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An overview of Yersinia enterocolitica and related species in samples of different origin from San Luis, Argentina
Journal Pre-proof An overview of Yersinia enterocolitica and related species in samples of different origin from San Luis, Argentina Cecilia Lucero Estrada, Gabriela Isabel Favier, María Esther Escudero PII:
S0740-0020(19)30955-4
DOI:
https://doi.org/10.1016/j.fm.2019.103345
Reference:
YFMIC 103345
To appear in:
Food Microbiology
Received Date: 11 June 2019 Revised Date:
5 September 2019
Accepted Date: 3 October 2019
Please cite this article as: Estrada, C.L., Favier, G.I., Escudero, M.E., An overview of Yersinia enterocolitica and related species in samples of different origin from San Luis, Argentina, Food Microbiology, https://doi.org/10.1016/j.fm.2019.103345. 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. © 2019 Published by Elsevier Ltd.
Highlights -
Animals and foods are important Y. enterocolitica sources in San Luis, Argentina.
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The 5% of samples analyzed throughout 30 years was Yersinia positive.
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183 Y. enterocolitica and 72 isolates of other Yersinia species were identified.
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Pathogenic Y. enterocolitica bioserotypes were recovered from foods.
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Biotype 1A was prevalent in Y. enterocolitica strains of various sources.
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An overview of Yersinia enterocolitica and related species in samples of different
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origin from San Luis, Argentina.
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Cecilia Lucero Estrada1,2,*, Gabriela Isabel Favier1, María Esther Escudero1
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1
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Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis,
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Argentina.
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2
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Investigaciones Científicas y Tecnológicas (IMIBIO-CONICET).
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Microbiología General, Facultad de Química, Bioquímica y Farmacia, Universidad
Instituto Multidisciplinario de Investigaciones Biológicas, San Luis- Consejo Nacional de
Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
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*
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Cecilia Lucero Estrada
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Tel. +54 0266 4520300 ext. 1611
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Fax +54 0266 4431301
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E-mail address: [email protected] (C. Lucero Estrada)
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Full postal address: Microbiología General, Facultad de Química, Bioquímica y Farmacia,
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Universidad Nacional de San Luis. Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San
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Luis, Argentina.
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Corresponding author:
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ABSTRACT
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This study is aimed at offering an overview of the prevalence of Yersinia
27
enterocolitica and related species in San Luis, Argentina, from samples of diverse origin
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received in our laboratory between 1984 and 2014, and providing an analysis of the
29
distribution of Yersinia isolates according to their isolation sources, highlighting
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bioserotypes and potential reservoirs and vehicles of transmission to humans. From a total
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of 4,572 samples of human, animal, food and environmental origins analyzed by traditional
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culture methods and molecular techniques, 229 (5%) samples were Yersinia positive. The
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highest frequency of Yersinia isolates was observed in environmental specimens (14.3%),
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followed by animal (9.2%), food (5%) and human (0.6%) samples. A total of 255 Yersinia
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isolates were characterized, including 183 Y. enterocolitica and 72 isolates of other Yersinia
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species. Biotype 1A associated to several serotypes was identified in Y. enterocolitica
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isolates from environment (100%), animals (95.5%), foods (71.7%) and human samples
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(40%); bioserotype 2/O:9 was identified in isolates from foods (25.5%), and biotype 3 was
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associated with strains from humans (60%), animals (4.5%) and foods (2.8%). This biotype
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included three strains O:3 and six strains O:5. The data highlight animals and foods as the
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main Y. enterocolitica sources in our region.
42 43 44 45 46 47 48
Keywords: Yersinia enterocolitica, enteropathogen, biotype, serotype, epidemiology
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1. Introduction Yersinia enterocolitica is the causative agent of a wide range of diseases, mainly
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gastrointestinal manifestations, in addition to severe complications such as mesenteric
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lymphadenitis, reactive arthritis and sepsis (Duan et al., 2017). Although foods from
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porcine origin are the main source of human infection, epidemiological studies highlight
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other animal carriers, as well as foods of various origins and the environment as possible
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sources of Y. enterocolitica infection for humans (Rahman et al., 2011). Due to its
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psychrotrophic growth, Y. enterocolitica is a hazardous organism in foods with extended
57
storage in chilling conditions (De Silvestri et al., 2018). Bioserotypes 1B/O:8, 2/O:5,27,
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2/O:9, 3/O:3 and 4/O:3 have been associated to human infections in different countries. The
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strains of five biotypes (1B, 2, 3, 4 and 5) may be carriers of a 70-kb virulence plasmid
60
(pYV) and chromosomally encoded virulence genes. Strains of biotype 1A include a wide
61
range of serotypes, which have been considered as nonpathogenic for a long time because
62
they lack the classical Y. enterocolitica virulence determinants (Joutsen et al., 2017). There
63
is epidemiological and experimental evidence, however, suggesting that some strains of this
64
biotype can cause gastrointestinal infections (Campioni and Falcão, 2014). Y. intermedia,
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Y. frederiksenii and Y. kristensenii are often categorized as Y. enterocolitica-like species
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and have been isolated from healthy and sick humans and animals, as well as from food and
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environmental sources (CDC, 2014). Their pathogenic potential might be related to the
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presence of some Y. enterocolitica virulence genes, although the existence of alternative
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virulence mechanisms has also been suggested (Imori et al., 2017).
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There is very little information about the epidemiology of Y. enterocolitica in
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Argentina, where it is considered a pathogen of moderate risk and limited dissemination
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(AAM, 2013). Y. enterocolitica has been sporadically recovered from animals, foods, water
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and human clinical samples in various regions of this country. The present study is aimed at
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offering an overview of the prevalence of Y. enterocolitica in San Luis, Argentina, from
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samples of diverse origin studied in our laboratory between 1984 and 2014, and providing
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an analysis of the distribution of Yersinia isolates according to their isolation sources,
77
highlighting bioserotypes and potential reservoirs and vehicles of transmission to humans.
78
79
2. Materials and methods
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2.1. Samples
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A total of 4,572 samples, including human stool samples, presumptive animal
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reservoirs, foods for human consumption and environmental samples, were collected
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between 1984 and 2014 in various locations of San Luis City and surrounding areas and
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investigated for Yersinia.
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Human stool samples (n = 801) were collected from pediatric and adult patients
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with enterocolitis symptoms attending four local clinical laboratories for microbiological
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analysis of feces in the 1989-1993, 2003-2004 and 2008-2011 periods. Animal samples (n
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= 620) were collected between 1984 and 2011 during surveys of Yersinia potential
89
reservoirs in San Luis City and upcoming rural areas. Samples of farm animals were
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randomly collected in slaughters, farms, feedlots and cattle markets. Samples of wild
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animals including wild boars (Sus scrofa), viscachas (Lagostomus maximum maximum),
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and trouts (Oncorhynchus mykiss) were provided by local hunters and fishers, and stools of
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different bird species (Passer domesticus, Columba maculosa, Sicalis luteosa, Molothrus
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rufoaxillaris, Cyanocompsa brissoni, Saltator aurantiirostris, Turdus chiguanco,
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Myiopsitta monacha, Pardalotus puntactus, Serinus canarius and Zonotrichia capensis)
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were collected in their natural habitats, immediately after defecation. Samples of foods
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intended for human consumption (n = 3,053) were purchased in local butcher shops, retail
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markets, fish shops and greengroceries between 1984 and 2014. Environmental samples (n
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= 98) included 84 surface water samples collected from three rivers (Grande, El Trapiche
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and El Volcán) and two lakes (La Florida and Potrero de los Funes) in the San Luis
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Province between 1990 and 1991, and 14 wastewater samples from a treatment plant of
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sewage effluents near San Luis City during 1991. All samples were packed in individual
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sterile containers, sent to the Laboratory of Microbiology of the National University of San
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Luis, and stored at 4 °C for up to 6 h before processing.
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Samples were investigated during periodical surveys with the purposes of
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establishing the frequency of Yersinia species in humans, animals, foods and environment
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for the San Luis region, and determining possible carriers, transmission vehicles and
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potential sources of infection.
109 110
2.2. Characterization of the Yersinia isolates
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Enrichment techniques and selective media for the isolation of Yersinia strains were
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applied according to international standards. Presumptive Yersinia colonies were examined
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by Gram staining and identified by classical biochemical assays (FDA, 2007). The final
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characterization of biotypes and serotypes was performed by Dr. E. Carniel, National
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Reference Center of Yersinia, Institute Pasteur, Paris, France.
116 117 118 119
2.3. Assessment of pathogenic potential of Y. enterocolitica strains In vitro phenotypic tests such as autoagglutination (AA) at 37 °C (Laird and Cavanaugh, 1980) and calcium-dependent growth (CA) (Riley and Toma, 1989) were
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performed for all Y. enterocolitica isolates in order to differentiate plasmid-bearing from
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plasmidless strains. In addition, virulence genetic markers such as virF, ail, ystA, ystB and
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myfA were assayed by PCR in strains isolated from 2005 to date and performed according
123
to Favier et al. (2005) and Lucero Estrada et al. (2011, 2012, 2015).
124 125
2.4. Statistical analysis
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Association between Yersinia species, their bioserotypes and isolation sources was
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assessed by using Chi-square test (Analytical Software, Tallahassee FL, USA). Statistical
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calculations were based on confidence level equal or higher than 95% (p ≤ 0.05 was
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considered statistically significant).
130 131
3. Results
132 133 134
3.1. Distribution of Yersinia of different sources in San Luis From a total of 4,572 samples from human, animal, food and environmental sources
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collected in San Luis, Argentina, 229 (5%) samples were Yersinia positive (Table 1). The
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highest frequency of Yersinia isolates was observed in environmental specimens (14.3%),
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followed by animal (9.2%), food (5%) and human (0.6%) samples (p ≤ 0.05). A total of 255
138
Yersinia isolates were identified, including 183 Y. enterocolitica strains recovered from
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3.6% of samples, and 72 isolates of other Yersinia species recovered from 1.4% of samples.
