Multidrug-Resistant Extraintestinal Pathogenic Escherichia coli Exhibits High Virulence in Calf Herds: A Case Report

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

In attachment are some comments in order to improve this Manuscript.

The paper presents an overview about the multidrug-resistant pathogenic bacteria Escherichia coli responsible for severe infectious diseases. After reading the submitted Manuscript, I consider that some sections can be improved. For this reason, I provide some comments that should be addressed. The following suggestions are presented:

Specific points

Abstract

Line 13-16: This part of the abstract would be better placed in the Background section. Also, specify what the aim of the study was.

Line 18: Indicate which methods were used for determination of antimicrobial activity, pathogenicity as well as isolation and identification.

Line 58: At the end of the introduction should state exactly what the aim of the study was?

Methods

Line 280: Antibiotic susceptibility testing: Specify which assay was used for determination of antibacterial activity of antibiotics. I suppose disk diffusion, and also briefly describe.

Line 301: Whole genome sequencing (WGS) and comprehensive analysis: How was DNA isolation and purification the sample of strain RZ-13 was performed for whole genome sequencing analysis. What is the origin of strain RZ-13, I supposed Escherichia coli, provide the full name of the strain.

In general the discussion should be improved.

The authors should compare the obtained results with similar findings.

Add a few sentences about contribution and innovative data from this paper.

Include future trends to keep working with the obtained data.

 

 

Author Response

Thank you very much for your valuable comments and suggestions. We have carefully considered each of your points, agreed with these views and revised them carefully. For your convenience, we put the newly changes below, and we hope it will satisfy you.

Comment 1: Line 13-16: This part of the abstract would be better placed in the Background section. Also, specify what the aim of the study was.

Response 1: The research background description is simplified and the research objectives are clarified. Line13-17: “We report a case of a calf herd infection by ExPEC with high rates of morbidity and mortality. The research purpose of this study was to thoroughly investigate the characteristics of the ExPEC responsible for the calf herd infection. Specifically, we aimed to understand the mechanisms underlying its multidrug resistance and high pathogenicity.”

Comment 2: Line 18: Indicate which methods were used for determination of antimicrobial activity, pathogenicity as well as isolation and identification.

Response 2: Modified. Line17-25: “Clinical samples were collected for isolation and identification of ExPECs, cultured on MacConkey agar, and further tested by PCR for the uidA gene, 16S rRNA gene sequencing and adhesion patterns on Hep-2 cells. The antimicrobial activity was determined using the disk diffusion method according to CLSI guidelines. Pathogenicity was assessed through experimental infection of Kunming mice, tracking survival and weight changes, and performing autopsies for bacterial counts and histopathological analysis. Additionally, whole genome sequencing and comprehensive analysis were performed, including multilocus sequence typing, serotyping, drug-resistance gene analysis, virulence factor analysis, metabolic pathway analysis, and enrichment analysis using various online tools and databases.”

Comment 3: Line 58: At the end of the introduction should state exactly what the aim of the study was?

Response 3: Modified. Line75-79: “This study would provide new data and valuable insights into the epidemiology, pathogenesis, and treatment strategies for ExPEC - induced infections in calves, ultimately contributing to the development of more effective control measures to prevent the spread of such infections and reduce the associated morbidity and mortality rates.”

Comment 4: Line 280: Antibiotic susceptibility testing: Specify which assay was used for determination of antibacterial activity of antibiotics. I suppose disk diffusion, and also briefly describe.

Response 4: The antimicrobial activity of antibiotics was determined using the disk diffusion method, with the steps of this method being briefly described in the original text. Line144-149 (Marked in red): “For the antimicrobial assay, a suspension of RZ-13 with a McFarland turbidity of 0.5 was uniformly spread onto the surface of agar plates (Beijing Coolaber), with CICC 24186 serving as the control strain. Antibiotic disks were then evenly placed on the agar plate surface. The plate was inverted and incubated at 37℃ for 16 to 18 hours. Following incubation, the diameters of the inhibition zones were measured to determine the antimicrobial activity of the tested antibiotics.”

Comment 5: Line 301: Whole genome sequencing (WGS) and comprehensive analysis: How was DNA isolation and purification the sample of strain RZ-13 was performed for whole genome sequencing analysis. What is the origin of strain RZ-13, I supposed Escherichia coli, provide the full name of the strain.

