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Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008–2018
Journal Pre-proof Prevalence and Genetic characteristics of Cronobacter spp. from Food and Human Clinical Stool Samples in Wenzhou, China 2008-2018 Yi Li, Leyi Zhang, Yuqin Hu, Chengji Hong, Airong Xie, Yuejin Wu, Zhihui, Shangguan, Lingling Mei, Biao Zhou, Yanjun Zhang, Lei Fang PII:
S0740-0020(20)30021-6
DOI:
https://doi.org/10.1016/j.fm.2020.103432
Reference:
YFMIC 103432
To appear in:
Food Microbiology
Received Date: 10 September 2019 Revised Date:
6 January 2020
Accepted Date: 15 January 2020
Please cite this article as: Li, Y., Zhang, L., Hu, Y., Hong, C., Xie, A., Wu, Y., Zhihui, Shangguan, Mei, L., Zhou, B., Zhang, Y., Fang, L., Prevalence and Genetic characteristics of Cronobacter spp. from Food and Human Clinical Stool Samples in Wenzhou, China 2008-2018, Food Microbiology, https:// doi.org/10.1016/j.fm.2020.103432. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
1 2
Prevalence and Genetic characteristics of Cronobacter spp. from Food and Human Clinical Stool Samples in Wenzhou, China 2008-2018.
3
Yi Li1†, Leyi Zhang1†, Yuqin Hu1, Chengji Hong1, Airong Xie1, Yuejin Wu1, Zhihui1
4
Shangguan1, Lingling Mei2, Biao Zhou2, Yanjun Zhang2, Lei Fang2*
5
1
Wenzhou Center for Disease Control and Prevention, Wenzhou, China
6
2
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China
7 8
* First and chief corresponding author: Dr. Lei Fang, Zhejiang Provincial Center
9
for Disease Control and Prevention, 3399 Bincheng Road, Hangzhou, 310051, China;
10
[email protected]; 86-15168287896
11 12
Second corresponding author: Dr. Yanjun Zhang, Zhejiang Provincial Center for
13
Disease Control and Prevention, 3399 Bincheng Road, Hangzhou, 310051, China;
14
[email protected]; 86-13516809119
15
†
16
Key words: Cronobacter, commercial foods, stool, antibiotic resistance, MLST,
17
PFGE
These authors contributed equally to this work.
1
18
ABSTRACT
19
Pathogenic Cronobacter species are responsible for life-threatening illness in neonates.
20
A ten-year comprehensive survey was conducted to examine the population structure
21
and antimicrobial resistant patterns of Cronobacter isolates from food (n=78) and
22
clinical (n=12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were
23
recovered from 2051 collected samples. The occurrence of Cronobacter spp. was
24
highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate
25
of 1% was found in powered infant and toddler formula (7/494), special medical
26
infant formula (1/95) and human stool samples (12/1024). Cronobacter strains
27
revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel
28
electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster
29
consisted of four strains. Multilocus sequence typing (MLST) method displayed 43
30
sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most
31
frequently identified. Meanwhile, two new sequence types were discovered and added
32
to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv,
33
amoxicillin,
34
chloramphenicol, as well as multidrug resistance, was noted. Taken together, this
35
large-scale surveillance study highlights the wide dissemination and diverse
36
molecular features of Cronobacter spp. in Wenzhou China.
ampicillin,
cefoxitin,
tetracycline,
streptomycin,
azithromycin,
2
37
INTRODUCTION
38
Cronobacter spp. are known as emerging opportunistic bacteria within the
39
family of Enterobacteriaceae. The genus Cronobacter has been reported to be
40
phenotypically and genetically diverse and has been proposed to contain seven
41
species: C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C.
42
universalis, and C. condimenti (Iversen et al., 2008; Joseph et al., 2012b). These
43
emerging pathogens cause severe and potentially life-threatening diseases, including
44
septicemia, infantile meningitis, or necrotizing enterocolitis with an overall mortality
45
rate around 27% worldwide (Biering et al., 1989; Simmons et al., 1989; Clark et al.,
46
1990; Giovannini et al., 2008; Friedemann, 2009). Cronobacter can infect individuals
47
of all age groups, while immunocompromised infants and children (<5 years of age)
48
are considered to be at greatest risk (Holý et al., 2014; Patrick et al., 2014; Alsonosi et
49
al., 2015).
50
The major food commodity associated with Cronobacter infections is powered
51
infant formula (PIF) (Clark et al., 1990). Other food products such as milk powders,
52
herbs, spices and rice seeds have also been reported as potential vectors for
53
Cronobacter-borne illness (Farber, 2004; Iversen et al., 2004a). In addition to its
54
ability to survive in low moisture food products, Cronobacter spp. have been
55
recovered from clinical specimens, soil and dried pellets of animal feed in the farm
56
environment (Søgaard and Kjaeldgaard, 1986; Kandhai et al., 2004). Due to its high
57
risk to infants and immunocompromised adults, Cronobacter have lately received
58
great attention among the scientific community, health care providers, and the food 3
59
industry.
60
Sporadic cases and outbreaks of Cronobacter illness have reportedly occurred in
61
developed countries including England, Belgium, Canada, France, Germany and
62
several states in United Sates. The reported cases of Cronobacter-related disease are
63
still limited in China, which may be due to lack of awareness and reporting of this
64
pathogen rather than the absence of illness related to this microorganism. Wenzhou is
65
a coastal city and recognized as one of the biggest food markets in eastern China. The
66
prevalence and genetic characteristics of major foodborne pathogens including Vibrio
67
parahaemolyticus, Shigella, Salmonella, Escherichia coli, and norovirus in
68
contaminated food sources and clinical specimens were regularly monitored and
69
reported in Wenzhou (Guo et al., 2018); however, Cronobacter contamination in local
70
food market and hospitals remain largely unknown. This study examines the
71
epidemiological distribution, genetic structure and antibiotic resistant patterns of
72
Cronobacter spp. in Wenzhou in order to promote more reliable source tracking of
73
contaminated foods and enhance the resolution of surveillance.
74
MATERIALS AND METHODS
75
Sample collection
76
From 2008 to 2018, 1027 commercially available food samples comprising 180
77
bands (Supplementary Table) were collected by Wenzhou CDC and tested for the
78
presence of Cronobacter spp., and the collection sources included special medical
79
infant formula, powered adult formula, powered infant and toddler formula, spices 4
80
and baby cereals. 1024 stool specimens were collected from sporadic diarrhea
81
outpatients in Wenzhou hospitals. As controls, reference strains of C. sakazakii CICC
82
21560 and C. muytjensii ATCC 51329 were provided by China Center of Industrial
83
Culture Collection.
84
Isolation and identification of Cronobacter spp.
85
Food samples were weighed (100 g), suspended in 900 ml sterile BPW
86
(Buffered Peptone Water), and homogenized for one minute according to the National
87
Food Safety Standard of China Food Microbiology Manual (GB 4789.40–2010). The
88
homogenates were incubated at 37°C for 18 h, and 1 ml pre-enriched sample was
89
subsequently transferred to 10 ml of mLST – Vm (modified Lauryl Sulfate Broth
90
supplemented with vancomycin 10ug/ml; Hopebio, China) and incubated by another
91
24 h at 44°C. Human stool samples were collected from Wenzhou Integrated
92
Traditional Chinese and Western Medicine Hospital and Second Affiliated Hospital of
93
Wenzhou Medical University and transported to the laboratory in Cary-Blair medium
94
within 4 h. Enrichments and human stool samples were streaked onto CHROMagarTM
95
Cronobacter (CHROMagar Microbiology, France). All presumptive positive colonies
96
were further confirmed as Cronobacter spp. by Vitek 2 identification cards and the
97
V7.01 identification database according to the manufacturer’s instructions
98
(BioMerieux, France). All isolates were stored at -80°C in trypticase soy broth (TSB)
99
containing 20% glycerol for later analysis.
100
Multilocus sequence typing 5
101
Multilocus sequence typing (MLST) was performed by the sequence analysis of
102
seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB and ppsA) according to
103
the protocol available on the PubMLST website (http://pubmlst.org/cronobacter/).
