IV 1 13 1/2a(11), 1/2c(1), 3a(1)
V 1 1 1/2a(1)
VI 6 14 1/2a(11), 1/2b(2),4b(1)
VII 3 5 1/2b(4), 4b(1)
VIII 3 41 1/2c(24), 1/2a(16), 4b(1)
Besides cefotaxime, among 90 isolates 33 (36.7%) displayed resistance
to one or more antimicrobials including 16 (17.8%) resistant to
additional one antibiotic; 10 (11.1%) to additional two antibiotics.
However, multiple resistances (resistance to 2 antibiotics) were
observed in 17 (18.9%) isolates. Among the commonly used
antimicrobials, ciprooxacin resistance in 17.8% (n = 16), tetracycline
resistance in 15.6% (n = 14) and streptomycin resistance in 12.2%
(n = 11) isolates were observed. A few isolates were resistant to
trimethoprim-sulfamethoxazole (n = 5), cephalothin (n = 3), ampi-
cillin (n = 2), chloramphenicol (n = 2), erythromycin (n = 2), genta-
micin (n = 1), penicillin (n = 1), rifampin (n = 2) and vancomycin
(n = 1). All isolates were susceptible to moxioxacin, levooxacin,
imipenem, ampicillin-sulbactam. Occurrence of multiple resistances
was more frequent among isolates belonging to serotypes 1/2a, 1/2c,
4b, while no such resistance could be observed among less frequent
isolates belonging to serotype 1/2b (Fig. 2).
It is worth noting that besides cefotaxime, resistance to at least 5
antimicrobials was observed in two isolates from quick-frozen food,
one (06CZ014) of 1/2a serotype isolated in 2006 that showed
resistance to cephalothin, tetracycline, doxycycline, vancomycin,
rifampin, chloramphenicol and streptomycin, while the other
(07LF005) of 1/2c serotype isolated in 2007 that showed resistance
to tetracycline, erythromycin, chloramphenicol, trimethoprim-sulfa-
methoxazole and streptomycin (Fig. 2).
3.4. Detection of class 1 integron, tetracycline resistance determinants
and Tn916
No class 1 integron bearing antibiotic resistance determinants
were detected among the L. monocytogenes isolates by our PCR
method. The molecular basis of tetracycline resistance among the
isolates was veried by checking the presence of tetracycline
resistance determinants, including tet(M), tet(L), tet(K), tet(S), and
tet(B) genes. Among 14 tetracycline-resistant isolates, 13 carried tet
(M) and one harbored tet(S), whereas none showed positive for the
other tet genes. However, when the tetracycline-susceptible isolates
were also screened for tet determinants no positive result was ob-
tained (data not shown). Furthermore, 9 of the 13 tet(M) positive
isolates possessed tet(M) gene associated with Tn916 conjugative
transposon. The existence of tet(M), tet(S) and Tn916 conjugative
transposon was also conrmed by sequencing analysis after cloning
of the PCR products. MIC values of tetracycline for the 14 isolates
carrying tet determinants ranged from 16 to 64 g/ml, although
most of them showed higher ( 64 g/ml) values. Tetracycline resis-
tance by tet determinants in these isolates accompanied with different
combination of multiple drug resistance mechanisms including resis-
tance to doxycycline (MIC 8 g/ml) in four isolates. The combination
of tet(M) along with Tn916 did not have any discernable inuence on
MIC values for tetracycline.
Food categories represented
quick-frozen food made of wheat our and rice
raw beef, cooked meats
raw pork, raw chicken, cooked meats, quick-frozen food made of wheat our and rice
quick-frozen food made of wheat our and rice, raw chicken, raw beef, seafood, cooked meats
cooked meats
quick-frozen food made of wheat our and rice, raw chicken, vegetables, raw pork, raw beef,
raw chicken, raw mutton
cooked meats, quick-frozen food made of wheat our and rice, raw chicken
raw pork, raw chicken, cooked meats, quick-frozen food made of wheat our and rice, seafood,
non-fermented bean products
 H. Yan et al. / International Journal of Food Microbiology 144 (2010) 310316
Fig. 1. Rates of antimicrobial resistance of L. monocytogenes isolates from food samples during 2005 to 2007. Antimicrobial resistance rates are presented for AMP, ampicillin; SAM,
ampicillin-sulbactam; CHL, chloramphenicol; CIP, ciprooxacin; CEF, cephalothin; CTX cefotaxime; DOX, doxycycline; ERY, erythromycin; GEN gentamicin; IPM imipenem; LVX
levooxacin; MOX, moxioxacin; PEN, penicillin; RIF, rifampin; STR, streptomycin; TET, tetracycline; VAN, vancomycin; SXT, trimethoprim-sulfamethoxazole.