Response 5: In the section of Whole genome sequencing (WGS) and comprehensive analysis, we described the steps of strain isolation and purification, as well as genomic DNA extraction. Line174-177 (Marked in red): “The preserved Escherichia coli RZ-13 strain was inoculated onto an agar plate and incubated in a 37°C constant-temperature incubator. After incubation, a single colony was selected and inoculated into LB liquid medium to obtain a bacterial suspension. 2 mL aliquot of the bacterial suspension was used for whole-genome DNA extraction.”

Line174：“Escherichia coli RZ-13 strain”

Comment 6:In general the discussion should be improved.

Response 6: The discussion has been improved as below.

(1) The authors should compare the obtained results with similar findings.

Line295-304: “A global analysis of antibiotic resistance in ExPEC revealed that the highest resistance levels were to sulfonamides (35%) and fluoroquinolones (32%), followed by 9% resistance to extended - spectrum third - and fourth - generation cephalosporins. Resistance to the last - resort antibiotics colistin and carbapenems was relatively low, at 0.7% and 0.1 - 0.2%, respectively [49]. In china, Isolates exhibited high resistance rates to tetracycline (95.5%), ampicillin (95.5%), piperacillin (90.1%), sulfamethoxazole - trimethoprim (89.9%), chloramphenicol (85.2%), gentamicin (70%), and fluoroquinolones (65%)[5]. Additionally, 51.8%, 13.4%, and 44.7% of isolates were resistant to cefotaxime, ceftazidime, and cefepime, respectively, which are third - and fourth - generation cephalosporins.”

(2) Add a few sentences about contribution and innovative data from this paper.

Line291-292: “firstly reported a case of MDR ExPEC leading to severe mortality in calf herds in China, and”

(3) Include future trends to keep working with the obtained data.

Line312-314: “This finding underscores the necessity for further investigation into the distribution and transfer capabilities of the antibiotic resistance genes in the RZ-13 strain.”

Line381-384: “Upon our recommendation, the farm has already implemented isolation of the diseased cattle, environmental disinfection, and harmless treatment of the dead cows. Subsequently, we will continue to monitor the farm's environment and diarrheic calves for multidrug - resistant strains.”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, thank you for the opportunity to read your case report. The discovery of MDR extraintestinal E. coli as the cause of diarrhea outbreak in calf herds is certainly significant, and the presented results can help in creating strategies for their prevention and quick and effective treatment.
However, in my opinion, additional clarifications are needed.

Major:

It would be more appropriate to call this study an Original Article, rather than a Case Report.
The Materials and Methods section should be placed before the results. This section should be clarified with additional information:

• it is not clear how many dead animal bodies were examined and how many samples (the authors mention 11 calves in several places, but the number 42 (line 68) is also mentioned. Please specify exactly and uniformly throughout the entire manuscript how many animals and how many samples (also specify exactly how many of which types of samples there were)
• add the names of the manufacturers of MacConkey agar medium and media for making antibiograms
• please explain why the two interpretive standards, CLSI and EUCAST, were used and add the years and references for both standards

Minor:

Title: E .coli (italic)

Abstract: line 12: delete a series of; line 14:delete and death; conclusion is weak and should be reformulated.

Introduction: line 32: delete losses and add "burden"; line 35:  delete "extraintestinal infections,leading to"; line 45: instead of "virulent traits" put "specific sequence types (ST)"; lines 48-51: rewrite this part; state mechanisms of resistance (e.g. beta-lactamase production) or state to which antibiotics resistance is increasing. Also, clarify the aim of the study. 

Case description: Please, move the information about antibiotic therapy from the discussion here (and add the doses and duration of treatment)

Results: Please explain why you named the isolate RZ-13, why not simply use the name ExPEC.

Lines 105-109: please, systematize antibiotics here and further in the text; group them by class. Also, you state that the isolate was sensitive only to cefoxitin, and further on in the text you say that it was resistant; which of these is correct?
Table 1 is unnecessary and can be deleted.

Line 158:please add full names for "KEGG" and "GO" (line 162)

Conclusion: line 321: instead of "a case of group infection" it would be better "an outbreak of infection"

 

 

Author Response

Thank you very much for your valuable comments and suggestions. We have carefully considered each of your points, agreed with these views and revised them carefully. For your convenience, we put the newly changes below, and we hope it will satisfy you.