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Y. enterocolitica positive samples were distributed as follows: animals (8.9%) > foods
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(3.4%) = environment (3.1%) > humans (0.6%) (p ≤ 0.05). Y. enterocolitica was the only
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species isolated from human samples. Other Yersinia species (Y. intermedia, Y.
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frederiksenii and Y. kristensenii) were detected as follows: environment (11.2%) > foods
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(1.6%) > animals (0.3%) (p ≤ 0.05). Y. intermedia was the predominant species with 63
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isolates, while only eight Y. frederiksenii isolates and one Y. kristensenii isolate were
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obtained.
147 148 149
3.2. Biotype and serotype distribution of Y. enterocolitica strains from different sources The biotype and serotype distribution of 183 Y. enterocolitica strains according to
150
their sources can be seen in Table 2. The most frequent biotype was 1A (147 isolates,
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80.3%) followed by biotypes 2 (27 isolates, 14.8%) and 3 (nine isolates, 4.9%) (p ≤ 0.05).
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Out of 19 serotypes associated to biotype 1A, the most frequent were: O:5 (35.5%), O:6
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(12.0%), O:6,30-6,31 (7.1%), O:7,8-8-8,19 (6.6%), O:41,42-41,43 (4.4%) and O:12,25-
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12,26 (2.2%). Six Y. enterocolitica 1A strains could not be serotyped. Regarding the
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biotype distribution according to the isolation sources, biotype 1A predominated in isolates
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from environmental origin (100%), followed by isolates from animal origin (95.5%) and
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food samples (71.7%). This biotype was present in two out of five isolates of human origin.
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Biotype 2 was identified in 25.5% isolates recovered from food samples (26 strains O:9 and
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one non-serotyped isolate), while biotype 3 was identified in strains from human (three out
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of five isolates), animal (4.5%) and food (2.8%) origin. This last biotype included three
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strains O:3 and six strains O:5. All 1A strains were negative for the phenotypic virulence
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markers and 31 of 62 studied strains were ystB+. As regards 2/O:9 strains, all of them were
163
positive for AA and CA. Among 27 isolates, 21 of them were characterized for virulence
164
genetic markers; 18 strains were virF+ myfA+ ail+ ystA+ and three strains were virF- myfA+
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ail+ ystA+. Similarly, all 3/O:3 and 3/O:5 isolates were positive for AA and CA. One Y.
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enterocolitica 3/O:3 strain was virF- ail+, and two of them were virF+ ail+. Among six
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3/O:5 isolates recovered from foods, only four strains were assayed for virulence genetic
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markers: three isolates were virF+ ail+ whereas the fourth one was virF- ail+ (Favier et al.,
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2005; Lucero et al., 2011; Lucero et al., 2012; Lucero et al., 2015).
170 171 172
3.3. Characteristics of Yersinia strains from human samples Eight hundred and one samples of human feces were analyzed for the presence of
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Yersinia species (Table 3), 0.6% of which were positive, yielding five Y. enterocolitica
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isolates. Of these, two Y. enterocolitica isolates were characterized as 1A/O:5 and
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1A/O:7,8-8-8,19, and the other three ones were identified as 3/O:3.
176 177
3.4. Distribution and characteristics of Yersinia strains of animal origin
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Table 4 shows the distribution and characteristics of Yersinia isolates in samples of
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animal origin. Samples of intestinal content and different body parts of farm animals (pigs,
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cows, goats and chicken) and wild animals (wild boars, fishes, rodents, and birds) were
181
investigated. Yersinia was positive in 8.9% of animal samples. Positive samples were more
182
frequent in farm animals (12.7%) with 64 isolates, than in wild animals (2%) with four
183
isolates (p ≤ 0.05). Y. enterocolitica (66 isolates, 97.1%) was found to be more frequent
184
than other Yersinia species (two isolates, 2.9%) (p ≤ 0.05). As regards farm animals, pigs
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yielded the highest number of Y. enterocolitica isolates, with 57 obtained from cecal
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contents and four from skin and bones. Out of these, 58 isolates were characterized as 1A
187
and three as 3/O:5. More than one strain per sample was found in pig cecal contents. Cows
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(2%) were also carriers of one Y. enterocolitica 1A isolate and one Y. frederiksenii isolate,
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followed by goats (1%) with one Y. enterocolitica 1A isolate. No isolations were obtained
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from pig stools or tonsils, or from chicken cecal contents. Among wild animals, three out of
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six wild boar samples were positive for Y. enterocolitica 1A. In contrast, a low isolation
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rate (1%) was observed in trout gills with one Y. intermedia isolate, whereas rodent and
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bird samples were negative.
194 195 196
3.5. Distribution and characteristics of Yersinia strains isolated from foods Yersinia prevalence in foods was 5% (Table 5). Yersinia species were most frequent
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in chicken (12.4%) and foods of bovine origin (10.2%), followed by hen eggs (3.8%),
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products of porcine origin (3.7%), products based on mixtures of porcine and bovine meat
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(2.4%), hake fillets (1.2%) and dairy products (0.7%) (p ≤ 0.05). No isolations were
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obtained from fresh vegetables.
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Positive chicken samples yielded 71 Yersinia isolates, with the following
202
distribution: entrails (22.5%), carcasses (15.3%), meatballs (12%), sausages (10.5%),
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hamburgers (10%) and skin (7.9%). Twenty-four Yersinia isolates were obtained from
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bovine ground meat (7.7%) and tongues (19.2). Foods of porcine origin yielded 32 Yersinia
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isolates, mainly from fresh sausages (8.9%) and tongues (4%) (p ≤ 0.05), followed by cold
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foods (1.1%) and frankfurter (1%). Yersinia detection in fresh sausages of porcine and
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bovine origin was 3.7%, whereas pre-cooked sausages were Yersinia negative.
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From a total of 156 Yersinia isolates recovered from food samples, Y. enterocolitica
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(106) predominated over Y. intermedia (47), Y. frederiksenii (2) and Y. kristensenii (1) (p ≤
210
0.05). Specifically, Y. enterocolitica (27 isolates) predominated over Y. intermedia (5
211
isolates) in foods of porcine origin and it was the only species recovered from foods of
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bovine origin (24 isolates), porcine and bovine foods (7), hen eggs (16), dairy products (2)
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and hake fillets (4). Y. enterocolitica recovery in liquid homogenates of egg yolk and white
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(6.7%) was higher than in eggshells (3.4%) (p ≤ 0.05). Two Y. enterocolitica strains were
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isolated from ice cream.
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Overall, Y. enterocolitica biotype 1A associated to different serotypes (76 isolates)
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was more frequent than biotype 2 (27 isolates) and biotype 3 (three isolates). Biotype 1A
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was predominant (40 isolates) over biotype 2 (16 isolates) in foods prepared with porcine
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and/or bovine meat. Conversely, bioserotype 2/O:9 was predominant (11 isolates from
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eggshell) over biotype 1A (five isolates from liquid egg) in hen eggs. In addition, chicken
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carcasses were strongly associated to Y. intermedia carriage (24 strains) followed by
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chicken skin and entrails (6 strains each) and other chicken samples (≤ 3 strains each). Y.
223
frederiksenni and Y. kristensenii were only recovered from chicken carcasses.
224 225 226 227
3.6.
Prevalence and characteristics of Yersinia strains isolated from
environmental sources The distribution and characteristics of 26 Yersinia strains isolated from
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environmental samples are shown in Table 6. Overall, Yersinia was detected in 14.3% of
229
the samples. The highest recovery frequency was observed in sewage samples (57.1%)
230
followed by surface water of rivers and lakes (7.1%). The frequency of the Yersinia species
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was as follows: Y. intermedia (15 strains) > Y. enterocolitica (6 strains) = Y. frederiksenii (5
232
strains) (p ≤ 0.05). All Y. enterocolitica isolates belonged to biotype 1A.
233 234
4. Discussion
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For many years, our laboratory has performed periodical surveys for Yersinia
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surveillance on suspect animal reservoirs and carriers, foods of different origin intended for
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human consumers, and samples of symptomatic patients. The accumulated data on Yersinia
238
positive samples collected over 30 years, and the characteristics of the isolates might offer
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an overview of reservoirs, sources of contamination and underdiagnosed cases of
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yersiniosis in this region.
241
The data highlight animals and foods as the main Y. enterocolitica sources and the
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predominance of biotype 1A associated to different serotypes in Y. enterocolitica isolates
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from all samples, excepting human stools. Biotype 1A has been implicated in foodborne
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and nosocomial outbreaks around the world (Bhagat and Virdi, 2011); however, its
245
pathogenicity mechanism in humans has not yet been elucidated. Severe pathogenesis in a
246
Galleria mellonella model with a lethal dose as low as 10 CFU, and the major role of
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flagella in the virulence phenotype have been observed for this biotype (Alenizi et al.,
248
2016). Strains of biotypes 2 and 3 were also recovered from samples of San Luis.
249
Although protocols recommended by international standards for Yersinia survey in samples
250
of diverse origin (FDA, 2007) were applied, bioserotype 4/O:3, which has been linked to
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approximately 80% of Y. enterocolitica human infections worldwide, with pigs as the main
252
reservoirs (Duan et al., 2017), was not identified in strains reported in the present study.
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The here reported percentage of Y. enterocolitica observed in feces of pediatric and
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adult patients in San Luis was compared to results from other Argentine regions. This value
255
was similar to that observed for feces of 1-12 years old patients (0.8%) in Neuquén
256
(González et al., 2010), and lower than 1.13% (Cortes et al., 2010) and 3.3% (Sánchez and
257
González, 2013) reported from fecal samples of pediatric patients from Córdoba. In
258
addition, Sánchez and González (2013) identified biotype 4 in all their isolates. To date, the
259
majority of Y. enterocolitica isolates from human diarrhea cases described in Argentina
260
have been identified as belonging to biotype 4. Thus, this biotype was identified in 43 Y.
