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. 2024 Jun 4;19(6):e0304621. doi: 10.1371/journal.pone.0304621

Antimicrobial resistance and genome characteristics of Salmonella enteritidis from Huzhou, China

Wei Yan 1, Deshun Xu 1,*, Liping Chen 1, Xiaofang Wu 1
Editor: Mabel Kamweli Aworh2
PMCID: PMC11149840  PMID: 38833480

Abstract

Salmonella enteritidis is a main pathogen responsible for sporadic outbreaks of gastroenteritis, and therefore is an important public health problem. This study investigated the drug resistance and genomic characteristics of S. enteritidis isolated from clinical and food sources in Huzhou, Zhejiang Province, China, from February 1, 2021, to December 30, 2023. In total, 43 S. enteritidis strains isolated during the study period were subjected to virulence gene, drug resistance gene, genetic correlation, antibiotic resistance, and multilocus sequence typing analyses. All 43 isolates were identified as ST11, and contained 108 virulence-related genes. Drug sensitivity analysis of the 43 isolates showed resistance rates of 100% to nalidixic acid and 90.70% to ampicillin and ampicillin/sulbactam. Multidrug resistance is a serious issue, with 81.40% of strains resistant to three or more antibacterial drugs. Genome sequencing indicated that S. enteritidis possessed 23 drug resistance genes, of which 14 were common to all 43 isolates. Phylogenetic analysis based on core genome single-nucleotide polymorphisms divided the 43 S. enteritidis strains into three clusters, with the 10 samples from an outbreak forming an independent branch located in cluster 3.

Introduction

Salmonella is a zoonotic pathogen, an important pathogenic factor responsible for foodborne diseases [1, 2]; it is the main cause of bacterial food poisoning [3]. Contaminated food and water or individuals infected with Salmonella can become the source of infection, posing a serious threat to the health humans and other animals [4]. More than 2600 serotypes or variants of Salmonella have been identified to date [5, 6], of which about 50 are closely related to human health. The clinical manifestations of Salmonella infection are complex and diverse, and can be divided into gastroenteritis, septicemia (typhoid), and local infection types [7]. Recent studies [8] have shown that the incidences of typhoid fever and paratyphoid fever have decreased, while some non-typhoidal Salmonella infections are showing upward trends. Non-typhoidal Salmonella is frequently associated with diarrheal illness or self-limiting gastroenteritis in humans [9, 10], and causes up to 80.3 million cases of gastrointestinal disease and 150 000 deaths each year around the world [11]. In China, 70–80% of cases of foodborne disease are caused by non-typhoidal Salmonella [12]. Salmonella enteritidis is the main Salmonella serotype, accounting for 40–60% of laboratory-confirmed Salmonella infections in recent years in various regions of the world [13, 14].

Antimicrobial resistance remains a serious challenge in the treatment and control of Salmonella enterica infections. Multidrug-resistant strains are linked to more severe disease outcomes [15] and can be passed along at various points from food production to consumption [16]. With the widespread use of antibiotics, the rate of drug-resistant Salmonella is also increasing, that bring great difficulty for clinical treatment, necessitating the study of drug resistance in these important foodborne pathogens [17, 18].

Whole-genome sequencing (WGS) has become an economically viable alternative to conventional typing methods for investigating disease outbreaks and public health surveillance [19]. Comparative genomics with WGS provides insight into the genomes of pathogenic bacteria, including identification of candidate drug compounds, potential virulence determinants, mechanisms of pathogenicity, and the evolution of pathogens. This technology has been instrumental in improving diagnostics and public health clinical microbiology [20], and is widely used to investigate outbreaks of pathogenic bacteria, including Bacillus cereus, Escherichia coli, Vibrio parahaemolyticus, and Salmonella species [21–24].

S. enteritidis is the most frequently isolated serovar from food sources patients with diarrhea in Huzhou, Zhejiang Province, China [25, 26]. As far as we know, there have been few previous reports on the drug resistance and genetic characteristics of S. enteritidis in the Huzhou area. In this study, we sequenced the whole genomes of S. enteritidis isolates from diarrhea patients in Huzhou. This study aims to explore the genetic type, distribution characteristics of resistance genes and virulence genes, as well as the evolutionary relationships of strains. The findings of this research can offer valuable insights for conducting food safety risk assessments, prevention and control measures against S. enteritidis in Huzhou, China.

Materials and methods

Ethics statement

This study was approved by the Human Research Ethics Committee of the Huzhou Center for Disease Control and Prevention (approval number: HZ2020007). The only human materials used in this study were stool samples obtained from patients for routine assessment. Each participant provided oral informed consent.

Bacterial isolates

In total, 41 S. enteritidis strains isolated from diarrhea patients from six active foodborne surveillance sentinel hospitals in Huzhou, Zhejiang Province, China, from February 1, 2021, to December 30, 2023 (9, 18, and 14 strains in 2021, 2022, and 2023, respectively), as well as two S. enteritidis strains isolated from food in November 2023 were examined. (S1 Table). Bacteria were isolated according to the methods described in Diagnostic Criteria for Infectious Diarrhea (WS271–2007) [27] and National Standards for Food Safety (GB4789.4–2016) [28]. All 43 strains of S. enteritidis were stored at –80°C in porcelain culture storage tubes (Qingdao Haibo, Qingdao, Shandong, China).