104
Genomic DNA was extracted by Rapid Bacterial Genomic DNA Isolation Kit (Omega
105
Bio-Teck, Dorvaville, USA) and amplified using the seven primer pairs described
106
previously (Baldwin et al., 2009). DNA extracts were sequenced by Qinke Biotech
107
Limited (Hangzhou, China). The nucleotide sequences for each locus were analyzed
108
with BioNumerics software version 7.5 (Applied Maths, Belgium) and compared to
109
published sequences on the PubMLST website. Sequence types (STs) were
110
determined according the obtained seven-digit allelic profiles. A phylogenetic tree was
111
generated using the unweighted pair-group method with arithmetic means (UPGMA)
112
and the minimum spanning tree (MST) methods. Species identification was
113
determined by classical MLST technique according to Joseph’s method (Joseph et al.,
114
2012a).
115
Pulsed-field Gel Electrophoresis subtyping of Cronobacter.
116
Pulsed-field gel electrophoresis was performed based on the CDC PulseNet
117
protocol for Cronobacter spp. Briefly, DNA was digested with 40 U of XbaI enzyme
118
(TaKaRa, Japan) at 36°C for 2 h. The separation of restriction fragments was
119
performed in 1% SeaKem gold agarose (Lonza, Switzerland) gels in 0.5x
120
Trisborate-EDTA (TBE) buffer (Milliporesigma, Burlington, MA) using the CHEF-
121
Mapper system (Bio-Rad), with the following parameters: initial switch time, 2.16 s;
122
final switch time, 63.8 s for 18.5 h at 6 V/cm and condensation temperature of 14°C. 6
123
The DNA banding patterns were visualized under UV light with GelDocTM
124
XR+system (Bio-Rad, Hercules, CA) after staining with GelRedTM (0.5 ug/ml;
125
Biotium, CA), and analyzed using BioNumerics software version 7.5 (Applied Maths,
126
Kortrijk, Belgium). XbaI-digested Salmonella enterica serovar Braenderup H9812
127
was used as the molecular weight standard. The dendrogram was created by UPGMA
128
with the Dice similarity coefficient and a position tolerance of 1.5%. Clusters were
129
defined based on an 85% similarity cutoff.
130
Antimicrobial susceptibility testing
131
The antibiotic susceptibility of 90 recovered Cronobacter isolates was
132
determined using Gram Negative MIC plates CMV3AGNF (Thermo Scientific,
133
Waltham,
134
trimethoprim/sulfamethoxazole, sulfanilamideisoxazole, gentamicin, ciprofloxacin,
135
nalidixic acid, tetracycline, streptomycin, azithromycin, cefotaxiv, chloramphenicol,
136
amoxicillin, ampicillin and cefoxitin. After incubation at 37°C for 18 h, the plates
137
were read and interpreted automatically by Sensititre Vision Digital MIC Viewing
138
System, and susceptibility or resistance pattern of the Cronobacter isolates to selected
139
antimicrobials was compared to the recorded concentration of the control organism
140
Escherichia coli ATCC 25922. Bacteria were classified as resistant, intermediate or
141
sensitive according to clinical and laboratory test Standard (Clinical and Laboratory
142
Standards Institute, 2019).
143
Results
MA)
against
fourteen
antibiotics,
including
ceftriaxone,
7
144
Prevalence of Cronobacter spp. in Wenzhou, China
145
As shown in Table 1, Cronobacter strains were isolated in Wenzhou retail foods
146
(n=78) and clinical samples (n=12) from 2008 to 2018. The highest percentage of
147
Cronobacter spp. isolates was found in spice samples (22%, 26/119), followed by
148
baby cereals (17%, 41/248), powered adult formula (4%, 3/71), special medical infant
149
formula (1%, 1/95), powered infant and toddler formula (1%, 7/494), and stool
150
specimens (1%, 12/1024).
151
Molecular characterization of Cronobacter spp. isolates
152
To investigate the population structure of Cronobacter spp. and relationship
153
between pathogenicity and specific lineages, seven housekeeping gene atpD, fusA,
154
glnS, gltB, gyrB, infB and ppsA were sequenced. MLST analysis was performed
155
according to Cronobacter PubMLST database (https://pubmlst.org/cronobacter/).
156
Species identification was determined by classical MLST technique according to
157
Joseph’s method (Joseph et al., 2012a). Comparison of the percentage samples for
158
each species was used to infer prevalence. C. sakazakii was recovered more
159
frequently (69%) than C. malonaticus (22%) and any other Cronobacter spp. (9%).
160
For example, thirty-one samples were positive in baby cereals for C. sakazakii, which
161
was significantly (P < 0.05) higher compared to C. malonaticus (n=8), C. muytjensii
162
(n=1) and C. dublinensis (n=1). All human stool samples and food samples were
163
negative for C. condimenti or C. turicensis, while four spice samples harbored both
164
species. Overall, a high phylogenetic diversity was observed among the ninety 8
165
isolates by PFGE analysis. Notably, 43 STs were overlaid onto the tree which
166
including two novel STs, ST 684 and ST 685 (Figure 1). There were 25 STs in C.
167
sakazakii isolates. ST1 was the dominant sequence type (12.3%, n=11), followed by
168
ST 64 (7.8%, n=7), ST4 = ST8 = ST148 (5.6%, n=5), ST40 (4.4%, n=4) and ST13
169
(3.3%, n=3). ST1, ST4, ST8, ST148, ST40 and ST13 were found in both retail foods
170
and clinical samples as baby cereal (n=18), human stool samples (n=7) and powered
171
infant and toddler formula (n=7) were found more frequently than other samples;
172
whereas ST64 were only recovered from baby cereals (n=6) and special medical
173
infant formula (n=1) but not clinical samples.
174
Moreover, the primary sequence type of C. malonaticus was ST201 (5.6%, n=5)
175
with two isolates collected from powered infant and toddler formula and three
176
positive clinical samples, followed by ST7 (4.4%, n=4), ST440 (2.2%, n=2) and
177
ST129 (2.2%, n=2). In addition, C. sakazakii CICC 21560 represented the same ST8
178
with six isolates collected from baby cereals (n=2), children clinical samples (n=1)
179
and spices (n=2), whereas C. muytjensii ATCC 51329 showed a distinct ST81 from all
180
the isolates in the study.
181
Ninety isolates of Cronobacter were subsequently examined for genetic
182
relatedness using a PFGE DNA fingerprinting technique, and four isolates could not
183
be digested with XbaI. Correspondingly, BioNumerics software analysis showed the
184
remaining 86 isolates indicating 75 distinguishable patterns at an 85% similarity
185
threshold (Figure 2). C. sakazakii and C. malonaticus strains formed 48 and 16
186
pulsotypes, respectively. Specifically, C. sakazakii ST1 (n=11) was further divided 9
187
into 9 pulsotypes, and C. sakazakii ST4 (n=5) contained 4 pulsotypes. Furthermore,
188
C. sakazakii ST64 (n=7) harbored 4 PFGE patterns, which were originated from baby
189
cereals (n=6) and special medical infant formula (n=1). All clinical isolates consisted
190
of C. sakazakii (n=6) and C. malonaticus (n=4) revealed 8 distinct patterns.
191
Antibiotic Susceptibility Analysis
192
The 90 Cronobacter spp. isolates were subjected to 14 antimicrobial
193
susceptibility tests (Table 2) using minimum broth dilution method. Most isolates
194
(84%) were sensitive or exhibited only intermediate resistance to all antibiotics
195
assessed.
196
trimethoprim/sulfamethoxazole, gentamicin, ciprofloxacin, and nalidixic acid.
197
Cronobacter
198
chloramphenicol (29%), followed by cefotaxiv (28%), cefoxitin (24%), amoxicillin
199
(12%), ampicillin (12%), streptomycin (11%), azithromycin (7%), tetracycline (6%)
200
and ceftriaxone (1%). There was no significant difference among C. sakazakii, C.
201
malonaticus, C. dublinensis, C. condimenti, C. turicensis and C. muytjensii. Fourteen
202
isolates (16%) showed multidrug resistance (resistant against two or more classes of
203
antimicrobials), including two isolates that were resistant to more than five antibiotics
204
and six isolates that were resistant to six antibiotics.