 

Major:

 

Comments 1: It would be more appropriate to call this study an Original Article, rather than a Case Report.The Materials and Methods section should be placed before the results. This section should be clarified with additional information

Response 1: We have followed your suggestion and placed the “Materials and Methods” section before the “Results” section. Regarding the modification of the article type, the final decision should be made by the editor of the journal, and we have no objections to this.

 

Comments 2: it is not clear how many dead animal bodies were examined and how many samples (the authors mention 11 calves in several places, but the number 42 (line 68) is also mentioned. Please specify exactly and uniformly throughout the entire manuscript how many animals and how many samples (also specify exactly how many of which types of samples there were)

Response 2: The number of dead animal bodies and samples were added in the Materials and methods section. Line 113-114: “We collected 66 clinical samples from 11 calves using aseptic procedures, including the heart, liver, spleen, lungs, kidneys, and intestines from each calf. ”

 

Comments 3: add the names of the manufacturers of MacConkey agar medium and media for making antibiograms

Response 3: The names of the manufacturers of MacConkey agar medium and media for making antibiograms have been added. Line 117: “MacConkey agar plate (Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd)”

Line145: “agar plates (Beijing Coolaber).”

 

Comments 4: please explain why the two interpretive standards, CLSI and EUCAST, were used and add the years and references for both standards

Response 4: During the antimicrobial susceptibility testing, we referred to both the CLSI and EUCAST standards, but ultimately, we based our results on the CLSI criteria. Therefore, we have removed “EUCAST” and retained “CLSI”. Line 156-159: “The antibiotic susceptibility of the tested bacteria was determined and interpreted in accordance with the Clinical & Laboratory Standards Institute (CLSI) guidelines (M100 - Ed35) .”

 

Minor:

 

Comments 5: Title: E .coli (italic)

Response 5: Title: Escherichia coli had been italicized.

 

Comments 6: Abstract: line 12: delete a series of; line 14:delete and death; conclusion is weak and should be reformulated.

Response 6: The phrase "a series of " in line 12 of the original text has been deleted. Line 12: “Extraintestinal pathogenic Escherichia coli (ExPEC) is a group of Escherichia coli strains that can cause severe infectious diseases outside the gastrointestinal tract.”

The word "death" in line 14 of the original text has been deleted. Line 13-14: “We report a case of a calf herd infection by ExPEC with high rates of morbidity and mortality.”

The conclusion in “Abstract” section has been reformulated. Line 31-35: “Our study isolated a multidrug-resistant ExPEC strain RZ-13, with a strong pathogenicity. This is a first case report of ExPEC leading to severe mortality in calf herds in China, underscoring the need for rational use of antibiotics to reduce the risk of generation and transmission of multi-resistant bacteria from food-producing animals to ensure food safety and public health.”

 

Comments 7: Introduction: line 32: delete losses and add "burden"; line 35: delete "extraintestinal infections,leading to"; line 45: instead of "virulent traits" put "specific sequence types (ST)";

Response 7:

• The word "losses" in line 32 of the original text has been deleted, and "burden"has been add Line 40: “Pathogenic Escherichia coli causes major economic burden worldwide, with high mortality rates.”
• "extraintestinal infections,leading to"in line 35of the original text has been deleted. Line 43-45: “Among them, ExPEC is a significant pathogen causing various diseases such as cystitis, pyelonephritis, and bacteremia, and can even pose a threat to human and animal life. ”
• Line53: "specific sequence types (ST)"in line45 of the original text has been instead of "virulent traits". Line 53: “specific sequence types (ST)”

 

Comments 8: lines 48-51: rewrite this part; state mechanisms of resistance (e.g. beta-lactamase production) or state to which antibiotics resistance is increasing. Also, clarify the aim of the study.

Response 8: I rewrote this part, Line 59-71: "The antimicrobial resistance mechanisms of ExPEC include reduced antibiotic uptake, enhanced efflux, target site modifications, or the production of inactivating enzymes [7]. Additionally, mobile genetic elements, such as plasmids and transposons, facilitate the spread of resistance genes, accelerating the dissemination of resistant bacteria The abuse of antibiotics has worsened the resistance issue in cattle populations. Existing studies indicate that the rumen of ruminants contains at least 4043 antibiotic resistance genes (ARGs), including genes for β-lactams (726 genes), glycopeptides (510 genes), tetracyclines (307 genes), and aminoglycosides (193 genes) [8]. Similarly, it has been reported that 97.3% of 37 Escherichia coli strains isolated from the rectum of diarrheal piglets exhibit resistance to at least four different antibiotics, with 28 strains carrying resistance genes to colistin [9]. It should be noted that antibiotic resistance is increasingly becoming a dynamic phenomenon, necessitating continuous updates to the knowledge in this field."