Antimicrobial susceptibility testing (AST)

The antimicrobial susceptibilities of the 43 isolates were tested using the broth microdilution method and strains were classified as sensitive, intermediate, or resistant according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints for Salmonella strains. E. coli ATCC 25922 was used as a control. The results were analyzed according to the CLSI breakpoints. The 17 antibacterial agents tested consisted of the macrolide azithromycin (AZM), the quinolone nalidixic acid (NAL), the fluoroquinolone ciprofloxacin (CIP), the aminoglycosides amikacin (AMI) and streptomycin (STR), the phenylpropanol chloramphenicol (CHL), the tetracyclines tetracycline (TET) and tigecycline (TIG), the folate pathway antagonist sulfamethoxazole (SMX); the lipopeptide polymyxin (PMX), and the β-lactams, ampicillin (AMP), ampicillin/sulbactam (AMS), ceftazidime (CAZ), ceftazidime/avibactam (CZA), cefotaxime (CTX), ertapenem (ETP), and meropenem (MEM).

Whole-genome sequencing and assembly

The genomes of S. enteritidis strains were extracted using a bacterial genomic DNA extraction kit and sequenced using a NextSeq 550 sequencer (Illumina, San Diego, CA, USA). Quality control analysis of raw genomic sequencing data was performed with FastQC (v0.11.9) and fastp (v0.23.2) was used to remove low-quality data. Assembly was performed with SPAdes_3. 14.1 (https://github.com/ablab/spades) after removal of fragments with < 1000 bp of overlap. Information such as genome size and GC content were obtained using RAST (https://rast.nmpdr.org/rast.cgi). Whole-genome sequencing was performed by Shanghai Berger Medical Technology Co., Ltd. (Shanghai, China).

Multilocus sequence typing

The results of whole-genome sequencing were typed for site sequence classification by multilocus sequence typing (MLST) with mlst v2.23.0 (https://github.com/tseemann/mlst). The numbers of housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) were obtained by alignment with the original allele sequences in the database and the corresponding sequence type (ST) was determined by combining them.

Analysis of drug resistance genes and virulence genes

Based on the results of whole-genome sequencing analysis, the CARD (https://card.mcmaster.ca/) and VFDB (http://www.mgc.ac.cn/VFs/) databases were used to predict the resistance genes and virulence genes using the parameters identity > 90.0% and coverage > 90.0%. Finally, comparative analysis was performed on genotype and drug resistance phenotypes.

Genetic correlation analysis

S. enterica Typhi CT18 (GenBank accession no. NC_003198) was used as the reference genome. The core single nucleotide polymorphism (SNP) data set of strains in this study was constructed using KSNP3.01. Sequence alignment and homology analysis were performed on 43 S. enteritidis strains using KSNP3.01 with 1000 bootstrap replicates. Phylogenetic trees were constructed using the maximum likelihood method.

Nucleotide sequence accession numbers

The sequences obtained in this study have been deposited in GenBank with accession numbers JBAKMS000000000–JBAKOH000000000 and JBAOJF000000000.

Results

Genome sequencing

The total genome length of 43 strains of S. enteritidis was 4,417,739–4,757,703 bp and the average GC content was 52.07–52.42% (S2 Table), consistent with the genomic characteristics of S.enteritidis.

MLST molecular typing

MLST analysis based on the whole genome sequence showed that all 43 S. enteritidis isolates belonged to ST11 type (S3 Table), and the seven housekeeping genes were aroC, dnaN, hemD, hisD, purE, sucA, and thrA.

Detection of AST and antimicrobial resistance genes

The 43 strains of S. enteritidis showed varying degrees of resistance to 17 antibiotics with resistance rates ranging from 2.33% to 100%. The highest resistance rate was observed for NAL (100%) followed by AMP and AMS (both 90.70%) (Table 1). In all, 35 of the 43 S. enteritidis strains were resistant to three or more antibacterial drugs, representing a total multidrug resistance rate of 81.40% (35/43). The 43 S. enteritidis strains showed 19 drug resistance spectra, among which AMP-AMS-NAL-STR-PMX and AMP-AMS-NAL-STR-TET-PMX, seen in eight S. enteritidis strains each, were predominant. S. enteritidis resistant to five antibiotics accounted for the greatest proportion among multidrug-resistant strains (31.43%, 11/35). The most resistant strain was detected in 2023 (S2023828), which was resistant to all 17 drugs examined (Table 2).

Table 1. Antimicrobial susceptibility testing of 43 Salmonella enteritidis strains with 17 antimicrobial agents.

antimicrobial sensitive (n,%) intermediate (n,%) resistant (n,%)
AMP 4(9.30) 0(0.00) 39(90.70)
AMS 4(9.30) 12(27.91) 27(62.80)
CAZ 40(90.2) 0(0.00) 3(6.98)
CZA 41(95.35) 0(0.00) 2(4.65)
CTX 36(83.72) 37(25.34) 7(16.28)
ETP 42(97.67) 0(0.00) 1(2.33)
MEM 42(97.67) 0(0.00) 1(2.33)
NAL 0(0.00) 0(0.00) 43(100)
CIP 42(97.67) 0(0.00) 1(2.33)
AZM 39(90.70) 0(0.00) 4(9.30)
AMI 42(97.67) 0(0.00) 1(2.33)
STR 10(23.26) 6(13.95) 27(62.79)
TET 27(62.80) 0(0.00) 16(37.21)
TIG 42(97.67) 0(0.00) 1(2.33)
CHL 39(90.70) 0(0.00) 4(9.30)
SXT 38(88.37) 0(0.00) 5(11.63)
PMX 12(27.91) 0(0.00) 31(72.09)
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Table 2. Drug resistance spectra of 43 Salmonella enteritidis strains to 17 antimicrobial agents.