261
enterocolitica isolates recovered from sporadic clinical cases in nine hospitals of five
262
Argentine provinces (Barcudi et al., 2014). Interestingly, Paz et al. (2004) identified one Y.
263
enterocolitica 1A/O:5 strain from diarrheic feces of a patient from Buenos Aires,
264
Argentina. In Uruguay, Y. enterocolitica isolates have been recovered from sporadic
265
clinical cases and one gastroenteritis outbreak in hospitalized infants; all strains except one
266
1/O:8 isolate from liver abscess, were identified as 4/O:3 (Mota et al., 2012). In Brazil, Y.
267
enterocolitica 4/O:3 was the predominant bioserotype in strains of human origin (Rusak et
268
al., 2014). The low isolation rate of Y. enterocolitica from clinical samples may be caused
269
by the presence of background microbiota and the limited sensitivity of culture methods
270
(Morka et al., 2018). Thus, Yersinia intestinal infections in humans might be
271
underdiagnosed in San Luis. In contrast with public health policies in other countries where
272
Y. enterocolitica is a notifiable gastrointestinal pathogen (Jeffs et al., 2019), the
273
surveillance of this microorganism and its report to the national net of epidemiology is not
274
mandatory in Argentina.
275
Y. enterocolitica can colonize a broad range of farm and wild animals. The study of
276
animal samples from San Luis revealed that the presence of Yersinia is higher in farm
277
animals than in wild animals, and highlighted pigs as the main reservoirs of Y.
278
enterocolitica in our region, followed by cows and goats. In a study carried out in China,
279
Liang et al. (2015) obtained the highest Y. enterocolitica prevalence among pigs (12.9%),
280
followed by chickens (4.5%), rodents (3.4%), cattle (2.7%) and sheep (0.8%). Biotype 1A
281
in association with different serotypes and bioserotype 3/O:5 predominated in Y.
282
enterocolitica isolates from pig samples in San Luis. By contrast, a high prevalence of
283
4/O:3 strains have been demonstrated in pigs worldwide (Rahman et al., 2011, Martins et
284
al., 2018, Raymond et al., 2018), and predominance of 2/O:9 and 3/O:3 strains has been
285
reported for pigs in England (Ortiz Martínez et al., 2010) and China (Liang et al., 2015),
286
respectively. This suggests that the association between Y. enterocolitica bioserotypes and
287
pigs might be country-dependent, due to specific breeding and eating habits (Le Guern et
288
al., 2016). Similarly to our results where the highest Y. enterocolitica recovery was
289
observed in pig cecal contents, Fois et al. (2018) obtained the best Y. enterocolitica
290
recovery from pig intestinal content in Italy.
291
Although it has not yet been possible to demonstrate whether cattle constitute
292
reservoirs or merely transient carriers of Y. enterocolitica, this microorganism has been
293
reported from cattle feces worldwide (Rahman et al., 2011). Detection frequencies of Y.
294
enterocolitica in cows and goats from San Luis were lower than those reported for cattle
295
feces (16%) in Ireland (O’Grady et al., 2016) and for goat flocks (14.9%) in New Zealand
296
(Lanada et al., 2005). All strains isolated from cows and goats in our region belonged to
297
biotype 1A, in contrast with 2/O:9 strains recovered from cows, goats and sheep in France
298
(Le Guern et al., 2016). It has been suggested that since these animals may share habitats,
299
pastures or drinking water in a certain geographical region, the transmission of Yersinia
300
among them is possible and the contamination fecal-oral may occur (Le Guern et al., 2016).
301
The presence of Y. enterocolitica in wild boar samples was demonstrated for the
302
first time in San Luis; however, a higher number of samples should be tested to determine
303
whether these animals are Yersinia usual reservoirs in our region, as has been reported in
304
other countries (Fredriksson-Ahomaa et al., 2011, Magistrali et al., 2014, Morka et al.,
305
2018). All other wild animal samples analyzed showed negative results for Yersinia.
306
Finally, as regards fish samples, Y. intermedia was recovered. As known, only
307
Yersinia ruckeri has been reported as a significant concern for fish farming around the
308
world (Wrobel et al., 2018, Delalay et al., 2019).
309
Y. enterocolitica 1A was predominant in foods from San Luis, followed by Y.
310
intermedia and Y. enterocolitica biotype 2. Similarly, 1A was reported to be the most
311
frequent biotype (59.7%) isolated from foods in France, followed by Y. intermedia (17.2%)
312
and Y. frederiksenii, Y. kristensenii and Y. bercovieri in lower percentages (Le Guern et al.,
313
2016). While Y. enterocolitica 4/O:3 was not isolated from foods in San Luis, this
314
bioserotype represented 3% of all isolates from foods in France, mainly recovered from
315
pork meat (Le Guern et al., 2016). The present study demonstrated high prevalence of
316
Yersinia species in chicken samples, with carcasses as the main sources. Y. enterocolitica
317
and Y. intermedia were the most frequent species in these samples. A previous study
318
performed in Argentina reported 10% of Y. enterocolitica or related species from chicken
319
carcasses in a processing plant of Buenos Aires, distributed as follows: 4.3% Y.
320
enterocolitica 1A/O:5, 1.4% Y. intermedia, and 4.3% Y. frederiksenii (Floccari et al., 2000).
321
Contamination of carcasses with Yersinia probably occurs by contamination in
322
slaughterhouses, processing plants, vehicles of transportation or commercial refrigerators
323
where other slaughtered animals are stored.
324
Low Yersinia recovery rates were observed in foods of bovine, porcine and
325
bovine/porcine origin. This can be accounted for the manufacturing and storing procedures
326
applied on these foods, which may destroy, inhibit or stress Yersinia cells thus preventing
327
their recovery by culture. Y. enterocolitica 1A isolates predominated in foods of this origin.
328
Strikingly, Y. enterocolitica 2/O:9 recovery from eggshells was observed. The presence of
329
Yersinia species in eggshells and liquid eggs seems to be uncommon. In USA, one Yersinia
330
spp isolate was reported from 84 washed and unwashed commercial shell eggs (Musgrove
331
et al., 2004). In Australia, six Y. enterocolitica isolates were obtained from eggshell surface
332
of 1,860 table eggs collected from layer flocks (Gole et al., 2013). These authors have
333
postulated that eggshells can get contaminated by any surface with which they come in
334
contact, being feces, water, caging material, nesting material, insects, hands, blood and soil
335
the most common sources of contamination. Contamination of the egg surface might also
336
occur from contact with other Y. enterocolitica contaminated products of animal origin
337
such as pork, during collection on farms or during transportation or handling in retail shops
338
(Favier et al., 2005). When the eggshell is broken, contamination of the egg content is
339
possible.
340
In San Luis, Yersinia recovery from dairy products and hake fillets was low and no
341
Yersinia isolate was detected in fresh vegetables. Previous studies performed in Buenos
342
Aires, Argentina, reported Y. enterocolitica 1A (5.5%), Y. frederiksenii (9.9%) and Y.
343
intermedia (3.6%) from raw cow milk (Mercado and Ibañez, 1986). Several investigations
344
have reported outbreaks of foodborne infections caused by consumption of Y. enterocolitica
345
contaminated dairy products in other countries (Rahimi et al., 2014). The low percentage of
346
Y. enterocolitica obtained in hake fillets from San Luis, matches reports of 2.7% Y.
347
enterocolitica 1A in retail seafoods from Germany (Li et al., 2018). No Yersinia isolate was
348
detected in fresh vegetables in San Luis. However, vegetables can be a source of human
349
infection by Y. enterocolitica (Lee et al., 2004; Sakai et al., 2005).
350
Our results point out foods of animal origin contaminated by pathogenic biotypes 2
351
and 3, such as porcine fresh sausages, porcine cold foods, bovine tongues, porcine and
352
bovine fresh sausages, hake fillets and eggshells. The Y. enterocolitica presence is
353
commonly associated to pork production (Laukkanen-Ninios et al., 2014), however, cross-
354
contamination from pork to foods of other animal origin is possible at farm level,
355
slaughterhouse, butcher shop or home (Fredriksson-Ahomaa et al., 2004). Y. enterocolitica,
356
as a typical psychrotrophic microorganism, multiplies in food (neutral pH, stored at 5°C)
357
from 10/mL to 2.8×107/mL in 5 days, and in products kept in a freezer at -18°C, and retains
358
pathogenic properties for several months (Bancerz-Kisiel and Szweda, 2015). Considering
359
the fact that raw or undercooked foods are the main source of yersiniosis in humans,
360
education for consumers might be an important resource to prevent Y. enterocolitica
361
infections (FDA, 2012). Good production and manufacturing practices for foods of animal
362
origin, Standardized Operating Procedures for Sanitation. (SOPS), and Hazard Analysis
363
and Critical Control Points (HACCP) are also recommended.
364
This study reports significant Yersinia recovery from environmental samples, with
365
Y. intermedia, Y. frederiksenii and Y. kristensenii predominating over Y. enterocolitica 1A.
366
These species were also recovered from 90% of sewage water samples in Buenos Aires
367
City, Argentina (Floccari et al., 2003). The presence of Y. enterocolitica in surface water is
368
a risk factor for human infections in recreational activities, crop irrigation or contaminated
369
drinking water. This microorganism has also been isolated from river water in Canada
370
(Cheyne et al., 2010), Poland (Terech-Majewska et al., 2016) and Argentina (Kuczynski,
371
2016). The Argentine strains have been shown to be resistant to adverse environment
372
factors and might survive in untreated drinking water (Kuczynski, 2016). Interestingly,
373
Floccari et al. (2003) demonstrated that the 16S rRNA gene sequence of five Y.
374
enterocolitica 1A strains isolated from sewage water in Buenos Aires, was specific for
375
the European subspecies Y. enterocolitica palearctica. These authors hypothesize that Y.
376
enterocolitica might have been imported into Argentina with animals, food, feed or even
377
humans and that it was possible that these strains adapted to adverse “living conditions”
378
in a new continent. The route of Y. enterocolitica transmission in our region is unknown,
379
but it could begin in the environment, from where the bacteria could be transmitted to
380
animals, spread in foods of animal origin, and finally, reach consumers. The stages of this
381
route would be related in different ways, giving rise to multiple possibilities of
382
contamination and infection.