The research objectives have been clarified. Line 75-79: "This study would provide new data and valuable insights into the epidemiology, pathogenesis, and treatment strategies for ExPEC - induced infections in calves, ultimately contributing to the development of more effective control measures to prevent the spread of such infections and reduce the associated morbidity and mortality rates."

 

Comments 9: Case description: Please, move the information about antibiotic therapy from the discussion here (and add the doses and duration of treatment)

Response 9: Case description has been revised. Line 86-93: “When symptoms are mild, oral rehydration salts (300 - 400 mL) are administered orally, along with oral sulfamidine tablets (20 - 30 tablets), or gentamicin injections (10 mg/kg, twice a day). If there is no significant improvement in symptoms after 2 days, intravenous fluid therapy is initiated, with intravenous injections of ampicillin (30 mg/kg, twice a day), and at the same time, intramuscular injections of ciprofloxacin or enrofloxacin (10 - 20 mL, once a day), or gentamicin injections (10 mg/kg, twice a day) are administered. This treatment continues until the calf recovers or dies.”

 

Comments 10: Results: Please explain why you named the isolate RZ-13, why not simply use the name ExPEC.

Response 10: ExPEC is a collective term for extraintestinal pathogenic Escherichia coli, not a specific species. Using RZ-13 can avoid confusion with other ExPEC isolates. This strain was isolated in Rizhao and is derived from the 13th isolate picked, hence the name was chosen based on the initials of the location (Rizhao) and the number 13.

 

Comments 11: Lines 105-109: please, systematize antibiotics here and further in the text; group them by class. Also, you state that the isolate was sensitive only to cefoxitin, and further on in the text you say that it was resistant; which of these is correct?

Response 11:

• Antibiotics have been categorized and described. Line149-156: “A variety of antibiotics were utilized in the drug sensitivity testing, including fluoroquinolones (norfloxacin at 10 μg, ofloxacin at 5 μg, and enrofloxacin at 10 μg), sulfonamides (sulfamethoxazole containing 23.75 μg of sulfonamide and 1.25 μg of trimethoprim), chloramphenicols (chloramphenicol at 30 μg), aminoglycosides (gentamicin at 10 μg and kanamycin at 30 μg), tetracyclines (doxycycline at 30 μg), cephalosporins (ceftriaxone at 30 μg, ceftazidime at 30 μg, cefalexin at 30 μg, and cefoxitin at 30 μg), and penicillins (ampicillin at 10 μg and amoxicillin at 20 μg)”

(2) There was an erroneous description, and corrections have been made. Line223-226:

“including fluoroquinolones (norfloxacin, ofloxacin, enrofloxacin), sulfonamides (sulfamethoxazole), chloramphenicols (chloramphenicol), aminoglycosides (gentamicin, kanamycin), tetracyclines (doxycycline), cephalosporins (ceftriaxone, ceftazidime, cephalexin), and penicillins (ampicillin, amoxicillin).”

 

Comments 12: Table 1 is unnecessary and can be deleted.

Response 12: Table 1 has been deleted.

 

Comments 13: Line 158:please add full names for "KEGG" and "GO" (line 162)

Response 13: When KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) are first mentioned, their full names should be provided.

Line 187: “Kyoto Encyclopedia of Genes and Genomes database (KEGG)”;

Line 187-188: “Gene Ontology database (GO)”

 

Comments 14: Conclusion: line 321: instead of "a case of group infection" it would be better "an outbreak of infection"

Response 14: "an outbreak of infection" has been instead of "a case of group infection".

Line 386: ”We report an outbreak of infection in calves accompanied by high incidence, high fatality rate and multiple drug-resistance.”

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The following points need to be addressed:

1. The italicized name of E. coli should be included in the title.

2. Many sentences in the introduction section are missing references.

3. Under "Case Description," clarify the criteria for case selection. Is there a reference that outlines the methodology followed by the author?

