Prevalence, serovars, and risk factors associated with the presence of Salmonella in pork sold in public markets in Quito, Ecuador

Background Salmonella enterica are bacteria that include more than 2,500 serovars. Most of these serovars have been linked to human foodborne illnesses, mainly related to poultry and pigs. Thus, these animals are considered the reservoirs of many Salmonella serovars and strains related to antibiotic resistance. This study aimed to determine the prevalence, serovars, β-lactam resistance genes, and the risk factors associated with Salmonella enterica in pork commercialized in open markets of Quito city. Methods For this, 165 pork meat samples were taken from municipal markets in three areas in the city. These samples were microbiologically processed following the ISO 6579-2014 standardized method. The polymerase chain reaction (PCR) test was used to identify Salmonella serotyping and resistance genes. Strains not identified by PCR were typed by the Kauffman White Le Minor scheme. A multivariate analysis was performed to identify risk factors associated with the presence of the microorganism. Results Salmonella prevalence in pork was 9.1%. Identified serovars were 4, [5], 12: i:- (53.3%), Infantis (33.3%), and Derby (13.4%). Furthermore, the β-lactam resistance genes bla CTX-M-65 could be identified in three S. infantis isolates. Multivariate analysis showed that temperature (above 8°C) and cutting surfaces (wood) presented significant association values. Conclusions In conclusion, pork in traditional markets of Quito is contaminated with Salmonella enterica, whose main serovars pose a public health concern, and shows beta-lactam resistance.


Background
Salmonella enterica are bacteria that include more than 2,500 serovars.Most of these serovars have been linked to human foodborne illnesses, mainly related to poultry and pigs.Thus, these animals are considered the reservoirs of many Salmonella serovars and strains related to antibiotic resistance.This study aimed to determine the prevalence, serovars, β-lactam resistance genes, and the risk factors associated with Salmonella enterica in pork commercialized in open markets of Quito city.

Methods
For this, 165 pork meat samples were taken from municipal markets in three areas in the city.These samples were microbiologically processed following the ISO 6579-2014 standardized method.The polymerase chain reaction (PCR) test was used to identify Salmonella serotyping and resistance genes.Strains not identified by PCR were typed by the Kauffman White Le Minor scheme.A multivariate analysis Xiaoping Liao, South China Agricultural 3.

Introduction
The consumption of undercooked food contaminated with non-typhoid Salmonella is one of the most important causes of human gastroenteritis worldwide.The World Health Organization (WHO), established that more than 2 billion people worldwide suffer from diarrheal disease annually. 1lmonella transmission to humans happens along the farm-to-fork via contaminated animal-and plant-derived foods, including poultry, eggs, fish, pork, beef, vegetables, fruits, nuts, and flour. 2 This contamination can occur at any stage in the production chain. 3,4Animals such as pigs can be infected or colonized by different Salmonella serovars, developing a disease or becoming a reservoir of this microorganism, excreting and spreading it. 57][8] It has to be considered that cross-contamination of food with Salmonella may occur by kitchen instruments, improper hygienic handling, etc. 9 Different Salmonella serovars can be implicated in a large number of human infections.Therefore, serovar identification is essential in the epidemiological surveillance of this pathogen. 10Several serovars have been reported in pork and pig production, associated with different geographical localization.Thus, S. Derby and S. Typhimurium were the most prevalent serovars in Europe, Oceania, Asia, and North America, 11 while S. Derby has been reported as the most pervasive serovar in pork in Latin America. 12sides, antimicrobial resistance has posed a critical problem for human and animal health in the last 20 years. 13he overuse of antibiotics in these two areas has contributed to the emergence of antibiotic resistance in foodborne bacteria. 146][17] Although the importance of non-typhoid Salmonella and its antimicrobial resistance is evident in food production, epidemiological information about the presence of this bacteria in pork is still limited in Ecuador. 18Therefore, this research aimed to estimate the prevalence of Salmonella serovars, antimicrobial resistance, and risk factors associated with Salmonella enterica in pork meat sold in markets in Quito-Ecuador.