205
DISCUSSION
All
isolates
strains
were
were
resistant
susceptible
or
showed
to
sulfanilamideisoxazole,
intermediate
resistance
to
206
Cronobacter spp. are opportunistic foodborne pathogen that are isolated from
207
environment samples and food products. Deciphering food sources as potential 10
208
reservoirs in driving the persistence and virulence potentials of Cronobacter spp. is
209
the key to mitigating potential Cronobacter-borne disease to humans. A continuous
210
surveillance was therefore conducted from Wenzhou sentinel hospitals and retail
211
foods for one decade.
212
C. sakazakii isolates were more widely distributed among retail foods and
213
human stool samples relative to the other pathogenic Cronobacter spp. which were
214
less prevalent in Wenzhou. These results support the premise that C. sakazakii is the
215
predominant foodborne pathogen among Cronobacter spp. (van Acker et al., 2001),
216
and other studies have examined prevalence of C. sakazakii elsewhere with similar
217
results as those reported herein (Brandão et al., 2017; Li et al., 2019). The presence of
218
C. sakazakii and C. malonaticus from clinical samples was previously noted (Cui et
219
al., 2014) and was reaffirmed by the present survey, as human stool samples from
220
diarrheal patients were exclusively defined as either C. sakazakii or C. malonaticus.
221
Thus, our results support the hypothesis that C. sakazakii and C. malonaticus may be
222
more closely associated with human host, while C. dublinensis, C. muytjensii and
223
other Cronobacter spp. are more likely inhabitants of environmental commensals with
224
unclear clinical significance (Iversen et al., 2004b; Schmid et al., 2009). The
225
understanding of true epidemiology and virulence potential for these organisms is still
226
poor and is likely to be complex, due to the diversity of these species. Grim and
227
colleagues found unique ferric dicitrate transport system in a small subset of C.
228
sakazakii and C. malonaticus but was absent in other Cronobacter species, indicating
229
iron acquisition system may contribute to the virulence of Cronobacter infections by 11
230
helping strains to acquire iron directly or indirectly from human hosts (Grim et al.,
231
2012). Nevertheless, the presence of other virulence factors including Cronobacter
232
plasminogen activator, type 6 secretion system, and associated putative adhesins
233
depended on species (Singh et al., 2015).
234
This survey revealed that a wide spectrum of food products was contaminated
235
with six species of Cronobacter spp. It is noteworthy that Cronobacter was isolated at
236
highest frequency (22%) in spice samples. These species were spread widely in spice
237
samples, suggesting high risk potential with the consumption of contaminated spices.
238
Spices have been reportedly implicated in foodborne outbreaks and recalls caused by
239
other enteric pathogens (Keller et al., 2013; Van Doren et al., 2013), as spices in the
240
desiccated state may provide appropriate conditions that allows the survival of
241
Cronobacter spp. as well as other foodborne pathogens such as Salmonella spp.,
242
Escherichia coli O157:H7, Bacillus cereus, and Clostridium perfringens ((CDC),
243
2010; Gurtler and Keller, 2019). In addition, 17% of baby cereal samples were
244
positive for Cronobacter spp., including C. sakazakii, C. malonaticus, C. muytjensii,
245
and C. dublinensis. The low prevalence of Cronobacter spp. in powered infant and
246
toddler formula (1%) contrasts with a prior report that Cronobacter found in 75% of
247
infant formula in United States (Clark et al., 1990). It should be noted that raw
248
ingredients, preparation equipment and personnel may contribute to the extrinsic
249
contamination of Cronobacter in food products. In a survey of milk power
250
manufacturing plant was reported to contaminated with C. sakazakii and in the
251
equipment of spray drying, fluidized bed drying, and packaging (Fei et al., 2015). 12
252
Inappropriate food safety practices may facilitate the emergence of new pathogenic
253
lineages from other nonpathogenic strains by horizontal transfer and gene
254
recombination. Meanwhile, the ability of Cronobacter to form biofilm enhanced their
255
persistence in adverse environmental conditions (Lehner et al., 2005). Therefore, new
256
food safety measurements and hygiene strategies are essential to reduce
257
Cronobacter-related contamination in food products as well as their environment.
258
Molecular based typing approaches demonstrated a high degree of diversity of
259
the strains tested. MLST revealed 25 STs for C. sakazakii out of 62 isolates. Seven of
260
which were previously associated with C. sakazakii infection (Joseph and Forsythe,
261
2011; Cui et al., 2014). All 20 C. malonaticus strains were segregated into 11 different
262
STs, including ST664 identified from a powered adult formula sample. Despite a low
263
contamination rate, the presence of C. malonaticus in powered adult formula poses a
264
potential risk to elderly people because C. malonaticus has been predominantly
265
associated with adult infection (Holý et al., 2014). Moreover, the identification of
266
novel ST685 from spice samples illustrates future surveillance on genetic
267
characteristic of Cronobacter spp. including new food routes/ related environment are
268
necessary, especially those from infections with unknown sources.
269
The 78 Cronobacter isolates cultured from food products, along with the
270
additional 12 clinical strains, were further investigated using PFGE. Although four
271
strains were untypeable, PFGE data offered superior ability for discrimination of
272
Cronobacter spp. compared to MLST analysis. For example, 37 patterns were found
273
among 41 baby cereal samples, while only 26 STs were discovered using MLST 13
274
method. Powered infant and toddler formula samples harbored seven pulsotypes but
275
only contained four STs. In particular, the observation of C. sakazakii MLST ST4 in
276
both of the baby cereal mixes and powered infant and toddler formula was highly
277
noteworthy because it has been proposed that C. sakazakii ST4 was especially
278
problematic to neonatal meningitis (Joseph and Forsythe, 2011; Hariri et al., 2013).
279
The identification of C. sakazakii ST12 in baby cereals was also significant, as it is a
280
specific clonal linage linked with necrotizing enterocolitis (Forsythe et al., 2014). To
281
the best of our knowledge, this is the first documented report demonstrating infant
282
supplementary food contaminated with C. sakazakii ST12. Our result provides
283
evidence that infants with the consumption of baby cereals or powered infant and
284
toddler formula posed potential risks and may thereby lead to severe disease.
285
Susceptibility tests revealed that all isolates were sensitive to the antibiotics in
286
the classes for inhibiting DNA or folic acid synthesis. However, several food samples
287
exhibited high or intermediate resistance to the antibiotics in the classes for cell wall
288
synthesis (cefotaxiv, cefoxitin, amoxicillin and ceftriaxone) and protein inhibitors
289
(chloramphenicol, streptomycin, azithromycin and tetracycline). These results
290
differed from previous reports that found Cronobacter spp. strains isolated from food
291
samples susceptible to almost all the antibiotics testes (Molloy et al., 2009; Terragno
292
et al., 2009; Brandão et al., 2017). Differences in antimicrobial resistance (AMR)
293
were also observed among strains of foods and clinical settings. Clinical samples only
294
showed resistance or reduced susceptibility to the antibiotics of cefotaxiv, cefoxitin,
295
and chloramphenicol, suggesting that traditional antibiotic regimen (ampicillin in 14
296
combination with either gentamicin or chloramphenicol) for Cronobacter infection
297
remain effective in Wenzhou. Multi-antimicrobial drug resistance characterized 16%
298
of the Cronobacter isolates, including fourteen spice isolates and one clinical sample,
299
indicating a potential public health risk. Although the rate of multi-drug resistance is
300
not high, the potential acquisition of antibiotic resistance by extensive use of
301
antibiotics in the farm or aquaculture may still exists. Therefore, the surveillance of
302
Cronobacter spp. in spices and other food products should be strengthened to
303
untangle the impact of microbial ecology to AMR gene transfer.
304
This study has provided scientific evidence on the contamination rate of
305
Cronobacter spp. from powered formula and other dried food products commercially
306
available in Wenzhou, China. Although the virulence potential of Cronobacter spp.
307
has not been fully elucidated, this systematic and long-term survey provided basis
308
guidance and vital knowledge to government agencies for performing risk assessment
309
and intervention strategies to control Cronobacter-related disease in human.
310
Acknowledgement
311
This work was funded by Major National Science and Technology Projects in the 13rd
312
Five-year Plan (2017ZX10103008-002), National Health Commission Scientific
313
Research Projects (WKJ-ZJ-1917), Ministry of Science and Technology of China (No.
314
2017YFC1601503), and Wenzhou Medical and Health Science Research Projects (No.