Antibiotic resistant species (species) Drug-resistant spectrum Number of isolate (n) The percentage (%)
1 NAL 3 6.98
2 AMP-NAL 4 9.30
NAL-STR 1 2.33
3 AMP-NAL-PMX 4 9.30
4 AMP-NAL-AMS-PMX 2 4.65
AMP-AMS-NAL-STR 2 4.65
AMP-NAL-STR-PMX 1 2.33
AMP-NAL-SXT-PMX 1 2.33
5 AMP-AMS-NAL-STR-TET 1 2.33
AMP-AMS-NAL-STR-PMX 8 18.60
AMP-AMS-NAL-CTX-TET 1 2.33
AMP-CAZ-CTX-NAL-PMX 1 2.33
6 AMP-AMS-NAL-STR-TET-PMX 8 18.60
AMP-AMS-NAL-STR-SXT 1 2.33
AMP-AMS-CTX-NAL-TET-PMX 1 2.33
9 AMP-AMS-CTX-NAL-AZM-STR-TET-CHL-PMX 1 2.33
AMP-AMS-CTX-NAL-AZM-STR-TET-CHL-SXT 1 2.33
12 AMP-AMS-CAZ-CZA-CTX-NAL-AZM-STR-TET-CHL-SXT-PMX 1 2.33
17 AMP-AMS-CAZ-CZA-CTX-ETP-MEM-NAL-CIP-AZM-AMI-STR-TET-TIG-CHL-SXT-PMX 1 2.33
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All 43 strains of S. enteritidis carried drug resistance genes, and a total of 23 drug resistance genes, were predicted by the analysis. Among these, 14 resistance genes were common to all 43 strains, i.e., bacA, mdtK, marA, AAC(6′)-Iy, cpxA, golS, mdsA, mdsB, mdsC, sdiA, acrB, emrR, H-NS, and CRP (Fig 1). The seven most common antimicrobial resistance genes were marA, sdiA, acrB, golS, mdsA, mdsB, and mdsC, of which marA was detected in the greatest number of strains (Table 3).

Fig 1. Drug resistance genes detected in the 43 S. enteritidis isolates examined in this study.

Fig 1

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Table 3. Drug resistance genotyping and drug resistance of Salmonella enteritidis strains.

GENE GENE_COUNT RESISTANCE_COUNT RESISTANCE
bacA 43 1 peptide
mdtK 43 1 fluoroquinolone
marA 43 12 carbapenem;cephalosporin;cephamycin;fluoroquinolone;glycylcycline;monobactam;penam;penem;phenicol;rifamycin;tetracycline;triclosan
AAC(6’)-Iy 43 1 aminoglycoside
cpxA 43 2 aminocoumarin;aminoglycoside
golS 43 7 carbapenem;cephalosporin;cephamycin;monobactam;penam;penem;phenicol
mdsA 43 7 carbapenem;cephalosporin;cephamycin;monobactam;penam;penem;phenicol
mdsB 43 7 carbapenem;cephalosporin;cephamycin;monobactam;penam;penem;phenicol
mdsC 43 7 carbapenem;cephalosporin;cephamycin;monobactam;penam;penem;phenicol
sdiA 43 8 cephalosporin;fluoroquinolone;glycylcycline;penam;phenicol;rifamycin;tetracycline;triclosan
acrB 43 8 cephalosporin;fluoroquinolone;glycylcycline;penam;phenicol;rifamycin;tetracycline;triclosan
emrR 43 1 fluoroquinolone
H-NS 43 6 cephalosporin;cephamycin;fluoroquinolone;macrolide;penam;tetracycline
CRP 43 3 fluoroquinolone;macrolide;penam
APH(6)-Id 28 1 aminoglycoside
APH(3’’)-Ib 28 1 aminoglycoside
TEM-1 26 4 cephalosporin;monobactam;penam;penem
sul2 17 1 sulfonamide
tet(A) 11 1 tetracycline
FosA3 2 1 fosfomycin
dfrA17 2 1 diaminopyrimidine
aadA5 2 1 aminoglycoside
CTX-M-14 1 1 cephalosporin
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Virulence genes

Analysis using the Virulence Factor Database (VFDB) showed that the 43 S. enteritidis strains possessed a total of 108 virulence genes and had 20 pathogenic mechanisms. Among them, 96 virulence genes were shared by the 43 strains, and the type III secretion system was the main pathogenic mechanism (Fig 2). (S4 Table and S5 Table)

Fig 2. Virulence factors detected in the 43 S. enteritidis isolates examined in this study.