Sample collection
Information of total markets in Quito city was provided by the city's major office report, where 56 local markets are established.According to the geographical distribution of these markets, 18 were selected as follows: 8 in the north of the city, 3 in the center of the city and 7 in the south of the city.The number of samples in every market was assigned according to the size of the market.Sampling and surveys were applied to the butchery employees who agreed to participate in the study.A total of 165 pork samples were collected in 11 traditional markets in three areas of Quito.Samples of 100 g of meat from 25 butcheries were collected between March and May 2021.Each sample was collected in sterile plastic flasks and transported to the laboratory in an icebox at 2-8°C.

Survey information
An epidemiological questionnaire to estimate the risk factors for pork contamination was developed and applied to the butchery employees Included variables were based in previous studies 3,19 and adapted for this study (Table 1).
Isolation and identification of Salmonella Salmonella was isolated and identified according to the ISO 6579: 2017 standardized method. 20Briefly, 25 g of pork and 225 ml of Buffered Peptone Water -BPW (BD Difco 218105 -USA) were placed in a sterile zip bag and homogenized to obtain a 1:10 suspension.The combination was incubated at 37°C AE 1°C for 18 h AE 2 h.After incubation, 0.1 ml of the pre-enriched culture as 1-3 equally spaced spots on the surface of Modified Semisolid Rappaport Vassidialis -MSRV plates (BD Difco 218681 -USA) and incubated at 41.5°C for 24 h AE 3 h.A loop of 1 μl was taken inside the edge of the opaque growth in MSRV and plated in the Xylose Lysine, Deoxycholate -XLD agar (BD Difco 278850 -USA) and incubated at 37°C for 24 AE 3 h.Typical colonies of Salmonella spp. in XLD (black center with a slightly transparent reddish area) were then identified by biochemical tests.These tests included Triple Sugar Agar -TSI (BD Difco 226540 -USA), Lysine Iron Agar -LIA (BD Difco 284920 -USA), Simmons Citrate BD (Difco 211620 -USA), and Urea broth REVISED Amendments from Version 2 This new version integrates some minor changes.
Any further responses from the reviewers can be found at the end of the article (BD Difco 227210 -USA).All the positive isolates were conserved at -70°C in a Brain Head Infusion medium (BD Difco 241830 -USA) with glycerol (Fisher Chemical G33500 -USA).

DNA extraction
DNA extraction was performed using the boiling method.Briefly, Salmonella isolates were plated in XLD agar and incubated at 37AE1°C for 24 hours.A typical Salmonella colony was transferred to a sterile Eppendorf tube containing 300 μl of 1X TE buffer (Tris base Promega H5131 -USA + EDTA Promega V4231 -USA) and lysed at 95°C for 20 minutes.The supernatant was collected and stored at -20°C.

Serovars identification
Serovars Infantis, Enteritidis, Typhimurium, and its monophasic variant (1,4, [5], 12: i:-) were identified by polymerase chain reaction (PCR), using the invA gene as housekeeping control.Primers and annealing temperature for these PCR protocols are described in Table 2. GoTaq ® Flexi DNA Polymerase (Promega M8291 -USA), nuclease-Free Water (Promega P1197 -USA), and dNTP Mix (Promega U1515 -USA) were used as PCR reagents on a SimpliAmp™ Thermal Cycler (Thermo Fisher A24811 -USA) to perform all reactions.According to the Kauffmann-White scheme, Salmonella isolates not typed by PCR were serotyped using the agglutination method. 19Briefly, each strain was recovered in Nutrient Agar (BD Difco 213000 -USA) and incubated for 16 to 20 h at 37°C AE 2.Then, the agglutination test was performed, confronting the bacterial suspension to specific antisera in a multi-cup plate.Positive agglutination is visualized by aggregate formation (more or less cottony appearance) exerting a moderate circular agitation of the plaque.Determination of somatic antigen (O antigen) requires the primary test of polyvalent sera (OMA, OMB, OMC, OMD, OME, OMF, and OMG) followed by monovalent ones (Remel™ Agglutinating Sera -UK).To determine flagellar antigen (H antigen), agglutination with one of the mixing sera for orientation (HMA, HMB, HMC) or the versatile serum H1 was observed (Remel™ Agglutinating Sera -UK).The determination of the flagellar antigen is obtained by successive elimination until detection of agglutination with one of the specific sera included in the mixing serum (search for major H then minor H).The combination of somatic antigen and flagellar antigen defines the serotype of the strain under study.The Kauffmann-White scheme gathers the groups and the corresponding sera.Ref. Antimicrobial susceptibility test Identification of antimicrobial resistance Salmonella isolates was carried out using the Kirby-Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines. 24The clinical cut-offs of M100 CLSI standards (CLSI, 2022) were used as references integrating intermedia values as resistant.Ref.