315
2019B03). Thank you to Dr. Anita C. Wright from University of Florida for assistance
316
in polishing the English of our manuscript.
15
317
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CDC, 2010. Salmonella montevideo infections associated with salami products made with contaminated imported black and red pepper --- United States, July 2009-April 2010. MMWR Morb. Mortal. Wkly Rep. 59, 1647-1650. https://www.cdc.gov/mmwr/preview/mmwrhtml/mm5950a3.htm. Alsonosi, A., Hariri, S., Kajsík, M., Oriešková, M., Hanulík, V., Röderová, M., Petrželová, J., Kollárová, H., Drahovská, H., Forsythe, S., Holý, O., 2015. The speciation and genotyping of Cronobacter isolates from hospitalized patients. Eur. J. Clin. Microbiol. Infect. Dis. 34, 1979-1988. http://dx.doi.org/10.1007/s10096-015-2440-8. Baldwin, A., Loughlin, M., Caubilla-Barron, J., Kucerova, E., Manning, G., Dowson, C., Forsythe, S., 2009. Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes. BMC Microbiol. 9, 223. http://dx.doi.org/10.1186/1471-2180-9-223. Biering, G., Karlsson, S., Clark, N.C., Jónsdóttir, K.E., Lúdvígsson, P., Steingrímsson, O., 1989. Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk. J. Clin. Microbiol. 27, 2054-2056. Brandão, M.L.L., Umeda, N.S., Jackson, E., Forsythe, S.J., de Filippis, I., 2017. Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods. Food Microbiol. 63, 129-138. https://doi.org/10.1016/j.fm.2016.11.011. Clark, N.C., Hill, B.C., O'Hara, C.M., Steingrimsson, O., Cooksey, R.C., 1990. Epidemiologic typing of Enterobacter sakazakii in two neonatal nosocomial outbreaks. Diagn. Microbiol. Infect. Dis. 13, 467-472. Cui, J., Du, X., Liu, H., Hu, G., Lv, G., Xu, B., Yang, X., Li, W., Cui, Z., 2014. The genotypic characterization of Cronobacter spp. isolated in China. PLoS One 9, e102179. https://doi.org/10.1371/journal.pone.0102179. Farber, J. M., 2004. Enterobacter sakazakii--new foods for thought? Lancet. 363, 5-6. https://doi.org/10.1016/S0140-6736(03)15244-0. Fei, P., Man, C., Lou, B., Forsythe, S. J., Chai, Y., Li, R., Niu, J., & Jiang, Y., 2015. Genotyping and source tracking of Cronobacter sakazakii and C. malonaticus isolates from powdered infant formula and an infant formula production factory in China. Appl. Environ. Microbiol. 81, 5430-5439. http://dx.doi.org/10.1128/AEM.01390-15.
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11. Friedemann, M., 2009. Epidemiology of invasive neonatal Cronobacter
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12. Giovannini, M., Verduci, E., Ghisleni, D., Salvatici, E., Riva, E., Agostoni, C., 2008. Enterobacter sakazakii: an emerging problem in pediatrics nutrition. J. Int. Med. Res. 36, 394-399. http://dx.doi.org/10.1177/147323000803600303.
362 363 364 365
13. Grim, C. J., Kothary, M. H., Gopinath, G., Jarvis, K. G., Beaubrun, J. J., McClelland, M., Tall, B. D., Franco, A. A., 2012. Identification and characterization of Cronobacter iron acquisition systems. Appl. Environ. Microbiol. 78, 6035-6050. http://dx.doi.org/10.1128/AEM.01457-12.
366 367 368
14. Guo, S., Lin, D., Wang, L. L., Hu, H., 2018. Monitoring the results of foodborne diseases in Sentinel Hospitals in Wenzhou city, China from 2014 to 2015. Iran J. Public Health. 47, 674-681.
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15. Gurtler, J. B., Keller, S. E., 2019. Microbiological safety of dried spices. Annu. Rev. Food Sci. Technol. 10, 409-427. http://dx.doi.org/10.1146/annurev-food-030216-030000.
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16. Hariri, S., Joseph, S., Forsythe, S. J., 2013. Cronobacter sakazakii ST4 strains and neonatal meningitis, United States. Emerg. Infect. Dis. 19, 175-177. http://dx.doi.org/10.3201/eid1901.120649.
375 376 377 378
17. Holy, O., Petrzelova, J., Hanulik, V., Chroma, M., Matouskova, I., Forsythe, S. J., 2014. Epidemiology of Cronobacter spp. isolates from patients admitted to the Olomouc University Hospital (Czech Republic). Epidemiol. Mikrobiol. Imunol. 63, 69-72.
379 380 381
18. Iversen, C., Druggan, P., Forsythe, S., 2004. A selective differential medium for Enterobacter sakazakii, a preliminary study. Int. J. Food Microbiol. 96, 133-139. http://dx.doi.org/10.1016/j.ijfoodmicro.2004.01.024.
382 383 384 385
19. Iversen, C., Lane, M., Forsythe, S. J., 2004. The growth profile, thermotolerance and biofilm formation of Enterobacter sakazakii grown in infant formula milk. Lett. Appl. Microbiol. 38, 378-382. http://dx.doi.org/10.1111/j.1472-765X.2004.01507.x.
386 387 388 389 390 391 392 393
20. Iversen, C., Mullane, N., McCardell, B., Tall, B. D., Lehner, A., Fanning, S., Stephan, R., Joosten, H., 2008. Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi 17
394 395
subsp. nov. Int. J. Syst. Evol. Microbiol. 58, 1442-1447. http://dx.doi.org/10.1099/ijs.0.65577-0.
396 397 398
21. Joseph, S., Forsythe, S. J., 2011. Predominance of Cronobacter sakazakii sequence type 4 in neonatal infections. Emerg. Infect. Dis. 17, 1713-1715. http://dx.doi.org/10.3201/eid1709.110260.
399 400 401 402 403
22. Joseph, S., Cetinkaya, E., Drahovska, H., Levican, A., Figueras, M. J., Forsythe, S. J., 2012. Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients. Int. J. Syst. Evol. Microbiol. 62, 1277-1283. http://dx.doi.org/10.1099/ijs.0.032292-0.
404 405 406
23. Joseph, S., Sonbol, H., Hariri, S., Desai, P., McClelland, M., Forsythe, S. J., 2012. Diversity of the Cronobacter genus as revealed by multilocus sequence typing. J. Clin. Microbiol. 50, 3031-3039. http://dx.doi.org/10.1128/JCM.00905-12.
407 408 409 410
24. Kandhai, M. C., Reij, M. W., Gorris, L. G., Guillaume-Gentil, O., van Schothorst, M., 2004. Occurrence of Enterobacter sakazakii in food production environments and households. Lancet. 363, 39-40. http://dx.doi.org/10.1016/S0140-6736(03)15169-0.
411 412 413
25. Keller, S. E., VanDoren, J. M., Grasso, E. M., Halik, L. A., 2013. Growth and survival of Salmonella in ground black pepper (Piper nigrum). Food Microbiol. 34, 182-188. http://dx.doi.org/10.1016/j.fm.2012.12.002.
414 415 416 417
26. Lehner, A., Tasara, T., & Stephan, R., 2004. 16S rRNA gene-based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification. BMC Microbiol. 4, 43. http://dx.doi.org/10.1186/1471-2180-4-43.
418 419 420 421 422
27. Lehner, A., Riedel, K., Eberl, L., Breeuwer, P., Diep, B., Stephan, R., 2005. Biofilm formation, extracellular polysaccharide production, and cell-to-cell signaling in various Enterobacter sakazakii strains: aspects promoting environmental persistence. J. Food Prot. 68, 2287-2294. http://dx.doi.org/10.4315/0362-028x-68.11.2287.
423 424 425 426 427
28. Li, C., Zeng, H., Zhang, J., He, W., Ling, N., Chen, M., Wu, S., Lei, T., Wu, H., Ye, Y., Ding, Y., Wang, J., Wei, X., Zhang, Y., Wu, Q., 2019. Prevalence, antibiotic susceptibility, and molecular characterization of Cronobacter spp. Isolated from edible mushrooms in China. Front Microbiol. 10, 283. http://dx.doi.org/10.3389/fmicb.2019.00283.