3.5. Genetic diversity analysis by pulsed-eld gel electrophoresis
PFGE was carried out for determination of genetic relatedness
among all the 90 L. monocytogenes isolates. Eighty-six isolates had
distinct electrophoretic patterns by this method while in the case
of four isolates, repeated attempts failed to obtain discrete PFGE
patterns. The 86 isolates could be categorized into 20 distinct PFGE
types (A to T) using Apal enzyme which were grouped into 8 clusters
(Fig. 2, Table 1). Each of the clusters contained 1 to 6 distinct PFGE
types, with an overall similarity value of 67% using the Dice cor-
relation coefcient and UPGMA. The largest group, cluster VII, con-
tained 41 isolates that belonged to serotype 1/2c (24 isolates), 1/2a
(16 isolates), and 4b (1 isolate) (Table 1). The most frequent PFGE
type was type T (31.4%, 27/86) that occurred in all 3 years followed by
type I (15.12%, 13/86) and type S (11.63%, 10/86). A source specic
pattern could not be distinguished, e.g., type T occurred in isolates
from 5 sources, including raw meats (n = 12), quick-frozen food
(n = 10), seafood (n = 3), and non-fermented bean products (n = 2)
isolated from seven different cities (Fig. 2, Table 1). Similarly other
PFGE types were also widely distributed and from different sources
(Fig. 2). PFGE types having low frequency (e.g., A, B, C, D, H, etc.) were
from specic sources or food types. However, when considering
individual food type the isolates belonged to different PFGE types,
e.g., among quick-frozen foods 9 patterns (A, G, I, K, L, O, P, S and T),
among raw pork samples 5 patterns (E, O, R, S and T) existed, etc.
A clear-cut association of PFGE types with specic serotypes was
not prominent, rather isolates classied as the same serotype could be
differentiated into different PFGE type, e.g., serotype 4b strains (n = 8)
displayed a total of 5 PFGE type, serotype 1/2a (n = 44) could be
grouped into eleven PFGE types, etc. (Fig. 2). However, most of the
isolates that had identical PFGE types belonged to the same serotype.
For example, the majority of isolates (88.9%, 24/27) in PFGE type T
belonged to serotype 1/2c, most isolates of type S, O, and R showed
serotype 1/2a, etc. (Fig. 2). Conversely, some isolates with the same
PFGE type belonged to different serotypes, e.g., type I isolates
belonged to three different serotypes (1/2a, 1/2c and 3a) (Fig. 2).
Some isolates collected from different sources but belonging to
same PFGE pattern and serotype had variable antimicrobial suscep-
tibility proles, e.g., strains belonging to PFGE type T and serotype 1/
2c (Fig. 2). This scenario was also reected among some isolates
collected from similar source, e.g., two isolates from raw chicken meat
with identical PFGE type (pattern I) and of the same serotype (1/2a)
were resistant to ciprooxacin, cefotaxime, tetracycline, and to cefo-
taxime, tetracycline, doxycline, respectively. The tetracycline resistant
isolates showed afliation to some extent to a particular PFGE type,
e.g., eight out of nine bearing both tet(M) and Tn916 were clustered in
PFGE type I while isolates having tet(M) but without Tn916 belonged
to PFGE type R.
313
4. Discussion
In this study, we report on the occurrence, antibiotic resistance
pattern and genetic diversity of L. monocytogenes isolated from
commonly consumed food items in nine cities of Hebei province,
China during 2005 to 2007. The prevalence of L. monocytogenes in food
products of several other provinces in China was 0.96% in 2000 and
1.74% in 2001 (Wang et al., 2004; Wu et al., 2003), thus our results
show a comparatively higher incidence (4.13%) in HeiBei province in
recent years. The variation in isolation rate among different cities
(Fig. 2) suggests preferential growth of the bacterium in certain places
which might be due to a differences in food-processing environment,
human activity, poultry and livestock farm management, agriculture
practice, bacteriophage abundance, type of food samples, sampling
seasons, and isolation methods etc. (Hansen et al., 2006). Although
the pathogen occurred to some extent among all the investigated food
types, a higher isolation rate among raw meat samples is consistent
with previous reports from China as well as other parts of the world
(Wu et al., 2003; Yang et al., 2008; Zhou et al., 2005). The present
study also indicates that quick frozen food made of wheat our and
rice can be potential sources of L. monocytogenes (Fig. 2, Table 1).