Fig 2

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Genetic correlation analysis

A maximum likelihood tree based on core SNPs was constructed using S. enterica Typhi CT18 (GenBank accession no. NC_003198) as the reference genome. The 43 strains of S. enteritidis were divided into three clusters, with cluster 1 containing 10 strains, cluster 2 containing 11 strains, and cluster 3 containing 22 strains. Two strains of S. enteritidis originating from food were located within cluster 3, and the 10 strains from concentrated outbreaks formed independent branches all of which were also within cluster 3 (Fig 3).

Fig 3. Phylogenetic tree based on core genome SNPs of 43 S. enteritidis isolates.

Fig 3

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Discussion

S. enteritis can cause severe invasive infections [29]. The main clinical manifestation is self-limited acute enteritis, but it can also develop into invasive infections outside the intestine and cause systemic disease [30]. Therefore, it is important to monitor drug resistance and analyze the genetic characteristics of S. enteritidis to not only provide a scientific basis for clinical treatment but also to support tracing of the sources of infection and establish a local Salmonella database.

Cephalosporins and quinolones are important antibiotics used to treat S. enteritidis infections [31]. However, there have been reports that some strains have developed resistance to these antibiotics [32]. The results of drug sensitivity testing in S. enteritidis in the Huzhou area indicate varying degrees of resistance to all 17 antibiotics examined. High levels of resistance were observed to NAL, AMP, AMS, and STR, consistent with previous reports [33–35], indicating that these agents are no longer suitable for the treatment of Salmonella infection in Huzhou area. In addition, multiple drug resistance of S. enteritidis is becoming increasingly problematic in the Huzhou area with 81.40% of strains showing resistance to three or more drugs, with marked resistance to NAL, AMP, and AMS. Twenty-six strains were resistant to five or more antibiotics, and the greatest spectrum of multiple drug resistance involved resistance to all 17 antibiotics. The emergence of multidrug resistance is likely due to the abuse of antibiotics and horizontal transmission of resistance genes. Therefore, it is necessary to improve prescription practices, reduce the occurrence of drug-resistant bacteria, and prevent the spread of drug-resistant strains.

The 43 strains were highly resistant to fluoroquinolones, β-lactams, and aminoglycosides, and their genotypes and phenotypes were essentially the same. Hyeon et al. [36] reported that some genes are usually not sufficient to cause changes in the resistance phenotype in Salmonella unless the strain has mutations that can increase their expression [37, 38]. Our results also show that the drug resistance genotype and phenotype were not consistent, for example, sul2 was detected in 17 strains, while only 5 strains showed SMX resistance. In addition, four strains were resistant to CHL, but no related drug resistance genes were detected. This may have been due to other unknown genes, and further study is warranted.

With advances in bioinformatics techniques, WGS has been widely used to analyze pathogenic bacteria due to its high resolution, good repeatability, and rapid and high-throughput characteristics [39].

It is also becoming increasingly common to use bioinformatics to predict serotypes based on Salmonella whole-genome sequence data. In this study, 43 S. enteritidis strains isolated in Huzhou in 2021–2023 were subjected to whole-genome sequencing, and compared to strains in public databases to construct a phylogenetic tree based on SNPs. The results divided the 43 strains of S. enteritidis into three clusters. Ten strains from outbreaks were clustered in independent branches. In addition, all three clusters contained strains from different regions and/or isolation times, indicating that S. enteritidis in Huzhou was composed of sporadic strains.

MLST is a classical bacterial molecular typing technique widely used to identify relationships among bacterial clones [40]. In this study, MLST analysis showed that all 43 strains of S. enteritidis were ST11 type, and included seven housekeeping genes, consistent with a previous report [41, 42]. The results indicated close correlations between sporadic S. enteritidis in Huzhou. In addition, the detection rate of S. enteritidis virulence factors was high, with 96 (88.89%) virulence genes shared by the 43 strains of S. enteritidis examined in this study.

Some studies have reported a positive correlation between virulence genes and the pathogenicity of Salmonella [43, 44]. The Salmonella plasmid virulence (spv) genes are commonly present on virulence plasmids of many Salmonella species, including spvRABCDE, which mainly mediates cytotoxicity during macrophage differentiation and apoptosis [45]. In this study, 34 strains of S. enteritidis were shown to carry spvRBC, suggesting a relatively high toxic plasmid carrying rate of S. enteritidis in Huzhou.

The major limitations of this study were that we analyzed only S. enteritidis and the number of strains was small. Further studies are required with a greater detection range and quantity of samples to evaluate the effects of S. enteritidis-related diseases and provide a scientific basis for the prevention and control of S. enteritidis infection in Huzhou.

Conclusion

We analyzed the characteristics of AST and WGS in S. enteritidis strains isolated from patients with diarrhea in Huzhou, China. All isolates were of the ST11 type, which were sporadic strains. The carrying rate of virulence genes was high, and drug resistance was serious, with many multidrug-resistant strains. Close attention should be paid to the emergence of drug-resistant strains and management of antibiotics should be strengthened. Our results provide a background for the prevention and treatment of foodborne diseases caused by S. enteritidis in Huzhou, Zhejiang Province, China.

Supporting information

S1 Table. The information of 43 Salmonella enteritidis isolates strain.

(DOCX)

pone.0304621.s001.docx (18.2KB, docx)
S2 Table. Genome assembly profile of Salmonella enteritidis.