4. In Table 1, what does "sensitive" mean? Does it refer to the concentration of antibiotics?

5. Provide a brief description of the role of each gene listed in Table 2, and include this information in a separate column.

6. Figure 6, titled "GO Enrichment Analysis of Virulence Genes in the RZ-13 Genome," should include a more comprehensive analysis of the presence of virulence genes in the genome.

7. The discussion section contains several sentences without references.

8. The Materials and Methods section is missing references. Including these is crucial to allow other researchers to replicate the experiments.

Author Response

Thank you very much for your valuable comments and suggestions. We have carefully considered each of your points, agreed with these views and revised them carefully. For your convenience, we put the newly changes below, and we hope it will satisfy you.

 

Comments 1: The italicized name of E. coli should be included in the title.

Response 1: Agree. Title: Escherichia coli had been italicized.

 

Comments 2: Many sentences in the introduction section are missing references.

Response 2: Agree. The part of Introduction section has been rewrited, and references in Introduction section have been supplementary.

Line 43-45: “Among them, ExPEC is a significant pathogen causing various diseases such as cystitis, pyelonephritis, and bacteremia, and can even pose a threat to human and animal life [4].”

Line 59-71: “The antimicrobial resistance mechanisms of ExPEC include reduced antibiotic uptake, enhanced efflux, target site modifications, or the production of inactivating enzymes [7]. Additionally, mobile genetic elements, such as plasmids and transposons, facilitate the spread of resistance genes, accelerating the dissemination of resistant bacteria The abuse of antibiotics has worsened the resistance issue in cattle populations. Existing studies indicate that the rumen of ruminants contains at least 4043 antibiotic resistance genes (ARGs), including genes for β-lactams (726 genes), glycopeptides (510 genes), tetracyclines (307 genes), and aminoglycosides (193 genes) [8]. Similarly, it has been reported that 97.3% of 37 Escherichia coli strains isolated from the rectum of diarrheal piglets exhibit resistance to at least four different antibiotics, with 28 strains carrying resistance genes to colistin [9]. It should be noted that antibiotic resistance is increasingly becoming a dynamic phenomenon, necessitating continuous updates to the knowledge in this field.”

 

Comments 3: Under "Case Description," clarify the criteria for case selection. Is there a reference that outlines the methodology followed by the author?

Response 3: Agree. We have augmented the description in this regard and cited relevant references. Line 81-83: “In the process of selecting cases and collecting information, we followed CARE guidelines [10] to support an increase in the accuracy, transparency, and usefulness of this case report and referred to similar case reports [11].”

 

Comments 4: In Table 1, what does "sensitive" mean? Does it refer to the concentration of antibiotics?

Response 4: Agree. The table 1 has been deleted according to other reviewer’s comment.

 

Comments 5: Provide a brief description of the role of each gene listed in Table 2, and include this information in a separate column.

Response 5: Agree. A brief description of the role of each gene has been provided and listed in a separate column of Table 1. Line 283:

Table 1. Antibiotic resistance genes in isolated strain RZ-13.

Category	Antibiotic resistant genes	Function
Quinolone	qepA1	Expel fluoroquinolone antibiotics from bacterial cells [32]
Sulfonamide	sul2	A variant that encodes dihydropteric acid synthase, making it less susceptible to sulfonamides antibiotics [33]
	sul3
Arsenic	arsC	Getting the arsenic out of the cell [34]
	arsR	Regulate the expression of related arsenic tolerance genes [35]
Lincosamide	lnu(F)	Protects ribosomes from the effects of lincoamide antibiotics
Efflux	mdtM	Encodes efflux pump proteins that remove a variety of antibiotics from cells [36]
	acrF
	emrD
Macrolide	mph(A)	Catalyzes the phosphorylation of macrolide antibiotics, conferring bacterial resistance to macrolides [37]
Rifamycin	arr-2	Encodes adenylate cyclase ribosyltransferase, which reduces antibiotic activity [38]
Tetracycline	tet(A)	Coded tetracycline efflux pump [39]
Beta-lactam	blaEC	Code for beta-lactamase, which inactivates antibiotics [40]
	blaCTX-M-55
	blaTEM
Tellurium	terD	Involved in the expulsion or isolation of tellurides [41]
	terZ
	terW
Phenicol	floR	Encodes a membrane transporter that excretes amidoalcohol antibiotics [42]
Aminoglycoside	rmtB1	Aminoglycoside antibiotics fail to bind to ribosomes [43][44]
	aac(3)-IId	Acetylated aminoglycoside antibiotics [43][44]
	aadA22	Adenosine aminoglycoside antibiotics [43][44]
	aph(6)-Id	Phosphorylated aminoglycoside antibiotics [43][44]
	aph(3')-Ia	Phosphorylated aminoglycoside antibiotics [43][44]
Trimethoprim	dfrA14	Synthesis of a variant of dihydrofolate reductase, thereby reducing the sensitivity of sulfonamides to its effects [45]
Ferrous-iron efflux pump FieF	fieF	Expel iron ions from the cell
Multiple antibiotic resistance protein MarA	marA	Multiple antibiotic resistant proteins [46]
Multiple antibiotic resistance protein MarR	marR	Regulates the expression of MarA and other drug resistance genes [46]