We could also recover L. monocytogenes isolates from cooked meat
products (3.31%); similar occurrence was also observed by various
authors (Baek et al., 2000; Uyttendaele et al., 1999). Some L.
monocytogenes strains may survive in biolms, persist in food
processing plants, and ultimately contaminate raw and ready-to-eat
products (Borucki et al., 2003). However, the pathogen can persist
in soilplant interface for long time and can multiply in livestock,
poultry and decaying plant products (Abadias et al., 2008; Crepet
et al., 2007). Agricultural run-offs as well as sewage or fecal con-
tamination of aquatic bodies can also aid in spreading of the pathogen
or association with sh and shellsh (Hansen et al., 2006). In case of
vegetables samples, low occurrence (3%) of L. monocytogenes have
been reported previously (Abadias et al., 2008; Crepet et al., 2007).
The majority of our L. monocytogenes isolates belonged to
serotypes 1/2a, 1/2c, and 1/2b (Fig. 2, Table 1), which is in agreement
with previous studies in USA, Japan, Chile and many European
countries (Kathariou, 2002; Parihar et al., 2008; Thvenot et al., 2006).
The predominant occurrence of serotype 1/2a indicates its good
ecological tness. Fugett et al. (2007) have reported that among food
samples lineage II-related isolates (1/2a, 1/2c, 3a) can outnumber
lineage I-related isolates (1/2b, 3b, 3c, 4b). L. monocytogenes isolates
belonging to serotype 4b occurred among 8.88% of our surveyed food
items, whereas in some countries there are reports of its higher
abundance (Kathariou, 2002; Zhang et al., 2007). Studies have shown
that more than 95% of human listeriosis cases are caused by 4b, 1/2a
and 1/2b serotypes (Fugett et al., 2007). The presence of these
epidemiologically important serotypes among almost all kind of food
314 H. Yan et al. / International Journal of Food Microbiology 144 (2010) 310316
 H. Yan et al. / International Journal of Food Microbiology 144 (2010) 310316
items indicates possible diverse sources of infection in Hebei province
of China and poses a potential public health risk.
The high prevalence of cefotaxime resistant L. monocytogenes
isolates (77.8%, Fig. 1) noted in the current study is also in con-
cordance with previous studies (Chao et al., 2007; Lyon et al., 2008).
This type of natural resistance has been related to the absence of
specic penicillin-binding proteins in the Listeria cytoplasmic mem-
brane (Heger et al., 1997). Apart from cefotaxime, comparatively
higher frequency of resistance to ciprooxacin (17.8%), tetracycline
(15.6%) and streptomycin (12.2%) than other antimicrobials was
observed. There are reports of frequent isolation of ciprooxacin,
tetracycline and streptomycin resistant L. monocytogenes strains from
food or other sources (Lyon et al., 2008; Yang et al., 2008; Zhang et al.,
2007). There is growing evidence that antibiotic use in agriculture is
affecting antibiotic resistance transfer in human pathogens through
the food chain (Wang et al., 2006). The occurrence of resistant
L. monocytogenes isolates against 14 out of 18 antimicrobials tested in
the present study (Fig. 1) and susceptibility of all isolates to 4 of them
(moxioxacin, levooxacin, imipenem, ampicillin-sulbactam) is im-
portant data for the physicians and epidemiologists. It may be
problematic to combat possible listeriosis in the study area because
of the emergence of some strains resistant to commonly used anti-
microbials, although majority of them were susceptible. A promising
alternative might be the use of new uoroquinolones (e.g., moxi-
oxacin) (Grayo et al., 2008). The emergence of trimethoprim-
sulfamethoxazole resistance in L. monocytogenes, as observed by us or
other researchers (e.g., Conter et al., 2009), is of particular importance
because the antimicrobial has been successfully used for listeriosis
patients with beta-lactams intolerance (Charpentier and Courvalin,
1999a; Hof, 2004b). Importantly, 18.9% of our isolates showed
resistance to multiple antimicrobials ( 2, besides cefotaxime),
including 2 isolates showing resistance to N5 antimicrobials. Ade-
quate knowledge about the emergence and spread of antimicrobial
resistant L. monocytogenes is required to tackle clinical cases because
of the severity of listeriosis which may often become fatal.