(DOCX)

pone.0304621.s002.docx (15.4KB, docx)
S3 Table. Results of MLST typing of Salmonella enteritidis genome.

(DOCX)

pone.0304621.s003.docx (20.3KB, docx)
S4 Table. Genomic virulence gene results of Salmonella enteritidis.

(DOCX)

pone.0304621.s004.docx (17KB, docx)
S5 Table. Salmonella enteritidis genome virulence gene pathogenic system.

(DOCX)

pone.0304621.s005.docx (13.5KB, docx)

Acknowledgments

We thank the outpatients, nurses, and clinicians of the participating hospitals for their cooperation with the study.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

"This work was supported by grants from Huzhou Science and Technology Bureau (Award Number: 2023GY08), the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Decision Letter 0

Mabel Kamweli Aworh

8 Apr 2024

PONE-D-24-09010Antimicrobial resistance and genome characteristics of Salmonella enteritidis from Huzhou,ChinaPLOS ONE

Dear Dr. Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Mabel Kamweli Aworh, DVM, MPH, PhD. FCVSN

Academic Editor

PLOS ONE

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Additional Editor Comments :

In addressing the reviewer's comments please address the following additional issues.

1. In your revision, please provide line numbers on the revised manuscript to make it easier for reviewers to review the revised manuscript.

2. Please engage the services of a native English speaker to revise the manuscript for typographical and grammatical errors. Delete any Chinese inscriptions in the manuscript, especially in the results section. Define any abbreviations upon first use in the manuscript.

3. Genes should be italicized and written in small letters all through the manuscript.

4. In discussing your results, do not repeat them; rather, provide a possible explanation for your findings while comparing them to the reports of other similar studies.

5. In the second paragraph of the discussion section, please italicize "Escherichia coli" and any other bacteria mentioned in the manuscript. Please italicize "Salmonella" all through the manuscript.

6. Highlight the main limitations of the current study in the last paragraph of the discussion section, please.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have a good study question but were not able to properly put it in writing. There is no justification for the study, the methodology was not properly referenced for reproducibility and there were several grammatical and writing errors. Also, all authors referenced in the article need to be fully stated in the reference section and not stating et al., in places where the full names are supposed to be written. Not to say that there were scientific names that were not italicized and confusing species of Salmonella in the result section. Authors need to proof-read, justify the document and pay attention to details.

Reviewer #2: General Comments:

Personally, I would like to see the letter of approval, especially since the author claims that the consent was given orally. The paper could benefit from a thorough revision, particularly in terms of punctuation. The lack of line numbers makes it challenging to pinpoint specific areas where punctuation is missing. Additionally, there are several acronyms that are not fully spelled out. I also noticed possible translation errors, as it appears the author may have translated the paper from another language to English. I recommend a careful review to ensure accurate translation. Finally, the author should pay much attention on their discussion because they did not discuss all results, and the conclusion is weak.

Abstract:

The authors have failed to state the purpose of their study, despite attempting to define the problem. This is a crucial element that could engage the reader’s interest in the article.

Introduction:

The sentence, “Multi-drug resistant bacteria and even super bacteria have emerged, which lead to the emergence of new epidemiological and clinical characteristics of the disease,” seems redundant and does not add value. It appears to repeat previously mentioned information. The preceding sentence could also be restructured and potentially supplemented with a citation.

Results:

It was disappointing to see that the authors did not reference the supplemental material, which, in my opinion, could have been effectively utilized in their results to strengthen their arguments.

Discussion:

The authors should consider using more direct language, as exemplified by their use of the vague term “some strains.” The discussion lacks depth and fails to highlight the practical implications of the results. There are no recommendations beyond drug resistance. Apparently, the genomic results are not discussed at all, and they applied genomic sequencing without a clear plan of how the results might be useful.

Conclusion:

The conclusion is weak. It merely restates the methods without summarizing the results or discussing their potential benefits to the community. It gives the impression that the authors were more interested in applying the methods and maintaining records than in the study’s purpose.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

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Attachment

Submitted filename: PONE-D-24-09010.pdf

pone.0304621.s006.pdf (1.2MB, pdf)
Attachment

Submitted filename: REVIEW genome characteristics of S.enteritidis.pdf

pone.0304621.s007.pdf (944.4KB, pdf)
Attachment

Submitted filename: PONE-D-24-09010 REVIEWERS COMMENT.pdf

pone.0304621.s008.pdf (118.7KB, pdf)
PLoS One. 2024 Jun 4;19(6):e0304621. doi: 10.1371/journal.pone.0304621.r002

Author response to Decision Letter 0

22 Apr 2024

Dear  editors: Thank you very much for your letter and the comments from the referees about our paper submitted to PLOS ONE (PONE-D-24-09010).

We have carefully addressed all of the comments from the reviewers, as outlined in the point-by-point responses attached below. We hope that you find the revised manuscript now acceptable for publication in PLOS ONE.

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

http://www.textcheck.com/certificate/axviDO

Thanks for your attention

Yours Sincerely

Deshun Xu

Responses to the editor’s comments:

(Q: as comments, A: as our responses)

Q: Can authors please justify the article?

Kindly proof read and ensure proper spacing after a comma or full stops. Pay attention to details!