428 429 430 431 432
29. Molloy, C., Cagney, C., O'Brien, S., Iversen, C., Fanning, S., Duffy, G., 2009. Surveillance and characterization by pulsed-field gel electrophoresis of Cronobacter spp. in farming and domestic environments, food production animals and retail foods. Int. J. Food Microbiol. 136, 198-203. http://dx.doi.org/10.1016/j.ijfoodmicro.2009.07.007. 18
433 434 435 436
30. Patrick, M. E., Mahon, B. E., Greene, S. A., Rounds, J., Cronquist, A., Wymore, K., Boothe, E., Lathrop, S., Palmer, A., Bowen, A., 2014. Incidence of Cronobacter spp. Infections, United States, 2003–2009. Emerg. Infect. Dis. 20, 1536-1539. http://dx.doi.org/10.3201/eid2009.140545.
437 438 439 440
31. Phillips, I., Casewell, M., Cox, T., De Groot, B., Friis, C., Jones, R., Nightingale, C., Preston, R., Waddell, J., 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J. Antimicrob. Chemother. 53, 28-52. http://dx.doi.org/10.1093/jac/dkg483.
441 442 443 444
32. Ruiz-Bolivar, Z., Neuque-Rico, M. C., Poutou-Pinales, R. A., Carrascal-Camacho, A. K., Mattar, S., 2011. Antimicrobial susceptibility of Listeria monocytogenes food isolates from different cities in Colombia. Foodborne Pathog. Dis. 8, 913-919. http://dx.doi.org/10.1089/fpd.2010.0813.
445 446 447 448
33. Schmid, M., Iversen, C., Gontia, I., Stephan, R., Hofmann, A., Hartmann, A., Jha, B., Eberl, L., Riedel, K., Lehner, A., 2009. Evidence for a plant-associated natural habitat for Cronobacter spp. Res. Microbiol. 160, 608-614. http://dx.doi.org/10.1016/j.resmic.2009.08.013.
449 450 451 452
34. Simmons, B. P., Gelfand, M. S., Haas, M., Metts, L., Ferguson, J., 1989. Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect. Control Hosp. Epidemiol. 10, 398-401.
453 454 455
35. Singh, N., Goel, G., Raghav, M., 2015. Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii. Virulence. 6, 433-440. http://dx.doi.org/10.1007/s00284-015-0816-8.
456 457 458
36. Sogaard, P., Kjaeldgaard, P., 1986. Two isolations of enteric group 69 from human clinical specimens. Acta Pathol. Microbiol. Immunol. Scand B. 94, 365-367.
459 460 461 462
37. Terragno, R., Salve, A., Pichel, M., Epszteyn, S., Brengi, S., Binsztein, N., 2009. Characterization and subtyping of Cronobacter spp. from imported powdered infant formulae in Argentina. Int. J. Food Microbiol. 136, 193-197. http://dx.doi.org/10.1016/j.ijfoodmicro.2009.10.013.
463 464 465 466
38. van Acker, J., de Smet, F., Muyldermans, G., Bougatef, A., Naessens, A., Lauwers, S., 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39, 293-297. http://dx.doi.org/10.1128/JCM.39.1.293-297.2001.
467 468 469 470
39. Van Doren, J. M., Neil, K. P., Parish, M., Gieraltowski, L., Gould, L. H., Gombas, K. L., 2013. Foodborne illness outbreaks from microbial contaminants in spices, 1973-2010. Food Microbiol. 36, 456-464. http://dx.doi.org/10.1016/j.fm.2013.04.014.
19
471
Figure legends
472
Figure 1 The minimum spanning tree of clinical and foodborne Cronobacter strains based on the allelic profiles. Cronobacter species are distinguished by color differences. Size of circle indicates number of isolates within the same ST, and thickness of the branches represents the degree of similarity among Cronobacters tested.
473 474 475 476 477 478
Figure 2 Dendrogram of 90 Cronobacter spp. based on XbaI-mediated PFGE profiles. Strain source, isolation date and STs are also included.
20
Table 1. Occurrence of Cronobacter species in food and clinical samples in Wenzhou Sample
No. of sample
Prevalence of positive samples1
C. sakazakii
C. malonaticus
C. muytjensii
C. dublinensis
C. condimenti
C. turicensis
Species isolated (Sample)
Special medical infant formula
95
1%
1
-
-
-
-
-
Powered adult formula
71
4%
2
1
-
-
-
-
Powered infant and toddler formula
494
1%
6
1
-
-
-
-
Spices
119
22%
14
6
1
1
2
2
Baby cereals
248
17%
31
8
1
1
-
-
Stool specimens
1024
1%
8
4
-
-
-
-
Total
2051
4%
62
20
2
2
2
2
1
Prevalence % = n positive / n tested
Table 2. Antibiotic susceptibility test result Antibiotic mechanism1
Resistant2 (%)
Intermediate (%)
Sensitive (%)
Ciprofloxacin
0
0
100
Nalidixic acid
0
0
100
Sulfanilamide Isoxazole
0
0
100
Trimethoprim/Sulfamethoxazole
0
0
100
Ceftriaxone
1
0
99
Cefotaxiv
8
20
72
Amoxicillin
10
2
88
Ampicillin
11
1
88
DNA synthesis inhibitors
Folic acid synthesis inhibitors
Cell wall synthesis
Cefoxitin
18
7
76
Gentamicin
0
0
100
Tetracycline
1
4
94
Streptomycin
3
8
89
Azithromycin
7
0
93
Chloramphenicol
9
20
71
Protein synthesis inhibitors
1
Antibiotics are classified based on the mechanism for antimicrobial activity.
2
Resistant, intermediate and sensitive percentages refer to the percentage of strains.
Highlights 1. Cronobacter was dispersed widely in various food products. 2. Consumption of spices may pose a high risk for Cronobacter infection. 3. Two STs (ST685 and ST684) were newly assigned. 4. The combination of MLST and PFGE methods revealed high genetic diversity in Cronobacter spp., as 43 sequence types and 75 clonal groups were displayed. 5. 16% of the Cronobacter isolates exhibited multidrug resistance.
S0740-0020(20)30021-6
DOI:
https://doi.org/10.1016/j.fm.2020.103432
Reference:
YFMIC 103432
To appear in:
Food Microbiology
Received Date: 10 September 2019 Revised Date:
6 January 2020
Accepted Date: 15 January 2020
Please cite this article as: Li, Y., Zhang, L., Hu, Y., Hong, C., Xie, A., Wu, Y., Zhihui, Shangguan, Mei, L., Zhou, B., Zhang, Y., Fang, L., Prevalence and Genetic characteristics of Cronobacter spp. from Food and Human Clinical Stool Samples in Wenzhou, China 2008-2018, Food Microbiology, https:// doi.org/10.1016/j.fm.2020.103432. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
1 2
Prevalence and Genetic characteristics of Cronobacter spp. from Food and Human Clinical Stool Samples in Wenzhou, China 2008-2018.
3
Yi Li1†, Leyi Zhang1†, Yuqin Hu1, Chengji Hong1, Airong Xie1, Yuejin Wu1, Zhihui1
4
Shangguan1, Lingling Mei2, Biao Zhou2, Yanjun Zhang2, Lei Fang2*
5
1
Wenzhou Center for Disease Control and Prevention, Wenzhou, China
6
2
Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China
7 8
* First and chief corresponding author: Dr. Lei Fang, Zhejiang Provincial Center
9
for Disease Control and Prevention, 3399 Bincheng Road, Hangzhou, 310051, China;
10
[email protected]; 86-15168287896
11 12
Second corresponding author: Dr. Yanjun Zhang, Zhejiang Provincial Center for
13
Disease Control and Prevention, 3399 Bincheng Road, Hangzhou, 310051, China;
14
[email protected]; 86-13516809119
15
†
16
Key words: Cronobacter, commercial foods, stool, antibiotic resistance, MLST,
17
PFGE
These authors contributed equally to this work.
1
18
ABSTRACT
19
Pathogenic Cronobacter species are responsible for life-threatening illness in neonates.