383 384
5. Conclusions The present study demonstrated that the main sources of Y. enterocolitica isolation
385 386
in San Luis are animals and foods, from which this organism could be transmitted to
387
humans. Biotype 1A was prevalent in Y. enterocolitica strains of various sources. The
388
pathogenic bioserotypes 2/O:9, 3/O:3 and 3/O:5 were demonstrated in food and clinical
389
samples. Sewage was a prominent reservoir of other Yersinia species. A close
390
correspondence was observed between Y. enterocolitica bioserotypes and isolation sources;
391
for instance, 2/O:9 was exclusively related to food isolates, 3/O:5 was linked to animal and
392
food isolates, and 3/O:3 was associated to human strains. The present analysis of the data
393
collected throughout 30 years provides an overview of Yersinia sources, reservoirs and
394
probable cases of undiagnosed yersiniosis in our region. The frequency of contacts between
395
sources of animal origin and humans as well as the consumption of contaminated foods
396
might contribute to the risk of infection caused by Y. enterocolitica. Since human clinical
397
cases associated to biotype 1A have been reported worldwide, the pathogenic potential of
398
each isolate 1A should be always investigated.
399 400
Declarations of interest
401
None
402 403
404
Acknowledgments
405
We wish to thank Dr. Ana María Stefanini de Guzman and her research group at
406
National University of San Luis, who were pioneers in the investigation of Yersinia
407
enterocolitica in San Luis, Argentina. This work was supported by the Science and
408
Technology Department, National University of San Luis, Argentina (Projects PROICO
409
8803, 2-0914 and 02-1518), Alexander von Humboldt Foundation from Germany, and the
410
National Agency for Scientific and Technological Promotion, Argentina (PICT-2015-
411
1722). We appreciate language revision by staff of Langue Institute, National University of
412
San Luis.
413 414
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Tabla 1. Isolation sources of Yersinia species from samples collected in San Luis from 1984 to 2014.
Species
Biotypes
Humans
Animals
Foods
Environment
Total
(B) Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Y. 1A enterocolitica
2/801 (0.2)
2
52/620 (8.4)
63
73/3053 (2.4)
76
3/98 (3.1)
6
130/4572 (2.8)
147
2-3
3/801 (0.4)
3
3/620 (0.5)
3
30/3053 (1.0)
30
0/98 (0)
0
36/4572 (0.8)
36
5/801 (0.6)
5
55/620 (8.9)
66
103/3053 (3.4)
106
3/98 (3.1)
6
166/4572 (3.6)
183
Subtotal
Y. intermedia
-
0/801 (0)
0
1/620 (0.2)
1
47/3053 (1.5)
47
8/98 (8.2)
15
56/4572 (1.2)
63
Y. frederiksenii
-
0/801 (0)
0
1/620 (0.2)
1
2/3053 (0.1)
2
3/98 (3.1)
5
6/4572 (0.1)
8
Y. kristensenii
-
0/801 (0)
0
0/620 (0)
0
1/3053 (0.03)
1
0/98 (0)
0
1/4572 (0.02)
1
Subtotal
0/801 (0)
0
2/620 (0.3)
2
50/3053 (1.6)
50
11/98 (11.2)
20
63/4572 (1.4)
72
Total
5/801 (0.6)
5
57/620 (9.2)
68
153/3053 (5.0)
156
14/98 (14.3)
26
229/4572 (5.0)
255
Table 2. Biotype and serotype distribution of Y. enterocolitica strains isolated from different sources.
Biotype (B) 1A
2
3
Serotype (O) 3,50,51 4,32-4,33 5 5-4,32-4.33 6 6,30 6,31 6,30-6,31 6,47 7,8 7,8-8-8,19 7,8-8-13-8,19 7-13 10-34 12,25-12,26 15,47 16,36 34,46 41,42-41,43 AA NA ND Subtotal 9 AA Subtotal 3 5 Subtotal Total
Humans N° of % strains 1 20 1 20 2 40 0 0 3 60 3 60 5 100
Animals N° of % strains 29 44.0 20 30.3 5 7.6 3 4.5 1 1.5 1 1.5 1 1.5 1 1.5 2 3.0 63 95.5 0 0% 3 4.5 3 4.5 66 100
Foods N° of % strains 1 0.9 2 1.9 31 29.2 1 0.9 2 1.9 2 1.9 1 0.9 8 7.5 1 0.9 1 0.9 8 7.5 1 0.9 4 3.8 1 0.9 6 5.7 2 1.9 3 2.8 1 0.9 76 71.7 26 24.5 1 0.9 27 25.5 3 2.8 3 2.8 106 100
Environment N° of % strains 4 66.7 1 16.7 1 16.7 6 100 0 0 0 0 6 100
Total N° of % strains 1 0.5 2 1.1 65 35.5 1 0.5 22 12.0 2 1.1 1 0.5 13 7.1 1 0.5 1 0.5 12 6.6 2 1.1 1 0.5 2 1.1 4 2.2 1 0.5 1 0.5 1 0.5 8 4.4 2 1.1 3 1.6 1 0.5 147 80.3 26 14.2 1 0.5 27 14.8 3 1.6 6 3.3 9 4.9 183 100
%: percentage of each Yersinia serotype in the total of Yersinia strains of the same origin. AA: autoagglutinated; NA: no agglutination; ND: not determined (the serotype was unavailable in the three cases).
Table 3. Distribution of Yersinia isolates in stool samples of human origin. Positive samples/ total of samples (%) 5/801 (0.6)
B1A
2
Y. enterocolitica isolates (n) B2 B3 Total
0
3
5
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
0
0
0
5
Table 4. Distribution of Yersinia isolates in samples of animal origin. Animals
Type of samples
Pigs
Cecal contents Stools Tonsils Skin and bones Cows Rectal contents Goats Cecal contents Chicken Cecal contents Subtotal of farm animals Wild boars Tonsils and tongues Trouts Gills Rodents (viscacha) Birds
Cecal contents Stools
Subtotal of wild animals Total
Positive samples/ total of samples (%) 46/129 (35.7) 0/2 (0) 0/6 (0) 4/30 (13.3) 2/100 (2) 1/100 (1) 0/50 (0) 53/417 (12.7) 3/6 (50) 1/100 (1) 0/31 (0) 0/66 (0) 4/203 (2) 57/620 (8.9)
B1A
Y. enterocolitica isolates (n) B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
54
0
3
57
0
0
0
57
0 0 4
0 0 0
0 0 0
0 0 4
0 0 0
0 0 0
0 0 0
0 0 4
1
0
0
1
0
1
0
2
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
60
0
3
63
0
1
0
64
3
0
0
3
0
0
0
3
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
3
1
0
0
4
63
0
3
66
1
1
0
68
Table 5. Distribution of Yersinia isolates in foods. Origin
Foods
Positive samples/ total of samples (%) Porcine Frankfurter 1/100 (1) Fresh 25/282 sausages (8.9) Cold foods 5/466 (1.1) Tongues 1/25 (4) Subtotal 32/873 (3.7) Bovine Ground meat 14/183 (7.7) Tongues 10/52 (19.2) Subtotal 24 /235 (10.2) Porcine and Fresh 7/191 bovine sausages (3.7) Pre-cooked 0/100 sausages (0) (“morcillas”) Subtotal 7/291 (2.4) Subtotal for bovine and 63/1404 porcine samples (4.5) Chicken Sausages 2/19 (10.5) Meatballs 3/25 (12)
Y. enterocolitica isolates (n) B1A B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. Total of kristensenii strains isolates (n)
1
0
0
1
0
0
0
1
11
7
2
20
5
0
0
25
1
4
0
5
0
0
0
5
1
0
0
1
0
0
0
1
14
11
2
27
5
0
0
32
14
0
0
14
0
0
0
14
9
1
0
10
0
0
0
10
23
1
0
24
0
0
0
24
3
4
0
7
0
0
0
7
0
0
0
0
0
0
0
0
3
4
0
7
0
0
0
7
40
16
2
58
5
0
0
63
1
0
0
1
1
0
0
2
1
0
0
1
0
0
3
2
Entrails
Hen eggs
Dairy products
Hake Fresh vegetables
9/40 (22.5) Hamburgers 3/30 (10) Carcasses 36/236 (15.3) Skin 17/216 (7.9) Subtotal 70/566 (12.4) Eggshell 11/322 (3.4) Liquid egg 3/45 (6.7) Subtotal 14/367 (3.8) Ice cream 2/203 (1) Goat cheese 0/30 (0) Raw milk 0/40 (0) Subtotal 2/273 (0.7) Fillets 4/335 (1.2) Tomatoes, 0/113 lettuce, (0) apples Total 153/3053 (5)
3
0
0
3
6
0
0
9
0
0
0
0
3
0
0
3
9
0
0
9
24
2
1
36
12
0
0
12
6
0
0
18
26
0
0
26
42
2
1
71
0
11
0
11
0
0
0
11
5
0
0
5
0
0
0
5
5
11
0
16
0
0
0
16
2
0
0
2
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
2
0
0
0
2
3
0
1
4
0
0
0
4
0
0
0
0
0
0
0
0
76
27
3
106
47
2
1
156
Table 6. Distribution of Yersinia isolates in environmental samples. Type of samples
Surface water (rivers and lakes) Sewage Total
Positive samples/ total of samples (%) 6/84 (7.1)
8/14 (57.1) 14/98 (14.3)
B1A
Y. enterocolitica isolates (n) B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
1
0
0
1
6
2
0
9
5
0
0
5
9
3
0
17
6
0
0
6
15
5
0
26
Declarations of interest None
S0740-0020(19)30955-4
DOI:
https://doi.org/10.1016/j.fm.2019.103345
Reference:
YFMIC 103345
To appear in:
Food Microbiology
Received Date: 11 June 2019 Revised Date:
5 September 2019
Accepted Date: 3 October 2019
Please cite this article as: Estrada, C.L., Favier, G.I., Escudero, M.E., An overview of Yersinia enterocolitica and related species in samples of different origin from San Luis, Argentina, Food Microbiology, https://doi.org/10.1016/j.fm.2019.103345. 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. © 2019 Published by Elsevier Ltd.