A: We had ensured proper spacing after a comma or full stops and revised other details in the manuscript.

INTRODUCTION

Q: Line 41 “Salmonella is a zoonotic pathogen. It is also an important pathogenic factor 42 causing foodborne diseases [2,3] and the main cause of bacterial food poisoning [4]. 43 Contaminated food and water source or infected individuals by Salmonella can 44 become the source of infection and spread to the outside, posing a serious threat to 45 human and animal health[1]”.

Any reason for starting the referencing numbers with 2, 3 and 4 before “1”? If none please correct it accordingly.

A: We have made corrections it accordingly in the manuscript.

Q: Line 48 – 49 “gastroenteritis type, septicemia type (typhoid type) and 49 local infection type” Kindly reference this statement

A: We have added references “Mingfeng SH, Rongxing C, Kai ZH, Hongjian CH. Etiological and clinical characteristics of 92 children with Salmonella enteritis[J].Chin J Nosocomiol, 2022, 32(14): 2212-2217.” in the manuscript.

Q: Authors need to state clearly the justification for the study as dearth of information of the said topic in the region studied.

A: We added justification for the study in the introduction: There have been no previous reports on the drug resistance and genetic characteristics of S. enteritidis in the Huzhou area。

METHODOLOGY

Q: The authors need to cite references for the methodology “as described by ……” for reproducibility.

A: We added 2 references in the manuscript: “ Bacteria were isolated according to the methods described in Diagnostic Criteria for Infectious Diarrhea (WS271-2007) and National Standards for Food Safety (GB4789.4-2016) ”.

Q: Lines 86 – 88 Ethics statement

This study was approved by the human research ethics committee of the Huzhou Center for Disease Control and Prevention.

kindly state the approval number

A: We added the approval number(HZ2020007) in the manuscript.

Q: Line 151 “and the 7 housekeeping genes were aroC 152 (5), dnaN (2), hemD (3), hisD (7), purE(6), sucA (6) and thrA (11).”

These values when added gives 40 as against 43 stated. Any reason for the discrepancy?Nothing was said about the statistical analysis in the methodology or none was carried out?

A: The data in parentheses represent an identifier for every unique allele sequence, rather than the number of strains, and were used to determine the ST genotype of the strain. We removed these data so as not to cause any misunderstanding.

Statistical analysis is rarely used in this paper.

RESULT

Q: Can authors confirm the species of Salmonella especially in the result because there are several confusing names?

A: We have made careful modifications as required.

Q: If authors are not stating a specific species or simple sp. there is no need italicizing the Salmonella

A: We have made careful modifications as required.

REFERENCES

Q: While writing the authors of articles cited, kindly write out the names of all authors and desist from using et al., in the reference section. Check online for the names of all authors. If truly these articles were downloaded and read before citing them, the authors should have all the names of the authors of articles cited in this study. See lines 286, 289, 301, 310, 314, 317, 320, 324, 327, 330, 333, 338, 342, 348, 351, 356, 362, 365, 368,371, 374, 381, 387

A: We have made careful modifications as required. The names of all authors have been written out in the reference section.

Q:5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

A: Supplementary Material had been submitted at the time of initial submission of the paper.

Q: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

A: We have carefully checked the references.

Additional Editor Comments :

In addressing the reviewer's comments please address the following additional issues.

Q: 1. In your revision, please provide line numbers on the revised manuscript to make it easier for reviewers to review the revised manuscript.

A: We have added line numbers to the manuscript as requested.

Q: 2. Please engage the services of a native English speaker to revise the manuscript for typographical and grammatical errors. Delete any Chinese inscriptions in the manuscript, especially in the results section. Define any abbreviations upon first use in the manuscript.

A: The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

http://www.textcheck.com/certificate/axviDO

Q: 3. Genes should be italicized and written in small letters all through the manuscript.

A: We have made the changes as requested.

Q: 4. In discussing your results, do not repeat them; rather, provide a possible explanation for your findings while comparing them to the reports of other similar studies.

A: We removed the same content as the results from the discussion and added comparisons with similar studies to the discussion.

Q: 5. In the second paragraph of the discussion section, please italicize "Escherichia coli" and any other bacteria mentioned in the manuscript. Please italicize "Salmonella" all through the manuscript.

A: We have made the changes as requested.

Q: 6. Highlight the main limitations of the current study in the last paragraph of the discussion section, please.

A: The main limitations of the current study were added in the last paragraph of the discussion section.

5. Review Comments to the Author

Q: Reviewer #1: The authors have a good study question but were not able to properly put it in writing. There is no justification for the study, the methodology was not properly referenced for reproducibility and there were several grammatical and writing errors. Also, all authors referenced in the article need to be fully stated in the reference section and not stating et al., in places where the full names are supposed to be written. Not to say that there were scientific names that were not italicized and confusing species of Salmonella in the result section. Authors need to proof-read, justify the document and pay attention to details.

A: We have carefully revised the above content as required.