 

 

Comments 6: Figure 6, titled "GO Enrichment Analysis of Virulence Genes in the RZ-13 Genome," should include a more comprehensive analysis of the presence of virulence genes in the genome.

Response 6: Agree. The title "GO Enrichment Analysis of Virulence Genes in the RZ-13 Genome" has been instead of "GO enrichment analysis of codon genes in the RZ-13 genome"（Line 287）

 

Comments 7: The discussion section contains several sentences without references.

Response 7: Agree. References in “Discussion” section have been supplementary.

Line 291-292: “...firstly reported a case of MDR ExPEC leading to severe mortality in calf herds in China, and...”

Line 295-304: “A global analysis of antibiotic resistance in ExPEC revealed that the highest resistance levels were to sulfonamides (35%) and fluoroquinolones (32%), followed by 9% resistance to extended - spectrum third - and fourth - generation cephalosporins. Resistance to the last - resort antibiotics colistin and carbapenems was relatively low, at 0.7% and 0.1 - 0.2%, respectively [49]. In china, Isolates exhibited high resistance rates to tetracycline (95.5%), ampicillin (95.5%), piperacillin (90.1%), sulfamethoxazole - trimethoprim (89.9%), chloramphenicol (85.2%), gentamicin (70%), and fluoroquinolones (65%)[5]. Additionally, 51.8%, 13.4%, and 44.7% of isolates were resistant to cefotaxime, ceftazidime, and cefepime, respectively, which are third - and fourth - generation cephalosporins.”

Line 305: “than most of others.”

Line 312-314: “This finding underscores the necessity for further investigation into the distribution and transfer capabilities of the antibiotic resistance genes in the RZ-13 strain.”

Line 381-384: “Upon our recommendation, the farm has already implemented isolation of the diseased cattle, environmental disinfection, and harmless treatment of the dead cows. Subsequently, we will continue to monitor the farm's environment and diarrheic calves for multidrug - resistant strains.”

 

Comments 8: The Materials and Methods section is missing references. Including these is crucial to allow other researchers to replicate the experiments.

Response 8: Agree. References in “Materials and Methods” section have been supplementary.

Line 118-120: “Several typical colonies were selected and enriched in LB broth (MDBio,Inc) at 37℃ overnight and stored at 4℃ for Gram staining and further detection[12].”

Line 126-128: “The results were compared with the National Center for Biotechnology Information (NCBI) database through BLAST [13].”

Line 141-142: “The adhesion phenotype was observed and recorded under a Fluorescent inverted microscope (Leica, DMIL, Germany) [14].”

Line 156-159: “The antibiotic susceptibility of the tested bacteria was determined and interpreted in accordance with the Clinical & Laboratory Standards Institute (CLSI) guidelines (M100 - Ed35) [15-23].”

Line 170-172: “The histopathological sections were sent to Wuhan Servicebio Technology Co., Ltd. The LD₅₀ was calculated using the Reed-Muench method [24].”

 

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This is a thorough case report suitable for the journal it was submitted to. The problematic of antibiotic resistance and its consequences in cattle breeding, among others, is very important and further signals must be drawn about the uncontrolled and excessive use of antibiotics that has led to such resistance from bacteria. 

There are several areas of improvement, some that have to do with style, others with content. I will present my comments on a line by line basis (line numbers refer to the pdf of the manuscript):

Title: Escherichia coli should be italicized

Line 81: had (not "has")

Line 91: were (not "was")

Line 93: exhibits (not "exhibit")

Figure 3 is incomplete. The curves with square, upwards triangle and downwards triangle symbols mentioned in the legend are not shown on the chart itself. Please address this!