Highlights -
Animals and foods are important Y. enterocolitica sources in San Luis, Argentina.
-
The 5% of samples analyzed throughout 30 years was Yersinia positive.
-
183 Y. enterocolitica and 72 isolates of other Yersinia species were identified.
-
Pathogenic Y. enterocolitica bioserotypes were recovered from foods.
-
Biotype 1A was prevalent in Y. enterocolitica strains of various sources.
1
An overview of Yersinia enterocolitica and related species in samples of different
2
origin from San Luis, Argentina.
3 4
Cecilia Lucero Estrada1,2,*, Gabriela Isabel Favier1, María Esther Escudero1
5
1
6
Nacional de San Luis, Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis,
7
Argentina.
8
2
9
Investigaciones Científicas y Tecnológicas (IMIBIO-CONICET).
10
Microbiología General, Facultad de Química, Bioquímica y Farmacia, Universidad
Instituto Multidisciplinario de Investigaciones Biológicas, San Luis- Consejo Nacional de
Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San Luis, Argentina.
11 12 13 14 15 16
*
17
Cecilia Lucero Estrada
18
Tel. +54 0266 4520300 ext. 1611
19
Fax +54 0266 4431301
20
E-mail address: [email protected] (C. Lucero Estrada)
21
Full postal address: Microbiología General, Facultad de Química, Bioquímica y Farmacia,
22
Universidad Nacional de San Luis. Ejército de los Andes 950, Bloque 1 Piso 1, 5700 San
23
Luis, Argentina.
24
Corresponding author:
25
ABSTRACT
26
This study is aimed at offering an overview of the prevalence of Yersinia
27
enterocolitica and related species in San Luis, Argentina, from samples of diverse origin
28
received in our laboratory between 1984 and 2014, and providing an analysis of the
29
distribution of Yersinia isolates according to their isolation sources, highlighting
30
bioserotypes and potential reservoirs and vehicles of transmission to humans. From a total
31
of 4,572 samples of human, animal, food and environmental origins analyzed by traditional
32
culture methods and molecular techniques, 229 (5%) samples were Yersinia positive. The
33
highest frequency of Yersinia isolates was observed in environmental specimens (14.3%),
34
followed by animal (9.2%), food (5%) and human (0.6%) samples. A total of 255 Yersinia
35
isolates were characterized, including 183 Y. enterocolitica and 72 isolates of other Yersinia
36
species. Biotype 1A associated to several serotypes was identified in Y. enterocolitica
37
isolates from environment (100%), animals (95.5%), foods (71.7%) and human samples
38
(40%); bioserotype 2/O:9 was identified in isolates from foods (25.5%), and biotype 3 was
39
associated with strains from humans (60%), animals (4.5%) and foods (2.8%). This biotype
40
included three strains O:3 and six strains O:5. The data highlight animals and foods as the
41
main Y. enterocolitica sources in our region.
42 43 44 45 46 47 48
Keywords: Yersinia enterocolitica, enteropathogen, biotype, serotype, epidemiology
49 50
1. Introduction Yersinia enterocolitica is the causative agent of a wide range of diseases, mainly
51
gastrointestinal manifestations, in addition to severe complications such as mesenteric
52
lymphadenitis, reactive arthritis and sepsis (Duan et al., 2017). Although foods from
53
porcine origin are the main source of human infection, epidemiological studies highlight
54
other animal carriers, as well as foods of various origins and the environment as possible
55
sources of Y. enterocolitica infection for humans (Rahman et al., 2011). Due to its
56
psychrotrophic growth, Y. enterocolitica is a hazardous organism in foods with extended
57
storage in chilling conditions (De Silvestri et al., 2018). Bioserotypes 1B/O:8, 2/O:5,27,
58
2/O:9, 3/O:3 and 4/O:3 have been associated to human infections in different countries. The
59
strains of five biotypes (1B, 2, 3, 4 and 5) may be carriers of a 70-kb virulence plasmid
60
(pYV) and chromosomally encoded virulence genes. Strains of biotype 1A include a wide
61
range of serotypes, which have been considered as nonpathogenic for a long time because
62
they lack the classical Y. enterocolitica virulence determinants (Joutsen et al., 2017). There
63
is epidemiological and experimental evidence, however, suggesting that some strains of this
64
biotype can cause gastrointestinal infections (Campioni and Falcão, 2014). Y. intermedia,
65
Y. frederiksenii and Y. kristensenii are often categorized as Y. enterocolitica-like species
66
and have been isolated from healthy and sick humans and animals, as well as from food and
67
environmental sources (CDC, 2014). Their pathogenic potential might be related to the
68
presence of some Y. enterocolitica virulence genes, although the existence of alternative
69
virulence mechanisms has also been suggested (Imori et al., 2017).
70
There is very little information about the epidemiology of Y. enterocolitica in
71
Argentina, where it is considered a pathogen of moderate risk and limited dissemination
72
(AAM, 2013). Y. enterocolitica has been sporadically recovered from animals, foods, water
73
and human clinical samples in various regions of this country. The present study is aimed at
74
offering an overview of the prevalence of Y. enterocolitica in San Luis, Argentina, from
75
samples of diverse origin studied in our laboratory between 1984 and 2014, and providing
76
an analysis of the distribution of Yersinia isolates according to their isolation sources,
77
highlighting bioserotypes and potential reservoirs and vehicles of transmission to humans.
78
79
2. Materials and methods
80
2.1. Samples
81
A total of 4,572 samples, including human stool samples, presumptive animal
82
reservoirs, foods for human consumption and environmental samples, were collected
83
between 1984 and 2014 in various locations of San Luis City and surrounding areas and
84
investigated for Yersinia.
85
Human stool samples (n = 801) were collected from pediatric and adult patients
86
with enterocolitis symptoms attending four local clinical laboratories for microbiological
87
analysis of feces in the 1989-1993, 2003-2004 and 2008-2011 periods. Animal samples (n
88
= 620) were collected between 1984 and 2011 during surveys of Yersinia potential
89
reservoirs in San Luis City and upcoming rural areas. Samples of farm animals were
90
randomly collected in slaughters, farms, feedlots and cattle markets. Samples of wild
91
animals including wild boars (Sus scrofa), viscachas (Lagostomus maximum maximum),
92
and trouts (Oncorhynchus mykiss) were provided by local hunters and fishers, and stools of
93
different bird species (Passer domesticus, Columba maculosa, Sicalis luteosa, Molothrus
94
rufoaxillaris, Cyanocompsa brissoni, Saltator aurantiirostris, Turdus chiguanco,
95
Myiopsitta monacha, Pardalotus puntactus, Serinus canarius and Zonotrichia capensis)
96
were collected in their natural habitats, immediately after defecation. Samples of foods
97
intended for human consumption (n = 3,053) were purchased in local butcher shops, retail
98
markets, fish shops and greengroceries between 1984 and 2014. Environmental samples (n
99
= 98) included 84 surface water samples collected from three rivers (Grande, El Trapiche
100
and El Volcán) and two lakes (La Florida and Potrero de los Funes) in the San Luis
101
Province between 1990 and 1991, and 14 wastewater samples from a treatment plant of
102
sewage effluents near San Luis City during 1991. All samples were packed in individual
103
sterile containers, sent to the Laboratory of Microbiology of the National University of San
104
Luis, and stored at 4 °C for up to 6 h before processing.
105
Samples were investigated during periodical surveys with the purposes of
106
establishing the frequency of Yersinia species in humans, animals, foods and environment
107
for the San Luis region, and determining possible carriers, transmission vehicles and
108
potential sources of infection.
109 110
2.2. Characterization of the Yersinia isolates
111
Enrichment techniques and selective media for the isolation of Yersinia strains were
112
applied according to international standards. Presumptive Yersinia colonies were examined
113
by Gram staining and identified by classical biochemical assays (FDA, 2007). The final
114
characterization of biotypes and serotypes was performed by Dr. E. Carniel, National
115
Reference Center of Yersinia, Institute Pasteur, Paris, France.
116 117 118 119
2.3. Assessment of pathogenic potential of Y. enterocolitica strains In vitro phenotypic tests such as autoagglutination (AA) at 37 °C (Laird and Cavanaugh, 1980) and calcium-dependent growth (CA) (Riley and Toma, 1989) were
120
performed for all Y. enterocolitica isolates in order to differentiate plasmid-bearing from
121
plasmidless strains. In addition, virulence genetic markers such as virF, ail, ystA, ystB and
122
myfA were assayed by PCR in strains isolated from 2005 to date and performed according
123
to Favier et al. (2005) and Lucero Estrada et al. (2011, 2012, 2015).
124 125
2.4. Statistical analysis
126
Association between Yersinia species, their bioserotypes and isolation sources was
127
assessed by using Chi-square test (Analytical Software, Tallahassee FL, USA). Statistical
128
calculations were based on confidence level equal or higher than 95% (p ≤ 0.05 was
129
considered statistically significant).
130 131
3. Results
132 133 134
3.1. Distribution of Yersinia of different sources in San Luis From a total of 4,572 samples from human, animal, food and environmental sources
135
collected in San Luis, Argentina, 229 (5%) samples were Yersinia positive (Table 1). The
136
highest frequency of Yersinia isolates was observed in environmental specimens (14.3%),
137
followed by animal (9.2%), food (5%) and human (0.6%) samples (p ≤ 0.05). A total of 255
138
Yersinia isolates were identified, including 183 Y. enterocolitica strains recovered from
139
3.6% of samples, and 72 isolates of other Yersinia species recovered from 1.4% of samples.