Statistical analysis
The free software R Studio (Version 1.4.1717) was used for statistical analysis.Descriptive statistics were used to facilitate the calculation of frequencies observed for each variable that would be used in the univariate analysis.For the bivariate analysis, we included variables of interest regarding the prevalence of Salmonella and then performed the chisquared test and Fisher's exact test.A univariate and multivariate logistic regression study was used to obtain odds ratios and 95% confidence intervals (CI).The level of significance was determined as p < 0.05.

Serovar identification of Salmonella
The PCR technique allowed the identification of five S. Infantis and eight monophasic variants of S. Typhimurium (1,4, [5],12:i:-) isolates.Additionally, two S. Derby isolates were characterized by the Kauffman White scheme.

Antimicrobial resistance analysis
Four antimicrobial resistance profiles were found in 11 isolates (Table 4).Three and seven isolates of S. Infantis and S. 1,4, [5],12:i:-presented multidrug-resistant patterns, respectively.The two S. Derby and two S. Infantis isolates showed to be susceptible to all tested antibiotics.
The highest resistance levels were present in three S. Infantis isolates with resistance phenotypes for 11 antibiotics.Additionally, all S. 1,4, [5],12:i:-isolates showed resistance to tetracycline and chloramphenicol (Table 5).The bla CTX-M-65 gene was identified in all the S. Infantis isolates resistant to cefotaxime.On the other hand, none of the studied β-lactamase genes were identified in S. 1,4, [5],12:i:-isolates resistant to β-lactams.

Risk factors for pork contamination
In bivariate analysis, the variable "Meat cutting surface wood" showed a significant association with Salmonella in pork (p = 0.0349).Multiple logistic regression analysis determined that the variable "Meat preservation temperature" (higher than 8°C) also showed significant values (p = 0.031) (Table 6).
Full responses for the survey, metadata, and susceptibility test reports are available in the Underlying data. 37