Among our 14 tetracycline resistant isolates, 13 carried tet(M) gene
and 1 carried tet(S) gene. Although a series of tetracycline resis-
tant genes have been reported in many bacteria, only tet(L), tet(M),
and tet(S) have been observed in L. monocytogenes (Charpentier and
Courvalin, 1999a). Acquisitions of genes resistant to antimicrobials
(e.g., tet, cat221, ermAM, aad6 genes for tetracycline, chloramphenicol;
erythromycin; streptomycin, etc.) in L. monocytogenes have been
related to horizontal gene transfer via self-transmissible plasmids or
conjugative transposons (e.g., Tn1545Tn916) from other antimicro-
bial resistant bacteria, particularly those living in gastrointestinal tract
(Charpentier and Courvalin, 1999a). We also checked for the presence
of tet(B) gene commonly associated with Gram-negative bacteria
(Chopra and Roberts, 2001) and tet(K) gene reported in L. innocua
(Charpentier and Courvalin, 1999a) but none of them was detected.
Moreover, the absence of the selective tet genes in our tetracycline-
susceptible isolates suggests that these genes do not remain in a silent
form in L. monocytogenes as observed in case of some Gram-positive
bacteria, e.g., Streptococcus pyogenes (Brenciani et al., 2007). We have
observed that 9 out of 13 tet(M) positive isolates also carried Tn916.
Thus, our data also indicate that L. monocytogenes could act as a
reservoir of mobile tet genes along the food chain.
The present study illustrates a potential public health risk of L.
monocytogenes in northern China because of its sporadic occurrence in
various food items, particularly in raw meats, quick-frozen food made
of wheat our and rice as well as ready-to-eat products. Importantly,
Fig. 2. PFGE patterns and antimicrobial resistance proles of L. monocytogenes isolates from food samples. Black dot indicates resistance to a particular antimicrobial. AMP, ampicillin;
SAM, Ampicillin-sulbactam; CHL, chloramphenicol; CIP, ciprooxacin; CEF, cephalothin; CTX cefotaxime; DOX, doxycycline; ERY, erythromycin; GEN gentamicin; IPM imipenem;
LVX levooxacin; MOX, moxioxacin; PEN, penicillin; RIF, rifampin; STR, treptomycin; TET, tetracycline; VAN, vancomycin; SXT, trimethoprim-sulfamethoxazole. Source
abbreviations are as follows: a, raw pork; b, raw beef; c, raw chicken; d, raw mutton; e, cooked meats, k, quick-frozen food made of wheat our and rice; l, non-fermented bean
products; g, seafood; j, vegetables. The numbers 1 to 9 indicate nine surveillance points of food samples source.
315
majority of isolates belonged to the serotypes (1/2a, 1/2b and 4b)
capable of causing disease and many showed resistance to commonly
used antibiotics. The combination of antibiotic susceptibility pheno-
types along with PFGE and serotype analyses enabled us to identify
differences among some isolates. This strategy can be used for tracing
the outbreak-causing L. monocytogenes strain to the source of con-
tamination. To reduce the risk of L. monocytogenes infection in
northern China appropriate sanitation practices in food industries
are required, e.g., hazard analysis critical control point programs
should be followed to minimize contamination from original sources
or during processing. Consumers should be very careful for high-risk
foods, and take proper care for prevention of the growth of the
organism, e.g., short-term refrigerated storage of perishable foods and
cooking before consumption. For better understanding of L. mono-
cytogenes contamination sources and prevention strategies, a large
scale future study is required in particular endemic areas with sam-
ples collected from the consumers, retail food shops, food-processing
plants as well as from other natural sources.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (20877028), the Special Grade of the Financial Support from
China Postdoctoral Science Foundation (200902327), the Guangdong
Nature Science Foundation of China (10451064101005159), and the
Fundamental Research Funds for the Central Universities, SCUT
(2009ZM0224).
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