140
Y. enterocolitica positive samples were distributed as follows: animals (8.9%) > foods
141
(3.4%) = environment (3.1%) > humans (0.6%) (p ≤ 0.05). Y. enterocolitica was the only
142
species isolated from human samples. Other Yersinia species (Y. intermedia, Y.
143
frederiksenii and Y. kristensenii) were detected as follows: environment (11.2%) > foods
144
(1.6%) > animals (0.3%) (p ≤ 0.05). Y. intermedia was the predominant species with 63
145
isolates, while only eight Y. frederiksenii isolates and one Y. kristensenii isolate were
146
obtained.
147 148 149
3.2. Biotype and serotype distribution of Y. enterocolitica strains from different sources The biotype and serotype distribution of 183 Y. enterocolitica strains according to
150
their sources can be seen in Table 2. The most frequent biotype was 1A (147 isolates,
151
80.3%) followed by biotypes 2 (27 isolates, 14.8%) and 3 (nine isolates, 4.9%) (p ≤ 0.05).
152
Out of 19 serotypes associated to biotype 1A, the most frequent were: O:5 (35.5%), O:6
153
(12.0%), O:6,30-6,31 (7.1%), O:7,8-8-8,19 (6.6%), O:41,42-41,43 (4.4%) and O:12,25-
154
12,26 (2.2%). Six Y. enterocolitica 1A strains could not be serotyped. Regarding the
155
biotype distribution according to the isolation sources, biotype 1A predominated in isolates
156
from environmental origin (100%), followed by isolates from animal origin (95.5%) and
157
food samples (71.7%). This biotype was present in two out of five isolates of human origin.
158
Biotype 2 was identified in 25.5% isolates recovered from food samples (26 strains O:9 and
159
one non-serotyped isolate), while biotype 3 was identified in strains from human (three out
160
of five isolates), animal (4.5%) and food (2.8%) origin. This last biotype included three
161
strains O:3 and six strains O:5. All 1A strains were negative for the phenotypic virulence
162
markers and 31 of 62 studied strains were ystB+. As regards 2/O:9 strains, all of them were
163
positive for AA and CA. Among 27 isolates, 21 of them were characterized for virulence
164
genetic markers; 18 strains were virF+ myfA+ ail+ ystA+ and three strains were virF- myfA+
165
ail+ ystA+. Similarly, all 3/O:3 and 3/O:5 isolates were positive for AA and CA. One Y.
166
enterocolitica 3/O:3 strain was virF- ail+, and two of them were virF+ ail+. Among six
167
3/O:5 isolates recovered from foods, only four strains were assayed for virulence genetic
168
markers: three isolates were virF+ ail+ whereas the fourth one was virF- ail+ (Favier et al.,
169
2005; Lucero et al., 2011; Lucero et al., 2012; Lucero et al., 2015).
170 171 172
3.3. Characteristics of Yersinia strains from human samples Eight hundred and one samples of human feces were analyzed for the presence of
173
Yersinia species (Table 3), 0.6% of which were positive, yielding five Y. enterocolitica
174
isolates. Of these, two Y. enterocolitica isolates were characterized as 1A/O:5 and
175
1A/O:7,8-8-8,19, and the other three ones were identified as 3/O:3.
176 177
3.4. Distribution and characteristics of Yersinia strains of animal origin
178
Table 4 shows the distribution and characteristics of Yersinia isolates in samples of
179
animal origin. Samples of intestinal content and different body parts of farm animals (pigs,
180
cows, goats and chicken) and wild animals (wild boars, fishes, rodents, and birds) were
181
investigated. Yersinia was positive in 8.9% of animal samples. Positive samples were more
182
frequent in farm animals (12.7%) with 64 isolates, than in wild animals (2%) with four
183
isolates (p ≤ 0.05). Y. enterocolitica (66 isolates, 97.1%) was found to be more frequent
184
than other Yersinia species (two isolates, 2.9%) (p ≤ 0.05). As regards farm animals, pigs
185
yielded the highest number of Y. enterocolitica isolates, with 57 obtained from cecal
186
contents and four from skin and bones. Out of these, 58 isolates were characterized as 1A
187
and three as 3/O:5. More than one strain per sample was found in pig cecal contents. Cows
188
(2%) were also carriers of one Y. enterocolitica 1A isolate and one Y. frederiksenii isolate,
189
followed by goats (1%) with one Y. enterocolitica 1A isolate. No isolations were obtained
190
from pig stools or tonsils, or from chicken cecal contents. Among wild animals, three out of
191
six wild boar samples were positive for Y. enterocolitica 1A. In contrast, a low isolation
192
rate (1%) was observed in trout gills with one Y. intermedia isolate, whereas rodent and
193
bird samples were negative.
194 195 196
3.5. Distribution and characteristics of Yersinia strains isolated from foods Yersinia prevalence in foods was 5% (Table 5). Yersinia species were most frequent
197
in chicken (12.4%) and foods of bovine origin (10.2%), followed by hen eggs (3.8%),
198
products of porcine origin (3.7%), products based on mixtures of porcine and bovine meat
199
(2.4%), hake fillets (1.2%) and dairy products (0.7%) (p ≤ 0.05). No isolations were
200
obtained from fresh vegetables.
201
Positive chicken samples yielded 71 Yersinia isolates, with the following
202
distribution: entrails (22.5%), carcasses (15.3%), meatballs (12%), sausages (10.5%),
203
hamburgers (10%) and skin (7.9%). Twenty-four Yersinia isolates were obtained from
204
bovine ground meat (7.7%) and tongues (19.2). Foods of porcine origin yielded 32 Yersinia
205
isolates, mainly from fresh sausages (8.9%) and tongues (4%) (p ≤ 0.05), followed by cold
206
foods (1.1%) and frankfurter (1%). Yersinia detection in fresh sausages of porcine and
207
bovine origin was 3.7%, whereas pre-cooked sausages were Yersinia negative.
208
From a total of 156 Yersinia isolates recovered from food samples, Y. enterocolitica
209
(106) predominated over Y. intermedia (47), Y. frederiksenii (2) and Y. kristensenii (1) (p ≤
210
0.05). Specifically, Y. enterocolitica (27 isolates) predominated over Y. intermedia (5
211
isolates) in foods of porcine origin and it was the only species recovered from foods of
212
bovine origin (24 isolates), porcine and bovine foods (7), hen eggs (16), dairy products (2)
213
and hake fillets (4). Y. enterocolitica recovery in liquid homogenates of egg yolk and white
214
(6.7%) was higher than in eggshells (3.4%) (p ≤ 0.05). Two Y. enterocolitica strains were
215
isolated from ice cream.
216
Overall, Y. enterocolitica biotype 1A associated to different serotypes (76 isolates)
217
was more frequent than biotype 2 (27 isolates) and biotype 3 (three isolates). Biotype 1A
218
was predominant (40 isolates) over biotype 2 (16 isolates) in foods prepared with porcine
219
and/or bovine meat. Conversely, bioserotype 2/O:9 was predominant (11 isolates from
220
eggshell) over biotype 1A (five isolates from liquid egg) in hen eggs. In addition, chicken
221
carcasses were strongly associated to Y. intermedia carriage (24 strains) followed by
222
chicken skin and entrails (6 strains each) and other chicken samples (≤ 3 strains each). Y.
223
frederiksenni and Y. kristensenii were only recovered from chicken carcasses.
224 225 226 227
3.6.
Prevalence and characteristics of Yersinia strains isolated from
environmental sources The distribution and characteristics of 26 Yersinia strains isolated from
228
environmental samples are shown in Table 6. Overall, Yersinia was detected in 14.3% of
229
the samples. The highest recovery frequency was observed in sewage samples (57.1%)
230
followed by surface water of rivers and lakes (7.1%). The frequency of the Yersinia species
231
was as follows: Y. intermedia (15 strains) > Y. enterocolitica (6 strains) = Y. frederiksenii (5
232
strains) (p ≤ 0.05). All Y. enterocolitica isolates belonged to biotype 1A.
233 234
4. Discussion
235
For many years, our laboratory has performed periodical surveys for Yersinia
236
surveillance on suspect animal reservoirs and carriers, foods of different origin intended for
237
human consumers, and samples of symptomatic patients. The accumulated data on Yersinia
238
positive samples collected over 30 years, and the characteristics of the isolates might offer
239
an overview of reservoirs, sources of contamination and underdiagnosed cases of
240
yersiniosis in this region.
241
The data highlight animals and foods as the main Y. enterocolitica sources and the
242
predominance of biotype 1A associated to different serotypes in Y. enterocolitica isolates
243
from all samples, excepting human stools. Biotype 1A has been implicated in foodborne
244
and nosocomial outbreaks around the world (Bhagat and Virdi, 2011); however, its
245
pathogenicity mechanism in humans has not yet been elucidated. Severe pathogenesis in a
246
Galleria mellonella model with a lethal dose as low as 10 CFU, and the major role of
247
flagella in the virulence phenotype have been observed for this biotype (Alenizi et al.,
248
2016). Strains of biotypes 2 and 3 were also recovered from samples of San Luis.
249
Although protocols recommended by international standards for Yersinia survey in samples
250
of diverse origin (FDA, 2007) were applied, bioserotype 4/O:3, which has been linked to
251
approximately 80% of Y. enterocolitica human infections worldwide, with pigs as the main
252
reservoirs (Duan et al., 2017), was not identified in strains reported in the present study.
253
The here reported percentage of Y. enterocolitica observed in feces of pediatric and
254
adult patients in San Luis was compared to results from other Argentine regions. This value
255
was similar to that observed for feces of 1-12 years old patients (0.8%) in Neuquén
256
(González et al., 2010), and lower than 1.13% (Cortes et al., 2010) and 3.3% (Sánchez and
257
González, 2013) reported from fecal samples of pediatric patients from Córdoba. In
258
addition, Sánchez and González (2013) identified biotype 4 in all their isolates. To date, the
259
majority of Y. enterocolitica isolates from human diarrhea cases described in Argentina
260
have been identified as belonging to biotype 4. Thus, this biotype was identified in 43 Y.