Discussion
This research aimed to determine the prevalence of Salmonella enterica in pork meat in public markets of Quito, Ecuador.Identification of Salmonella serovars, antimicrobial resistance profiles, and risk factors associated with the presence of the microorganism were included in this work.
In the present study, the prevalence of Salmonella in pork was 9.1% (95% CI = 5.6 -14.5).Another study in Quito reported a higher Salmonella occurrence in pork in 2016. 38This decrease in Salmonella occurrence could indicate that some interventions for improving market sanitary conditions could have been implemented.Several studies around the world have reported different levels of Salmonella in pork.In Europe, the prevalence of Salmonella in pork was 0.64% in 2019 (n=20 613), 39 while in Asian countries, the prevalence of Salmonella varies from 14.1% to 57.74%. 3,40,41Overall, variations of Salmonella prevalence within countries or among regions of the world could be associated with the implementation of specific technologies in meat treatment or the existence of specific regulations that could be put in place in Ecuadorian markets. 42Nontheless, it must be mentioned that the epidemiology of Salmonella in raw meat is a multifactorial phenomenom and other local factors could influence the occurrence of this pathogen. 5,11lmonella Typhimurium monophasic variant (S. 1,4, [5],12:i:-) was the predominant serovar in this study (53.4%), becoming the first report of this serovar in pig meat in Ecuador.
It has been mentioned that isolation of S. 1,4, [5],12:i:-has increased worldwide during the last 20 years. 43Pigs are ranked as the main reservoir of this serovar in the European Union. 44Furthermore, S. 1,4, [5],12:i:-has become one of the most common variants of Salmonella in pork production, 45,46 being considered an emerging serovar in many countries. 46he present study demonstrates that this serovar may be relevant in the epidemiology of Salmonella linked to food contamination in Ecuador.
Concerning S. Infantis, our results differ from those reported by Mejia et al., 18 who showed this serovar as the most isolated one in pig meat samples in retail markets in Ecuador.Since the later study samples were taken from supermarkets, this difference could be determined by the pig meat supply chain.In this regard, pork in traditional markets comes from many small pig production farms.On the other hand, pork from supermarkets comes from a few companies with intensive industrial production.These facts could explain the differences between studies.Moreover, S. Infantis has been reported as one of the most frequent serovars in industrial poultry production in many countries, [47][48][49] indicating that this serovar is well adapted to industrial conditions. 50,51Since S. Infantis has been reported to cause human diseases, 52 the epidemiology of this serotype in pork and its relationship with other meat products (e.g., poultry) should be further investigated.The same criterion should be considered when approaching the epidemiological surveillance of S. Derby, since this Salmonella serovar has also been described as an important foodborne pathogen.Antimicrobial resistance (AMR) of Salmonella isolates from pork and pig production has been reported in other countries at regional and global levels showing multidrug-resistant phenotypes. 54,55However, AMR varies according to Salmonella serotypes.In this study, S. Infantis was the least susceptible serotype with resistance patterns of up to seven antimicrobial families.In the same way, S. Infantis isolates with high rates of AMR have been reported in the poultry industry of Ecuador, 51,56 which has been linked to the overuse of antimicrobials in this sector. 57However, multidrugresistant S. Infantis has been reported worldwide, demonstrating that multi-resistance is an important feature of this serovar, possibly linked to specific plasmids.Thus, the capacity of S. Infantis to acquire genetic determinants of resistance has been associated with the "plasmid of emerging S. enterica Infantis" (pESI) and related plasmids (pESI-like plasmids). 58[63] The same can be said for S. 1,4, [5],12:i:-whose majority of isolates showed multidrug-resistant patterns.[66] High levels of AMR Salmonella have been related to the incorrect use of antibiotics in animal production.The administration of cephalosporins and other beta-lactams in pig production has seen to increase in many countries, like France, 67 India, 68 and Brazil. 69Moreover, in Ecuador, the misuse of antibiotics in animal production systems has been reported. 70,71This aspect has particular relevance since beta-lactams are widely used in animal husbandry. 72,73It must also be considered that ESBL genes could determine failure in treating diseased people with complicated infections caused by Salmonella and other bacteria to which horizontal transference of genes may occur. 74In the present work, we found that meat preservation above 8°C (OR = 1.15) and wooden surfaces for meat cutting (OR = 59.31) were risk factors for Salmonella in pork meat.This is unsurprising since the lack of an appropriate cool chain allows the growth of contaminating microorganisms and pathogens in food products. 75][78] Whether Salmonella isolates recuperated in our study originated in the primary sector or are the result of such crosscontamination events should be further studied, targeting samples from pig production farms and conducting analysis of genomic clonality.
In conclusion, pork expended in traditional markets of Quito showed contamination of Salmonella enterica, whose main serovars are of public health concern.Beta-lactam-resistance in Salmonella isolates is also remarkable, which could become a risk for pork consumers.

Underlying data
Figshare: PCR images of Prevalence, serovars, and risk factors associated with the presence of Salmonella in pork sold in public markets in Quito, Ecuador.https://doi.org/10.6084/m9.figshare.24195030.v1. 37is project contains the following underlying data: • CTX-M-9 RAW.jpegThis work is very relevant since it aimed to determine the prevalence, serovars, β-lactam resistance genes, and the risk factors associated with Salmonella enterica in pork since these animals are considered the reservoirs of many Salmonella serovars and strains related to antibiotic resistance, and this information is still limited in Ecuador.Identification of Salmonella serovars, antimicrobial resistance profiles, and risk factors associated with the presence of the microorganism were included in this work.
It is recommended to make the following modifications: The conclusion of the summary is obvious and uninformative, it is recommended to write one that better describes the results.
In the introduction, if the information in pork is limited in Ecuador, put a reference to how little is known or in any case change it to non-existent information.
Since they amplified beta-lactamase, it is necessary to justify in the introduction why these were specifically sought.
In materials and methods, change minutes and hours by min and h respectively.In PCR amplifications, include the amount of DNA contained in the assay.If possible, update the bibliography since there are some references that are too old.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Molecular epidemiology of Salmonella, Acinetobacter and Mycobacterium tuberculosis I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
In the discussion, the fifth paragraph, I don't understand the statement "because the surveillance of Salmonella serovars shapes the epidemiology of these pathogens in foodstuffs."Please give some clarification.
Please verify references 6 and 8.I think they are incompletely written.Next, we will answer your questions and recommendations regarding the evaluated article.a) A major issue is related to the serotyping methodology.The PCR reaction for serotyping should be backed up with the traditional Kauffmann-White scheme or with whole-genome sequencing.Because PCR may have some false positive results (as indicated in the cited reference), all isolates should have been confirmed with KW.Why did the authors do it partially?