Reviewer #2: General Comments:

Q: Personally, I would like to see the letter of approval, especially since the author claims that the consent was given orally. The paper could benefit from a thorough revision, particularly in terms of punctuation. The lack of line numbers makes it challenging to pinpoint specific areas where punctuation is missing. Additionally, there are several acronyms that are not fully spelled out. I also noticed possible translation errors, as it appears the author may have translated the paper from another language to English. I recommend a careful review to ensure accurate translation. Finally, the author should pay much attention on their discussion because they did not discuss all results, and the conclusion is weak.

A: We had submitted the human research ethics committee of the Huzhou Center for Disease Control and Prevention(approval number: HZ2020007). The name of file is “approval document(English)”. And asked to modify the relevant content.

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

http://www.textcheck.com/certificate/axviDO

Abstract:

Q: The authors have failed to state the purpose of their study, despite attempting to define the problem. This is a crucial element that could engage the reader’s interest in the article.

A: the purpose of study were added in abstract: This study investigated the drug resistance and genomic characteristics of S. enteritidis isolated from clinical and food sources in Huzhou, Zhejiang Province, China, from February 1 2021, to December 30, 2023.

Introduction:

Q:The sentence, “Multi-drug resistant bacteria and even super bacteria have emerged, which lead to the emergence of new epidemiological and clinical characteristics of the disease,” seems redundant and does not add value. It appears to repeat previously mentioned information. The preceding sentence could also be restructured and potentially supplemented with a citation.

A: We removed “Multi-drug resistant bacteria and even super bacteria have emerged, which lead to the emergence of new epidemiological and clinical characteristics of the disease,” and the sentence was also rearranged.

Results:

Q: It was disappointing to see that the authors did not reference the supplemental material, which, in my opinion, could have been effectively utilized in their results to strengthen their arguments.

A: Supplementary material file had been submitted at the time of initial submission of the paper.

Discussion:

Q: The authors should consider using more direct language, as exemplified by their use of the vague term “some strains.” The discussion lacks depth and fails to highlight the practical implications of the results. There are no recommendations beyond drug resistance. Apparently, the genomic results are not discussed at all, and they applied genomic sequencing without a clear plan of how the results might be useful.

A: By analyzing the relationship between drug resistance genes and drug resistance phenotypes, we found that 12 strains, including S2021237, carried sul2 gene, but were sensitive to trimethoprim-sulfamethoxazole. In addition, 4 strains, including S2023209, were resistant to chloramphenicol but no chloramphenicol resistance genes were found. At the same time, we removed the expression of ambiguous terms in the text.

Conclusion:

Q: The conclusion is weak. It merely restates the methods without summarizing the results or discussing their potential benefits to the community. It gives the impression that the authors were more interested in applying the methods and maintaining records than in the study’s purpose.

A: We reformatted the conclusion section.

Attachment

Submitted filename: Response to Reviewers.doc

pone.0304621.s009.doc (69.6KB, doc)

Decision Letter 1

Mabel Kamweli Aworh

9 May 2024

PONE-D-24-09010R1Antimicrobial Resistance and Genome Characteristics of Salmonella enteritidis  from Huzhou, ChinaPLOS ONE

Dear Dr. Xu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mabel Kamweli Aworh, DVM, MPH, PhD. FCVSN

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have responded to all questions raised earlier and the manuscript can be accepted for publication.

Reviewer #2: I appreciate the current revised version, as it addresses most of the problems stated in the first version. I would also recommend it for publication.

A. However, the authors made a strong statement in lines 75 and 76: "There have been no previous reports on drug resistance and genetic characteristics of S enteriditis in the Huzhou area." Unless the authors are 100% sure there is none, I suggest revising this statement.

Based on a few searches, I realized that some co-authors have indeed contributed to previous works that include drug resistance in the Huzhou area. Nevertheless, these studies did not specifically investigate the genetic characteristics of S. enteriditis.

See articles:

1. Prevalence and Serotyping of Salmonella in Retail Food in Huzhou, China (D. Xu et al., 2024)

2. Characterization of Clinical Salmonella entericas Strains in Huzhou, China (D. Xu et al., 2022)

B. Furthermore, a review (though it is not a big issue) of the known genetic characteristics of S. enteriditis in other areas of China would be appreciated.

Example articles:

1. The Resistance and Virulence Characteristics of Salmonella Enteritidis Strain Isolated from Patients with Food Poisoning Based on the Whole-Genome Sequencing and Quantitative Proteomic Analysis (B. Xu et al., 2023)

2. Isolation, Identification, Antimicrobial Resistance, Genotyping, and Whole-Genome Sequencing Analysis of Salmonella Enteritidis Isolated from a Food-Poisoning Incident (Hou et al., 2024)

Bibliography:

Hou, Z., Xu, B., Liu, L., Yan, R., & Zhang, J. (2024). Isolation, Identification, Antimicrobial Resistance, Genotyping, and Whole-Genome Sequencing Analysis of Salmonella Enteritidis Isolated from a Food.Poisoning Incident. Polish Journal of Microbiology, 73(1), 69–89. https://doi.org/10.33073/PJM-2024-008

Xu, B., Hou, Z., Liu, L., Yan, R., Zhang, J., Wei, J., Du, M., Xuan, Y., Fan, L., & Li, Z. (2023). The Resistance and Virulence Characteristics of Salmonella Enteritidis Strain Isolated from Patients with Food Poisoning Based on the Whole-Genome Sequencing and Quantitative Proteomic Analysis. Infection and Drug Resistance, 16, 6567–6586. https://doi.org/10.2147/IDR.S411125