261
enterocolitica isolates recovered from sporadic clinical cases in nine hospitals of five
262
Argentine provinces (Barcudi et al., 2014). Interestingly, Paz et al. (2004) identified one Y.
263
enterocolitica 1A/O:5 strain from diarrheic feces of a patient from Buenos Aires,
264
Argentina. In Uruguay, Y. enterocolitica isolates have been recovered from sporadic
265
clinical cases and one gastroenteritis outbreak in hospitalized infants; all strains except one
266
1/O:8 isolate from liver abscess, were identified as 4/O:3 (Mota et al., 2012). In Brazil, Y.
267
enterocolitica 4/O:3 was the predominant bioserotype in strains of human origin (Rusak et
268
al., 2014). The low isolation rate of Y. enterocolitica from clinical samples may be caused
269
by the presence of background microbiota and the limited sensitivity of culture methods
270
(Morka et al., 2018). Thus, Yersinia intestinal infections in humans might be
271
underdiagnosed in San Luis. In contrast with public health policies in other countries where
272
Y. enterocolitica is a notifiable gastrointestinal pathogen (Jeffs et al., 2019), the
273
surveillance of this microorganism and its report to the national net of epidemiology is not
274
mandatory in Argentina.
275
Y. enterocolitica can colonize a broad range of farm and wild animals. The study of
276
animal samples from San Luis revealed that the presence of Yersinia is higher in farm
277
animals than in wild animals, and highlighted pigs as the main reservoirs of Y.
278
enterocolitica in our region, followed by cows and goats. In a study carried out in China,
279
Liang et al. (2015) obtained the highest Y. enterocolitica prevalence among pigs (12.9%),
280
followed by chickens (4.5%), rodents (3.4%), cattle (2.7%) and sheep (0.8%). Biotype 1A
281
in association with different serotypes and bioserotype 3/O:5 predominated in Y.
282
enterocolitica isolates from pig samples in San Luis. By contrast, a high prevalence of
283
4/O:3 strains have been demonstrated in pigs worldwide (Rahman et al., 2011, Martins et
284
al., 2018, Raymond et al., 2018), and predominance of 2/O:9 and 3/O:3 strains has been
285
reported for pigs in England (Ortiz Martínez et al., 2010) and China (Liang et al., 2015),
286
respectively. This suggests that the association between Y. enterocolitica bioserotypes and
287
pigs might be country-dependent, due to specific breeding and eating habits (Le Guern et
288
al., 2016). Similarly to our results where the highest Y. enterocolitica recovery was
289
observed in pig cecal contents, Fois et al. (2018) obtained the best Y. enterocolitica
290
recovery from pig intestinal content in Italy.
291
Although it has not yet been possible to demonstrate whether cattle constitute
292
reservoirs or merely transient carriers of Y. enterocolitica, this microorganism has been
293
reported from cattle feces worldwide (Rahman et al., 2011). Detection frequencies of Y.
294
enterocolitica in cows and goats from San Luis were lower than those reported for cattle
295
feces (16%) in Ireland (O’Grady et al., 2016) and for goat flocks (14.9%) in New Zealand
296
(Lanada et al., 2005). All strains isolated from cows and goats in our region belonged to
297
biotype 1A, in contrast with 2/O:9 strains recovered from cows, goats and sheep in France
298
(Le Guern et al., 2016). It has been suggested that since these animals may share habitats,
299
pastures or drinking water in a certain geographical region, the transmission of Yersinia
300
among them is possible and the contamination fecal-oral may occur (Le Guern et al., 2016).
301
The presence of Y. enterocolitica in wild boar samples was demonstrated for the
302
first time in San Luis; however, a higher number of samples should be tested to determine
303
whether these animals are Yersinia usual reservoirs in our region, as has been reported in
304
other countries (Fredriksson-Ahomaa et al., 2011, Magistrali et al., 2014, Morka et al.,
305
2018). All other wild animal samples analyzed showed negative results for Yersinia.
306
Finally, as regards fish samples, Y. intermedia was recovered. As known, only
307
Yersinia ruckeri has been reported as a significant concern for fish farming around the
308
world (Wrobel et al., 2018, Delalay et al., 2019).
309
Y. enterocolitica 1A was predominant in foods from San Luis, followed by Y.
310
intermedia and Y. enterocolitica biotype 2. Similarly, 1A was reported to be the most
311
frequent biotype (59.7%) isolated from foods in France, followed by Y. intermedia (17.2%)
312
and Y. frederiksenii, Y. kristensenii and Y. bercovieri in lower percentages (Le Guern et al.,
313
2016). While Y. enterocolitica 4/O:3 was not isolated from foods in San Luis, this
314
bioserotype represented 3% of all isolates from foods in France, mainly recovered from
315
pork meat (Le Guern et al., 2016). The present study demonstrated high prevalence of
316
Yersinia species in chicken samples, with carcasses as the main sources. Y. enterocolitica
317
and Y. intermedia were the most frequent species in these samples. A previous study
318
performed in Argentina reported 10% of Y. enterocolitica or related species from chicken
319
carcasses in a processing plant of Buenos Aires, distributed as follows: 4.3% Y.
320
enterocolitica 1A/O:5, 1.4% Y. intermedia, and 4.3% Y. frederiksenii (Floccari et al., 2000).
321
Contamination of carcasses with Yersinia probably occurs by contamination in
322
slaughterhouses, processing plants, vehicles of transportation or commercial refrigerators
323
where other slaughtered animals are stored.
324
Low Yersinia recovery rates were observed in foods of bovine, porcine and
325
bovine/porcine origin. This can be accounted for the manufacturing and storing procedures
326
applied on these foods, which may destroy, inhibit or stress Yersinia cells thus preventing
327
their recovery by culture. Y. enterocolitica 1A isolates predominated in foods of this origin.
328
Strikingly, Y. enterocolitica 2/O:9 recovery from eggshells was observed. The presence of
329
Yersinia species in eggshells and liquid eggs seems to be uncommon. In USA, one Yersinia
330
spp isolate was reported from 84 washed and unwashed commercial shell eggs (Musgrove
331
et al., 2004). In Australia, six Y. enterocolitica isolates were obtained from eggshell surface
332
of 1,860 table eggs collected from layer flocks (Gole et al., 2013). These authors have
333
postulated that eggshells can get contaminated by any surface with which they come in
334
contact, being feces, water, caging material, nesting material, insects, hands, blood and soil
335
the most common sources of contamination. Contamination of the egg surface might also
336
occur from contact with other Y. enterocolitica contaminated products of animal origin
337
such as pork, during collection on farms or during transportation or handling in retail shops
338
(Favier et al., 2005). When the eggshell is broken, contamination of the egg content is
339
possible.
340
In San Luis, Yersinia recovery from dairy products and hake fillets was low and no
341
Yersinia isolate was detected in fresh vegetables. Previous studies performed in Buenos
342
Aires, Argentina, reported Y. enterocolitica 1A (5.5%), Y. frederiksenii (9.9%) and Y.
343
intermedia (3.6%) from raw cow milk (Mercado and Ibañez, 1986). Several investigations
344
have reported outbreaks of foodborne infections caused by consumption of Y. enterocolitica
345
contaminated dairy products in other countries (Rahimi et al., 2014). The low percentage of
346
Y. enterocolitica obtained in hake fillets from San Luis, matches reports of 2.7% Y.
347
enterocolitica 1A in retail seafoods from Germany (Li et al., 2018). No Yersinia isolate was
348
detected in fresh vegetables in San Luis. However, vegetables can be a source of human
349
infection by Y. enterocolitica (Lee et al., 2004; Sakai et al., 2005).
350
Our results point out foods of animal origin contaminated by pathogenic biotypes 2
351
and 3, such as porcine fresh sausages, porcine cold foods, bovine tongues, porcine and
352
bovine fresh sausages, hake fillets and eggshells. The Y. enterocolitica presence is
353
commonly associated to pork production (Laukkanen-Ninios et al., 2014), however, cross-
354
contamination from pork to foods of other animal origin is possible at farm level,
355
slaughterhouse, butcher shop or home (Fredriksson-Ahomaa et al., 2004). Y. enterocolitica,
356
as a typical psychrotrophic microorganism, multiplies in food (neutral pH, stored at 5°C)
357
from 10/mL to 2.8×107/mL in 5 days, and in products kept in a freezer at -18°C, and retains
358
pathogenic properties for several months (Bancerz-Kisiel and Szweda, 2015). Considering
359
the fact that raw or undercooked foods are the main source of yersiniosis in humans,
360
education for consumers might be an important resource to prevent Y. enterocolitica
361
infections (FDA, 2012). Good production and manufacturing practices for foods of animal
362
origin, Standardized Operating Procedures for Sanitation. (SOPS), and Hazard Analysis
363
and Critical Control Points (HACCP) are also recommended.
364
This study reports significant Yersinia recovery from environmental samples, with
365
Y. intermedia, Y. frederiksenii and Y. kristensenii predominating over Y. enterocolitica 1A.
366
These species were also recovered from 90% of sewage water samples in Buenos Aires
367
City, Argentina (Floccari et al., 2003). The presence of Y. enterocolitica in surface water is
368
a risk factor for human infections in recreational activities, crop irrigation or contaminated
369
drinking water. This microorganism has also been isolated from river water in Canada
370
(Cheyne et al., 2010), Poland (Terech-Majewska et al., 2016) and Argentina (Kuczynski,
371
2016). The Argentine strains have been shown to be resistant to adverse environment
372
factors and might survive in untreated drinking water (Kuczynski, 2016). Interestingly,
373
Floccari et al. (2003) demonstrated that the 16S rRNA gene sequence of five Y.
374
enterocolitica 1A strains isolated from sewage water in Buenos Aires, was specific for
375
the European subspecies Y. enterocolitica palearctica. These authors hypothesize that Y.