Response:
PCR used in this research has been extensively proved as an appropriate method for the tested serotypes in this research (Infantis, Enteritidis, Typhimurium, and (1,4, [5], 12: i:-)).Moreover, we have proved its specificity compared with WGS in more than 800 Salmonella isolates of our collection.Therefore, we think that it is a reliable method to access the serotype of Salmonella isolates of this study.In Ecuador, there is not a reference laboratory for Salmonella serotyping.Therefore, performing KW in all isolates is cumbersome and costly and was not included in this project.It is important to mention that WGS was later performed in these isolates confirming the serotyping results.Information of WGS is not included in this manuscript since it is part of another publication.
b) Conclusions, both in the abstract and within the main text, should address the main findings of this work.

Response:
We changed conclusions in lines 30-33.
c) The cited references need an update.Just one of them is from 2023, and some data is definitely old.For instance, the first paragraph of the introduction indicates cases from 2010.

Response:
We updated the references in lines 41-43 and 45-47.References for other parts of the manuscript are updated.
d) The text should be revised by a native English speaker.There are several mistakes throughout the manuscript.

Response:
The text has been revised.
e) What was the criterion for the selection of markets?Clarification is required.What was the criterion for the selection of butcheries and interviewed employees?
Response: Clarification added in 73 -78 f) Normally, variables included in epidemiological surveys are selected from previous reports or locally identified during in situ observations.What were the sources for the included variables?
Response: Clarification added in 84 -85 g) The Kauffmann-White serotyping is a standard procedure that does not require a detailed description.A reference is enough.The same occurs with the Kirby-Bauer method.
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Table 1 .
Variables included in the epidemiological survey.

Table 2 .
Primers used for Salmonella serovars identification.
The antibiotics used were: Ampicillin (Oxoid, USA; 10 μg), Cefoxitin (Oxoid CT0003B -USA; 30 μg), Cefotaxime (BD 231606 -USA; 30 μg), The E. coli ATCC 25922 was used as control strain.PCR further studied isolates with phenotypic resistance to beta-lactams to detect bla CTX-M , bla TEM , bla CMY , and bla SHV genes.Additionally, the bla CTX-M subgroups were identified for later sequencing.Primers and annealing temperature for these PCR protocols are described in Table3.PCR reagents and equipment used for serotyping by PCR were used in this step.
38l amplified PCR products were exanimated by electrophoresis in a 1% Agarose, LE, Analytical Grade (Promega V3125 -USA) on 0.5X TAE Buffer (Promega V4281 -USA) and stained with SYBR™ Safe DNA Gel Stain (Invitrogen S33102 -USA).Amplicons' size was estimated by 100 bp Plus DNA Ladder (Bioneer D-1035 -Korea).Gel casting and electrophoresis were performed in a horizontal electrophoresis system (CBS Scientific HSU-014, EPS-300X -USA).Specific bands were visualized on ENDURO™ UV Transilluminator (Labnet U1001 -USA).Raw and edited images of all PCR products are available in the Underlying data.37AllbetalactamesePCR products were sent for sequencing to Macrogen Inc. (Seul-South Korea), and obtained sequences were aligned against reference sequences by the web tool ResFinder 4.0.38

Table 3 .
Primers used for Beta-lactams identification.

Table 5 .
Resistance rates for tested antibiotics.

Table 6 .
Logistic regression model analysis of variables with significant values.

the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests:
No competing interests were disclosed.