20
A ten-year comprehensive survey was conducted to examine the population structure
21
and antimicrobial resistant patterns of Cronobacter isolates from food (n=78) and
22
clinical (n=12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were
23
recovered from 2051 collected samples. The occurrence of Cronobacter spp. was
24
highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate
25
of 1% was found in powered infant and toddler formula (7/494), special medical
26
infant formula (1/95) and human stool samples (12/1024). Cronobacter strains
27
revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel
28
electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster
29
consisted of four strains. Multilocus sequence typing (MLST) method displayed 43
30
sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most
31
frequently identified. Meanwhile, two new sequence types were discovered and added
32
to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv,
33
amoxicillin,
34
chloramphenicol, as well as multidrug resistance, was noted. Taken together, this
35
large-scale surveillance study highlights the wide dissemination and diverse
36
molecular features of Cronobacter spp. in Wenzhou China.
ampicillin,
cefoxitin,
tetracycline,
streptomycin,
azithromycin,
2
37
INTRODUCTION
38
Cronobacter spp. are known as emerging opportunistic bacteria within the
39
family of Enterobacteriaceae. The genus Cronobacter has been reported to be
40
phenotypically and genetically diverse and has been proposed to contain seven
41
species: C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C.
42
universalis, and C. condimenti (Iversen et al., 2008; Joseph et al., 2012b). These
43
emerging pathogens cause severe and potentially life-threatening diseases, including
44
septicemia, infantile meningitis, or necrotizing enterocolitis with an overall mortality
45
rate around 27% worldwide (Biering et al., 1989; Simmons et al., 1989; Clark et al.,
46
1990; Giovannini et al., 2008; Friedemann, 2009). Cronobacter can infect individuals
47
of all age groups, while immunocompromised infants and children (<5 years of age)
48
are considered to be at greatest risk (Holý et al., 2014; Patrick et al., 2014; Alsonosi et
49
al., 2015).
50
The major food commodity associated with Cronobacter infections is powered
51
infant formula (PIF) (Clark et al., 1990). Other food products such as milk powders,
52
herbs, spices and rice seeds have also been reported as potential vectors for
53
Cronobacter-borne illness (Farber, 2004; Iversen et al., 2004a). In addition to its
54
ability to survive in low moisture food products, Cronobacter spp. have been
55
recovered from clinical specimens, soil and dried pellets of animal feed in the farm
56
environment (Søgaard and Kjaeldgaard, 1986; Kandhai et al., 2004). Due to its high
57
risk to infants and immunocompromised adults, Cronobacter have lately received
58
great attention among the scientific community, health care providers, and the food 3
59
industry.
60
Sporadic cases and outbreaks of Cronobacter illness have reportedly occurred in
61
developed countries including England, Belgium, Canada, France, Germany and
62
several states in United Sates. The reported cases of Cronobacter-related disease are
63
still limited in China, which may be due to lack of awareness and reporting of this
64
pathogen rather than the absence of illness related to this microorganism. Wenzhou is
65
a coastal city and recognized as one of the biggest food markets in eastern China. The
66
prevalence and genetic characteristics of major foodborne pathogens including Vibrio
67
parahaemolyticus, Shigella, Salmonella, Escherichia coli, and norovirus in
68
contaminated food sources and clinical specimens were regularly monitored and
69
reported in Wenzhou (Guo et al., 2018); however, Cronobacter contamination in local
70
food market and hospitals remain largely unknown. This study examines the
71
epidemiological distribution, genetic structure and antibiotic resistant patterns of
72
Cronobacter spp. in Wenzhou in order to promote more reliable source tracking of
73
contaminated foods and enhance the resolution of surveillance.
74
MATERIALS AND METHODS
75
Sample collection
76
From 2008 to 2018, 1027 commercially available food samples comprising 180
77
bands (Supplementary Table) were collected by Wenzhou CDC and tested for the
78
presence of Cronobacter spp., and the collection sources included special medical
79
infant formula, powered adult formula, powered infant and toddler formula, spices 4
80
and baby cereals. 1024 stool specimens were collected from sporadic diarrhea
81
outpatients in Wenzhou hospitals. As controls, reference strains of C. sakazakii CICC
82
21560 and C. muytjensii ATCC 51329 were provided by China Center of Industrial
83
Culture Collection.
84
Isolation and identification of Cronobacter spp.
85
Food samples were weighed (100 g), suspended in 900 ml sterile BPW
86
(Buffered Peptone Water), and homogenized for one minute according to the National
87
Food Safety Standard of China Food Microbiology Manual (GB 4789.40–2010). The
88
homogenates were incubated at 37°C for 18 h, and 1 ml pre-enriched sample was
89
subsequently transferred to 10 ml of mLST – Vm (modified Lauryl Sulfate Broth
90
supplemented with vancomycin 10ug/ml; Hopebio, China) and incubated by another
91
24 h at 44°C. Human stool samples were collected from Wenzhou Integrated
92
Traditional Chinese and Western Medicine Hospital and Second Affiliated Hospital of
93
Wenzhou Medical University and transported to the laboratory in Cary-Blair medium
94
within 4 h. Enrichments and human stool samples were streaked onto CHROMagarTM
95
Cronobacter (CHROMagar Microbiology, France). All presumptive positive colonies
96
were further confirmed as Cronobacter spp. by Vitek 2 identification cards and the
97
V7.01 identification database according to the manufacturer’s instructions
98
(BioMerieux, France). All isolates were stored at -80°C in trypticase soy broth (TSB)
99
containing 20% glycerol for later analysis.
100
Multilocus sequence typing 5
101
Multilocus sequence typing (MLST) was performed by the sequence analysis of
102
seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB and ppsA) according to
103
the protocol available on the PubMLST website (http://pubmlst.org/cronobacter/).
104
Genomic DNA was extracted by Rapid Bacterial Genomic DNA Isolation Kit (Omega
105
Bio-Teck, Dorvaville, USA) and amplified using the seven primer pairs described
106
previously (Baldwin et al., 2009). DNA extracts were sequenced by Qinke Biotech
107
Limited (Hangzhou, China). The nucleotide sequences for each locus were analyzed
108
with BioNumerics software version 7.5 (Applied Maths, Belgium) and compared to
109
published sequences on the PubMLST website. Sequence types (STs) were
110
determined according the obtained seven-digit allelic profiles. A phylogenetic tree was
111
generated using the unweighted pair-group method with arithmetic means (UPGMA)
112
and the minimum spanning tree (MST) methods. Species identification was
113
determined by classical MLST technique according to Joseph’s method (Joseph et al.,
114
2012a).
115
Pulsed-field Gel Electrophoresis subtyping of Cronobacter.
116
Pulsed-field gel electrophoresis was performed based on the CDC PulseNet
117
protocol for Cronobacter spp. Briefly, DNA was digested with 40 U of XbaI enzyme
118
(TaKaRa, Japan) at 36°C for 2 h. The separation of restriction fragments was
119
performed in 1% SeaKem gold agarose (Lonza, Switzerland) gels in 0.5x
120
Trisborate-EDTA (TBE) buffer (Milliporesigma, Burlington, MA) using the CHEF-
121
Mapper system (Bio-Rad), with the following parameters: initial switch time, 2.16 s;
122
final switch time, 63.8 s for 18.5 h at 6 V/cm and condensation temperature of 14°C. 6
123
The DNA banding patterns were visualized under UV light with GelDocTM
124
XR+system (Bio-Rad, Hercules, CA) after staining with GelRedTM (0.5 ug/ml;
125
Biotium, CA), and analyzed using BioNumerics software version 7.5 (Applied Maths,
126
Kortrijk, Belgium). XbaI-digested Salmonella enterica serovar Braenderup H9812
127
was used as the molecular weight standard. The dendrogram was created by UPGMA
128
with the Dice similarity coefficient and a position tolerance of 1.5%. Clusters were
129
defined based on an 85% similarity cutoff.
130
Antimicrobial susceptibility testing
131
The antibiotic susceptibility of 90 recovered Cronobacter isolates was
132
determined using Gram Negative MIC plates CMV3AGNF (Thermo Scientific,
133
Waltham,
134
trimethoprim/sulfamethoxazole, sulfanilamideisoxazole, gentamicin, ciprofloxacin,
135
nalidixic acid, tetracycline, streptomycin, azithromycin, cefotaxiv, chloramphenicol,
136
amoxicillin, ampicillin and cefoxitin. After incubation at 37°C for 18 h, the plates
137
were read and interpreted automatically by Sensititre Vision Digital MIC Viewing
138
System, and susceptibility or resistance pattern of the Cronobacter isolates to selected
139
antimicrobials was compared to the recorded concentration of the control organism
140
Escherichia coli ATCC 25922. Bacteria were classified as resistant, intermediate or
141
sensitive according to clinical and laboratory test Standard (Clinical and Laboratory
142
Standards Institute, 2019).