Xu, D., Chen, L., Lu, Z., & Wu, X. (2024). Prevalence and Serotyping of Salmonella in Retail Food in Huzhou China. Journal of Food Protection, 87(2), 100219. https://doi.org/10.1016/J.JFP.2024.100219

Xu, D., Ji, L., Yan, W., & Chen, L. (2022). Characterization of Clinical Salmonella entericas Trains in Huzhou, China. Canadian Journal of Infectious Diseases and Medical Microbiology, 2022. https://doi.org/10.1155/2022/7280376

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2024 Jun 4;19(6):e0304621. doi: 10.1371/journal.pone.0304621.r004

Author response to Decision Letter 1

13 May 2024

Dear  editors: Thank you very much for your letter and the comments from the referees about our paper submitted to PLOS ONE (PONE-D-24-09010).

We have carefully addressed all of the comments from the reviewers, as outlined in the point-by-point responses attached below. We hope that you find the revised manuscript now acceptable for publication in PLOS ONE.

Thanks for your attention

Yours Sincerely

Deshun Xu

Responses to the editor’s comments:

(Q: as comments, A: as our responses)

Q: However, the authors made a strong statement in lines 75 and 76: "There have been no previous reports on drug resistance and genetic characteristics of S enteriditis in the Huzhou area." Unless the authors are 100% sure there is none, I suggest revising this statement.

A: We have put "There have been no previous reports on drug resistance and genetic characteristics of S enteriditis in the Huzhou area. "changed to “As far as we know, there have been few previous reports on the drug resistance and genetic characteristics of S.enteritidis in the Huzhou area”.

Q: Based on a few searches, I realized that some co-authors have indeed contributed to previous works that include drug resistance in the Huzhou area. Nevertheless, these studies did not specifically investigate the genetic characteristics of S. enteriditis.

See articles:

1. Prevalence and Serotyping of Salmonella in Retail Food in Huzhou, China (D. Xu et al., 2024)

2. Characterization of Clinical Salmonella entericas Strains in Huzhou, China (D. Xu et al., 2022)

A: These two manuscripts are previous research conducted by our research group. One manuscript focuses on the Prevalence and Serotyping of Salmonella in Retail Food in Huzhou, while the other paper examines the Characterization of Clinical Salmonella entericas Strains in Huzhou. However, it is important to note that these studies did not specifically investigate the genetic characteristics of S. enteriditis.

Q: B. Furthermore, a review (though it is not a big issue) of the known genetic characteristics of S. enteriditis in other areas of China would be appreciated.

Example articles:

1. The Resistance and Virulence Characteristics of Salmonella Enteritidis Strain Isolated from Patients with Food Poisoning Based on the Whole-Genome Sequencing and Quantitative Proteomic Analysis (B. Xu et al., 2023)

2. Isolation, Identification, Antimicrobial Resistance, Genotyping, and Whole-Genome Sequencing Analysis of Salmonella Enteritidis Isolated from a Food-Poisoning Incident (Hou et al., 2024)

A: These two manuscripts have been cited in the article, with References 35 and 42 respectively.

Attachment

Submitted filename: Response to Reviewers.doc

pone.0304621.s010.doc (21KB, doc)

Decision Letter 2

Mabel Kamweli Aworh

15 May 2024

Antimicrobial Resistance and Genome Characteristics of Salmonella enteritidis  from Huzhou, China

PONE-D-24-09010R2

Dear Dr. Xu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mabel Kamweli Aworh, DVM, MPH, PhD. FCVSN

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All reviewer's comments have been addressed.

Reviewers' comments:

Acceptance letter

Mabel Kamweli Aworh

22 May 2024

PONE-D-24-09010R2

PLOS ONE

Dear Dr. Xu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Mabel Kamweli Aworh

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. The information of 43 Salmonella enteritidis isolates strain.

    (DOCX)

    pone.0304621.s001.docx (18.2KB, docx)
    S2 Table. Genome assembly profile of Salmonella enteritidis.

    (DOCX)

    pone.0304621.s002.docx (15.4KB, docx)
    S3 Table. Results of MLST typing of Salmonella enteritidis genome.

    (DOCX)

    pone.0304621.s003.docx (20.3KB, docx)
    S4 Table. Genomic virulence gene results of Salmonella enteritidis.

    (DOCX)

    pone.0304621.s004.docx (17KB, docx)
    S5 Table. Salmonella enteritidis genome virulence gene pathogenic system.

    (DOCX)

    pone.0304621.s005.docx (13.5KB, docx)
    Attachment

    Submitted filename: PONE-D-24-09010.pdf

    pone.0304621.s006.pdf (1.2MB, pdf)
    Attachment

    Submitted filename: REVIEW genome characteristics of S.enteritidis.pdf

    pone.0304621.s007.pdf (944.4KB, pdf)
    Attachment

    Submitted filename: PONE-D-24-09010 REVIEWERS COMMENT.pdf

    pone.0304621.s008.pdf (118.7KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.doc

    pone.0304621.s009.doc (69.6KB, doc)
    Attachment

    Submitted filename: Response to Reviewers.doc

    pone.0304621.s010.doc (21KB, doc)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

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