376
enterocolitica might have been imported into Argentina with animals, food, feed or even
377
humans and that it was possible that these strains adapted to adverse “living conditions”
378
in a new continent. The route of Y. enterocolitica transmission in our region is unknown,
379
but it could begin in the environment, from where the bacteria could be transmitted to
380
animals, spread in foods of animal origin, and finally, reach consumers. The stages of this
381
route would be related in different ways, giving rise to multiple possibilities of
382
contamination and infection.
383 384
5. Conclusions The present study demonstrated that the main sources of Y. enterocolitica isolation
385 386
in San Luis are animals and foods, from which this organism could be transmitted to
387
humans. Biotype 1A was prevalent in Y. enterocolitica strains of various sources. The
388
pathogenic bioserotypes 2/O:9, 3/O:3 and 3/O:5 were demonstrated in food and clinical
389
samples. Sewage was a prominent reservoir of other Yersinia species. A close
390
correspondence was observed between Y. enterocolitica bioserotypes and isolation sources;
391
for instance, 2/O:9 was exclusively related to food isolates, 3/O:5 was linked to animal and
392
food isolates, and 3/O:3 was associated to human strains. The present analysis of the data
393
collected throughout 30 years provides an overview of Yersinia sources, reservoirs and
394
probable cases of undiagnosed yersiniosis in our region. The frequency of contacts between
395
sources of animal origin and humans as well as the consumption of contaminated foods
396
might contribute to the risk of infection caused by Y. enterocolitica. Since human clinical
397
cases associated to biotype 1A have been reported worldwide, the pathogenic potential of
398
each isolate 1A should be always investigated.
399 400
Declarations of interest
401
None
402 403
404
Acknowledgments
405
We wish to thank Dr. Ana María Stefanini de Guzman and her research group at
406
National University of San Luis, who were pioneers in the investigation of Yersinia
407
enterocolitica in San Luis, Argentina. This work was supported by the Science and
408
Technology Department, National University of San Luis, Argentina (Projects PROICO
409
8803, 2-0914 and 02-1518), Alexander von Humboldt Foundation from Germany, and the
410
National Agency for Scientific and Technological Promotion, Argentina (PICT-2015-
411
1722). We appreciate language revision by staff of Langue Institute, National University of
412
San Luis.
413 414
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Tabla 1. Isolation sources of Yersinia species from samples collected in San Luis from 1984 to 2014.
Species
Biotypes
Humans
Animals
Foods
Environment
Total
(B) Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Positive samples/ total of samples (%)
N° of strains
Y. 1A enterocolitica
2/801 (0.2)
2
52/620 (8.4)
63
73/3053 (2.4)
76
3/98 (3.1)
6
130/4572 (2.8)
147
2-3
3/801 (0.4)
3
3/620 (0.5)
3
30/3053 (1.0)
30
0/98 (0)
0
36/4572 (0.8)
36
5/801 (0.6)
5
55/620 (8.9)
66
103/3053 (3.4)
106
3/98 (3.1)
6
166/4572 (3.6)
183
Subtotal
Y. intermedia
-
0/801 (0)
0
1/620 (0.2)
1
47/3053 (1.5)
47
8/98 (8.2)
15
56/4572 (1.2)
63
Y. frederiksenii
-
0/801 (0)
0
1/620 (0.2)
1
2/3053 (0.1)
2
3/98 (3.1)
5
6/4572 (0.1)
8
Y. kristensenii
-
0/801 (0)
0
0/620 (0)
0
1/3053 (0.03)
1
0/98 (0)
0
1/4572 (0.02)
1
Subtotal
0/801 (0)
0
2/620 (0.3)
2
50/3053 (1.6)
50
11/98 (11.2)
20
63/4572 (1.4)
72
Total
5/801 (0.6)
5
57/620 (9.2)
68
153/3053 (5.0)
156
14/98 (14.3)
26
229/4572 (5.0)
255
Table 2. Biotype and serotype distribution of Y. enterocolitica strains isolated from different sources.
Biotype (B) 1A
2
3
Serotype (O) 3,50,51 4,32-4,33 5 5-4,32-4.33 6 6,30 6,31 6,30-6,31 6,47 7,8 7,8-8-8,19 7,8-8-13-8,19 7-13 10-34 12,25-12,26 15,47 16,36 34,46 41,42-41,43 AA NA ND Subtotal 9 AA Subtotal 3 5 Subtotal Total
Humans N° of % strains 1 20 1 20 2 40 0 0 3 60 3 60 5 100
Animals N° of % strains 29 44.0 20 30.3 5 7.6 3 4.5 1 1.5 1 1.5 1 1.5 1 1.5 2 3.0 63 95.5 0 0% 3 4.5 3 4.5 66 100
Foods N° of % strains 1 0.9 2 1.9 31 29.2 1 0.9 2 1.9 2 1.9 1 0.9 8 7.5 1 0.9 1 0.9 8 7.5 1 0.9 4 3.8 1 0.9 6 5.7 2 1.9 3 2.8 1 0.9 76 71.7 26 24.5 1 0.9 27 25.5 3 2.8 3 2.8 106 100
Environment N° of % strains 4 66.7 1 16.7 1 16.7 6 100 0 0 0 0 6 100
Total N° of % strains 1 0.5 2 1.1 65 35.5 1 0.5 22 12.0 2 1.1 1 0.5 13 7.1 1 0.5 1 0.5 12 6.6 2 1.1 1 0.5 2 1.1 4 2.2 1 0.5 1 0.5 1 0.5 8 4.4 2 1.1 3 1.6 1 0.5 147 80.3 26 14.2 1 0.5 27 14.8 3 1.6 6 3.3 9 4.9 183 100
%: percentage of each Yersinia serotype in the total of Yersinia strains of the same origin. AA: autoagglutinated; NA: no agglutination; ND: not determined (the serotype was unavailable in the three cases).
Table 3. Distribution of Yersinia isolates in stool samples of human origin. Positive samples/ total of samples (%) 5/801 (0.6)
B1A
2
Y. enterocolitica isolates (n) B2 B3 Total
0
3
5
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
0
0
0
5
Table 4. Distribution of Yersinia isolates in samples of animal origin. Animals
Type of samples
Pigs
Cecal contents Stools Tonsils Skin and bones Cows Rectal contents Goats Cecal contents Chicken Cecal contents Subtotal of farm animals Wild boars Tonsils and tongues Trouts Gills Rodents (viscacha) Birds
Cecal contents Stools
Subtotal of wild animals Total
Positive samples/ total of samples (%) 46/129 (35.7) 0/2 (0) 0/6 (0) 4/30 (13.3) 2/100 (2) 1/100 (1) 0/50 (0) 53/417 (12.7) 3/6 (50) 1/100 (1) 0/31 (0) 0/66 (0) 4/203 (2) 57/620 (8.9)
B1A
Y. enterocolitica isolates (n) B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
54
0
3
57
0
0
0
57
0 0 4
0 0 0
0 0 0
0 0 4
0 0 0
0 0 0
0 0 0
0 0 4
1
0
0
1
0
1
0
2
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
60
0
3
63
0
1
0
64
3
0
0
3
0
0
0
3
0
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
3
1
0
0
4
63
0
3
66
1
1
0
68
Table 5. Distribution of Yersinia isolates in foods. Origin
Foods
Positive samples/ total of samples (%) Porcine Frankfurter 1/100 (1) Fresh 25/282 sausages (8.9) Cold foods 5/466 (1.1) Tongues 1/25 (4) Subtotal 32/873 (3.7) Bovine Ground meat 14/183 (7.7) Tongues 10/52 (19.2) Subtotal 24 /235 (10.2) Porcine and Fresh 7/191 bovine sausages (3.7) Pre-cooked 0/100 sausages (0) (“morcillas”) Subtotal 7/291 (2.4) Subtotal for bovine and 63/1404 porcine samples (4.5) Chicken Sausages 2/19 (10.5) Meatballs 3/25 (12)
Y. enterocolitica isolates (n) B1A B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. Total of kristensenii strains isolates (n)
1
0
0
1
0
0
0
1
11
7
2
20
5
0
0
25
1
4
0
5
0
0
0
5
1
0
0
1
0
0
0
1
14
11
2
27
5
0
0
32
14
0
0
14
0
0
0
14
9
1
0
10
0
0
0
10
23
1
0
24
0
0
0
24
3
4
0
7
0
0
0
7
0
0
0
0
0
0
0
0
3
4
0
7
0
0
0
7
40
16
2
58
5
0
0
63
1
0
0
1
1
0
0
2
1
0
0
1
0
0
3
2
Entrails
Hen eggs
Dairy products
Hake Fresh vegetables
9/40 (22.5) Hamburgers 3/30 (10) Carcasses 36/236 (15.3) Skin 17/216 (7.9) Subtotal 70/566 (12.4) Eggshell 11/322 (3.4) Liquid egg 3/45 (6.7) Subtotal 14/367 (3.8) Ice cream 2/203 (1) Goat cheese 0/30 (0) Raw milk 0/40 (0) Subtotal 2/273 (0.7) Fillets 4/335 (1.2) Tomatoes, 0/113 lettuce, (0) apples Total 153/3053 (5)
3
0
0
3
6
0
0
9
0
0
0
0
3
0
0
3
9
0
0
9
24
2
1
36
12
0
0
12
6
0
0
18
26
0
0
26
42
2
1
71
0
11
0
11
0
0
0
11
5
0
0
5
0
0
0
5
5
11
0
16
0
0
0
16
2
0
0
2
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
2
0
0
0
2
3
0
1
4
0
0
0
4
0
0
0
0
0
0
0
0
76
27
3
106
47
2
1
156
Table 6. Distribution of Yersinia isolates in environmental samples. Type of samples
Surface water (rivers and lakes) Sewage Total
Positive samples/ total of samples (%) 6/84 (7.1)
8/14 (57.1) 14/98 (14.3)
B1A
Y. enterocolitica isolates (n) B2 B3 Total
Y. intermedia isolates (n)
Y. frederiksenii isolates (n)
Y. kristensenii isolates (n)
Total of strains
1
0
0
1
6
2
0
9
5
0
0
5
9
3
0
17
6
0
0
6
15
5
0
26
Declarations of interest None