143
Results
MA)
against
fourteen
antibiotics,
including
ceftriaxone,
7
144
Prevalence of Cronobacter spp. in Wenzhou, China
145
As shown in Table 1, Cronobacter strains were isolated in Wenzhou retail foods
146
(n=78) and clinical samples (n=12) from 2008 to 2018. The highest percentage of
147
Cronobacter spp. isolates was found in spice samples (22%, 26/119), followed by
148
baby cereals (17%, 41/248), powered adult formula (4%, 3/71), special medical infant
149
formula (1%, 1/95), powered infant and toddler formula (1%, 7/494), and stool
150
specimens (1%, 12/1024).
151
Molecular characterization of Cronobacter spp. isolates
152
To investigate the population structure of Cronobacter spp. and relationship
153
between pathogenicity and specific lineages, seven housekeeping gene atpD, fusA,
154
glnS, gltB, gyrB, infB and ppsA were sequenced. MLST analysis was performed
155
according to Cronobacter PubMLST database (https://pubmlst.org/cronobacter/).
156
Species identification was determined by classical MLST technique according to
157
Joseph’s method (Joseph et al., 2012a). Comparison of the percentage samples for
158
each species was used to infer prevalence. C. sakazakii was recovered more
159
frequently (69%) than C. malonaticus (22%) and any other Cronobacter spp. (9%).
160
For example, thirty-one samples were positive in baby cereals for C. sakazakii, which
161
was significantly (P < 0.05) higher compared to C. malonaticus (n=8), C. muytjensii
162
(n=1) and C. dublinensis (n=1). All human stool samples and food samples were
163
negative for C. condimenti or C. turicensis, while four spice samples harbored both
164
species. Overall, a high phylogenetic diversity was observed among the ninety 8
165
isolates by PFGE analysis. Notably, 43 STs were overlaid onto the tree which
166
including two novel STs, ST 684 and ST 685 (Figure 1). There were 25 STs in C.
167
sakazakii isolates. ST1 was the dominant sequence type (12.3%, n=11), followed by
168
ST 64 (7.8%, n=7), ST4 = ST8 = ST148 (5.6%, n=5), ST40 (4.4%, n=4) and ST13
169
(3.3%, n=3). ST1, ST4, ST8, ST148, ST40 and ST13 were found in both retail foods
170
and clinical samples as baby cereal (n=18), human stool samples (n=7) and powered
171
infant and toddler formula (n=7) were found more frequently than other samples;
172
whereas ST64 were only recovered from baby cereals (n=6) and special medical
173
infant formula (n=1) but not clinical samples.
174
Moreover, the primary sequence type of C. malonaticus was ST201 (5.6%, n=5)
175
with two isolates collected from powered infant and toddler formula and three
176
positive clinical samples, followed by ST7 (4.4%, n=4), ST440 (2.2%, n=2) and
177
ST129 (2.2%, n=2). In addition, C. sakazakii CICC 21560 represented the same ST8
178
with six isolates collected from baby cereals (n=2), children clinical samples (n=1)
179
and spices (n=2), whereas C. muytjensii ATCC 51329 showed a distinct ST81 from all
180
the isolates in the study.
181
Ninety isolates of Cronobacter were subsequently examined for genetic
182
relatedness using a PFGE DNA fingerprinting technique, and four isolates could not
183
be digested with XbaI. Correspondingly, BioNumerics software analysis showed the
184
remaining 86 isolates indicating 75 distinguishable patterns at an 85% similarity
185
threshold (Figure 2). C. sakazakii and C. malonaticus strains formed 48 and 16
186
pulsotypes, respectively. Specifically, C. sakazakii ST1 (n=11) was further divided 9
187
into 9 pulsotypes, and C. sakazakii ST4 (n=5) contained 4 pulsotypes. Furthermore,
188
C. sakazakii ST64 (n=7) harbored 4 PFGE patterns, which were originated from baby
189
cereals (n=6) and special medical infant formula (n=1). All clinical isolates consisted
190
of C. sakazakii (n=6) and C. malonaticus (n=4) revealed 8 distinct patterns.
191
Antibiotic Susceptibility Analysis
192
The 90 Cronobacter spp. isolates were subjected to 14 antimicrobial
193
susceptibility tests (Table 2) using minimum broth dilution method. Most isolates
194
(84%) were sensitive or exhibited only intermediate resistance to all antibiotics
195
assessed.
196
trimethoprim/sulfamethoxazole, gentamicin, ciprofloxacin, and nalidixic acid.
197
Cronobacter
198
chloramphenicol (29%), followed by cefotaxiv (28%), cefoxitin (24%), amoxicillin
199
(12%), ampicillin (12%), streptomycin (11%), azithromycin (7%), tetracycline (6%)
200
and ceftriaxone (1%). There was no significant difference among C. sakazakii, C.
201
malonaticus, C. dublinensis, C. condimenti, C. turicensis and C. muytjensii. Fourteen
202
isolates (16%) showed multidrug resistance (resistant against two or more classes of
203
antimicrobials), including two isolates that were resistant to more than five antibiotics
204
and six isolates that were resistant to six antibiotics.
205
DISCUSSION
All
isolates
strains
were
were
resistant
susceptible
or
showed
to
sulfanilamideisoxazole,
intermediate
resistance
to
206
Cronobacter spp. are opportunistic foodborne pathogen that are isolated from
207
environment samples and food products. Deciphering food sources as potential 10
208
reservoirs in driving the persistence and virulence potentials of Cronobacter spp. is
209
the key to mitigating potential Cronobacter-borne disease to humans. A continuous
210
surveillance was therefore conducted from Wenzhou sentinel hospitals and retail
211
foods for one decade.
212
C. sakazakii isolates were more widely distributed among retail foods and
213
human stool samples relative to the other pathogenic Cronobacter spp. which were
214
less prevalent in Wenzhou. These results support the premise that C. sakazakii is the
215
predominant foodborne pathogen among Cronobacter spp. (van Acker et al., 2001),
216
and other studies have examined prevalence of C. sakazakii elsewhere with similar
217
results as those reported herein (Brandão et al., 2017; Li et al., 2019). The presence of
218
C. sakazakii and C. malonaticus from clinical samples was previously noted (Cui et
219
al., 2014) and was reaffirmed by the present survey, as human stool samples from
220
diarrheal patients were exclusively defined as either C. sakazakii or C. malonaticus.
221
Thus, our results support the hypothesis that C. sakazakii and C. malonaticus may be
222
more closely associated with human host, while C. dublinensis, C. muytjensii and
223
other Cronobacter spp. are more likely inhabitants of environmental commensals with
224
unclear clinical significance (Iversen et al., 2004b; Schmid et al., 2009). The
225
understanding of true epidemiology and virulence potential for these organisms is still
226
poor and is likely to be complex, due to the diversity of these species. Grim and
227
colleagues found unique ferric dicitrate transport system in a small subset of C.
228
sakazakii and C. malonaticus but was absent in other Cronobacter species, indicating
229
iron acquisition system may contribute to the virulence of Cronobacter infections by 11
230
helping strains to acquire iron directly or indirectly from human hosts (Grim et al.,
231
2012). Nevertheless, the presence of other virulence factors including Cronobacter
232
plasminogen activator, type 6 secretion system, and associated putative adhesins
233
depended on species (Singh et al., 2015).
234
This survey revealed that a wide spectrum of food products was contaminated
235
with six species of Cronobacter spp. It is noteworthy that Cronobacter was isolated at
236
highest frequency (22%) in spice samples. These species were spread widely in spice
237
samples, suggesting high risk potential with the consumption of contaminated spices.
238
Spices have been reportedly implicated in foodborne outbreaks and recalls caused by
239
other enteric pathogens (Keller et al., 2013; Van Doren et al., 2013), as spices in the
240
desiccated state may provide appropriate conditions that allows the survival of
241
Cronobacter spp. as well as other foodborne pathogens such as Salmonella spp.,
242
Escherichia coli O157:H7, Bacillus cereus, and Clostridium perfringens ((CDC),
243
2010; Gurtler and Keller, 2019). In addition, 17% of baby cereal samples were
244
positive for Cronobacter spp., including C. sakazakii, C. malonaticus, C. muytjensii,
245
and C. dublinensis. The low prevalence of Cronobacter spp. in powered infant and
246
toddler formula (1%) contrasts with a prior report that Cronobacter found in 75% of
247
infant formula in United States (Clark et al., 1990). It should be noted that raw
248
ingredients, preparation equipment and personnel may contribute to the extrinsic
249
contamination of Cronobacter in food products. In a survey of milk power
250
manufacturing plant was reported to contaminated with C. sakazakii and in the
251
equipment of spray drying, fluidized bed drying, and packaging (Fei et al., 2015). 12
252
Inappropriate food safety practices may facilitate the emergence of new pathogenic
253
lineages from other nonpathogenic strains by horizontal transfer and gene
254
recombination. Meanwhile, the ability of Cronobacter to form biofilm enhanced their
255
persistence in adverse environmental conditions (Lehner et al., 2005). Therefore, new
256
food safety measurements and hygiene strategies are essential to reduce
257
Cronobacter-related contamination in food products as well as their environment.
258
Molecular based typing approaches demonstrated a high degree of diversity of
259
the strains tested. MLST revealed 25 STs for C. sakazakii out of 62 isolates. Seven of
260
which were previously associated with C. sakazakii infection (Joseph and Forsythe,
261
2011; Cui et al., 2014). All 20 C. malonaticus strains were segregated into 11 different
262
STs, including ST664 identified from a powered adult formula sample. Despite a low
263
contamination rate, the presence of C. malonaticus in powered adult formula poses a
264
potential risk to elderly people because C. malonaticus has been predominantly
265
associated with adult infection (Holý et al., 2014). Moreover, the identification of
266
novel ST685 from spice samples illustrates future surveillance on genetic
267
characteristic of Cronobacter spp. including new food routes/ related environment are
268
necessary, especially those from infections with unknown sources.
269
The 78 Cronobacter isolates cultured from food products, along with the
270
additional 12 clinical strains, were further investigated using PFGE. Although four
271
strains were untypeable, PFGE data offered superior ability for discrimination of
272
Cronobacter spp. compared to MLST analysis. For example, 37 patterns were found
273
among 41 baby cereal samples, while only 26 STs were discovered using MLST 13
274
method. Powered infant and toddler formula samples harbored seven pulsotypes but
275
only contained four STs. In particular, the observation of C. sakazakii MLST ST4 in
276
both of the baby cereal mixes and powered infant and toddler formula was highly
277
noteworthy because it has been proposed that C. sakazakii ST4 was especially
278
problematic to neonatal meningitis (Joseph and Forsythe, 2011; Hariri et al., 2013).
279
The identification of C. sakazakii ST12 in baby cereals was also significant, as it is a
280
specific clonal linage linked with necrotizing enterocolitis (Forsythe et al., 2014). To
281
the best of our knowledge, this is the first documented report demonstrating infant
282
supplementary food contaminated with C. sakazakii ST12. Our result provides
283
evidence that infants with the consumption of baby cereals or powered infant and
284
toddler formula posed potential risks and may thereby lead to severe disease.
285
Susceptibility tests revealed that all isolates were sensitive to the antibiotics in
286
the classes for inhibiting DNA or folic acid synthesis. However, several food samples
287
exhibited high or intermediate resistance to the antibiotics in the classes for cell wall
288
synthesis (cefotaxiv, cefoxitin, amoxicillin and ceftriaxone) and protein inhibitors
289
(chloramphenicol, streptomycin, azithromycin and tetracycline). These results
290
differed from previous reports that found Cronobacter spp. strains isolated from food
291
samples susceptible to almost all the antibiotics testes (Molloy et al., 2009; Terragno
292
et al., 2009; Brandão et al., 2017). Differences in antimicrobial resistance (AMR)
293
were also observed among strains of foods and clinical settings. Clinical samples only
294
showed resistance or reduced susceptibility to the antibiotics of cefotaxiv, cefoxitin,
295
and chloramphenicol, suggesting that traditional antibiotic regimen (ampicillin in 14
296
combination with either gentamicin or chloramphenicol) for Cronobacter infection
297
remain effective in Wenzhou. Multi-antimicrobial drug resistance characterized 16%
298
of the Cronobacter isolates, including fourteen spice isolates and one clinical sample,
299
indicating a potential public health risk. Although the rate of multi-drug resistance is
300
not high, the potential acquisition of antibiotic resistance by extensive use of
301
antibiotics in the farm or aquaculture may still exists. Therefore, the surveillance of
302
Cronobacter spp. in spices and other food products should be strengthened to
303
untangle the impact of microbial ecology to AMR gene transfer.
304
This study has provided scientific evidence on the contamination rate of
305
Cronobacter spp. from powered formula and other dried food products commercially
306
available in Wenzhou, China. Although the virulence potential of Cronobacter spp.
307
has not been fully elucidated, this systematic and long-term survey provided basis
308
guidance and vital knowledge to government agencies for performing risk assessment
309
and intervention strategies to control Cronobacter-related disease in human.
310
Acknowledgement
311
This work was funded by Major National Science and Technology Projects in the 13rd
312
Five-year Plan (2017ZX10103008-002), National Health Commission Scientific
313
Research Projects (WKJ-ZJ-1917), Ministry of Science and Technology of China (No.
314
2017YFC1601503), and Wenzhou Medical and Health Science Research Projects (No.
315
2019B03). Thank you to Dr. Anita C. Wright from University of Florida for assistance
316
in polishing the English of our manuscript.
15
317
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19
471
Figure legends
472
Figure 1 The minimum spanning tree of clinical and foodborne Cronobacter strains based on the allelic profiles. Cronobacter species are distinguished by color differences. Size of circle indicates number of isolates within the same ST, and thickness of the branches represents the degree of similarity among Cronobacters tested.
473 474 475 476 477 478
Figure 2 Dendrogram of 90 Cronobacter spp. based on XbaI-mediated PFGE profiles. Strain source, isolation date and STs are also included.
20
Table 1. Occurrence of Cronobacter species in food and clinical samples in Wenzhou Sample
No. of sample
Prevalence of positive samples1
C. sakazakii
C. malonaticus
C. muytjensii
C. dublinensis
C. condimenti
C. turicensis
Species isolated (Sample)
Special medical infant formula
95
1%
1
-
-
-
-
-
Powered adult formula
71
4%
2
1
-
-
-
-
Powered infant and toddler formula
494
1%
6
1
-
-
-
-
Spices
119
22%
14
6
1
1
2
2
Baby cereals
248
17%
31
8
1
1
-
-
Stool specimens
1024
1%
8
4
-
-
-
-
Total
2051
4%
62
20
2
2
2
2
1
Prevalence % = n positive / n tested
Table 2. Antibiotic susceptibility test result Antibiotic mechanism1
Resistant2 (%)
Intermediate (%)
Sensitive (%)
Ciprofloxacin
0
0
100
Nalidixic acid
0
0
100
Sulfanilamide Isoxazole
0
0
100
Trimethoprim/Sulfamethoxazole
0
0
100
Ceftriaxone
1
0
99
Cefotaxiv
8
20
72
Amoxicillin
10
2
88
Ampicillin
11
1
88
DNA synthesis inhibitors
Folic acid synthesis inhibitors
Cell wall synthesis
Cefoxitin
18
7
76
Gentamicin
0
0
100
Tetracycline
1
4
94
Streptomycin
3
8
89
Azithromycin
7
0
93
Chloramphenicol
9
20
71
Protein synthesis inhibitors
1
Antibiotics are classified based on the mechanism for antimicrobial activity.
2
Resistant, intermediate and sensitive percentages refer to the percentage of strains.
Highlights 1. Cronobacter was dispersed widely in various food products. 2. Consumption of spices may pose a high risk for Cronobacter infection. 3. Two STs (ST685 and ST684) were newly assigned. 4. The combination of MLST and PFGE methods revealed high genetic diversity in Cronobacter spp., as 43 sequence types and 75 clonal groups were displayed. 5. 16% of the Cronobacter isolates exhibited multidrug resistance.