Although regular readers of this journal are likely familiar with such acronyms, it would be helpful for the broader readership to include a list of all abbreviations used and their meaning (KEGG, GO, DMEM etc.)

Line 165: "resistance" (not "resistant")

Lines 172-175: All this information belongs to the Introduction rather than the Discussion section. I recommend moving it to the Intro and developing it a bit further using multiple literature references. The introduction in its current version is too brief. The causes of antibiotic resistance in relation to cattle or more broadly should be discussed in full to better place into context the motivation that stands behind this particular case report.

Lines 179-180: comma symbols are unusual

Line 204: the (no capital T needed in this case)

Line 236: no capital V needed in Veterinarians

Line 247: no need to underline the author name Tianshi Xiao

Line 259: "mL" (not "ml") is the internationally recognized symbol for milliliter

Line 258: How many hearts, livers, spleens etc. were harvested and analysed? What was the sample size in each case and for each organ? These are important details for the statistical analyses you report in a later section (section 5.5).

Experimental section: For all chemicals and preparations used in all experimental protocols, suppliers must be  indicated as well as purity or at least reagent grade (e.g. PBS, agar, CO2 gas, formaldehyde, crystal violet, Zoleti, all antibiotics mentioned etc.). Similar information must be provided for all biological supplies acquired commercially (e.g Hep-2 cells, LB broth etc.)

Line 279: Provide all technical details regarding the microscope used (type, maker, place, country, typical settings used)

Line 300: Cite a reference where details regarding this method can be found

Add DOIs to all references in the reference list

Author Response

Thank you very much for your valuable comments and suggestions. We have carefully considered each of your points, agreed with these views and revised them carefully. For your convenience, we put the newly changes below, and we hope it will satisfy you.

 

Comments 1: Title: Escherichia coli should be italicized

Response 1: Agree. Title: Escherichia coli had been italicized.

 

Comments 2: Line 81: had (not "has")

Response 2: Agree. “has” had been instead of “had”. Line 110: ”The outbreak records implied that the E. coli causing the outbreak was highly virulent and pathogenic, and had widespread drug resistance.”

 

Comments 3: Line 91: were (not "was")

Response 3: Agree. “was”had been instead of “were”. Line 206: “The toxin typing results showed that no toxin genes in the national standard were detected, indicating that RZ-13 is an atypical pathogenic E. coli.”

 

Comments 4: Line 93: exhibits (not "exhibit")

Response 4: Agree. “exhibit”had been instead of “exhibits”. Line 208: “The Hep-2 cell adhesion assay results showed that RZ-13 strain exhibits less obvious pattern of "stacked bricks" similar to the Enteroaggregative Escherichia coli (EAEC) reference strain CICC 24186 on Hep-2 cells (Figure 2).”

 

Comments 5: Figure 3 is incomplete. The curves with square, upwards triangle and downwards triangle symbols mentioned in the legend are not shown on the chart itself. Please address this!

Response 5: Agree. We modified Figure 3 as mentioned in Line XX, although the distinctions are not easy to make out due to overlapping. Line 252:

 

 

Comments 6: Although regular readers of this journal are likely familiar with such acronyms, it would be helpful for the broader readership to include a list of all abbreviations used and their meaning (KEGG, GO, DMEM etc.)

Response 6: Agree. The full form of KEGG, GO and DMEM had been supplemented.

Line 186-187: “Kyoto Encyclopedia of Genes and Genomes database (KEGG)” ;

Line 187-188: “Gene Ontology database (GO)” ;

Line 136-137: “Dulbecco's Modified Eagle Medium (DMEM)”.

 

Comments 7: Line 165: "resistance" (not "resistant")

Response 7: Agree. “resistant”has been instead of “resistance”. Line 283: “ Table 1. Antibiotic resistance genes in isolated strain RZ-13. .”

 

Comments 8: Lines 172-175: All this information belongs to the Introduction rather than the Discussion section. I recommend moving it to the Intro and developing it a bit further using multiple literature references. The introduction in its current version is too brief. The causes of antibiotic resistance in relation to cattle or more broadly should be discussed in full to better place into context the motivation that stands behind this particular case report.

Response 8: Agree. The sentence “The abuse of antibiotics causes increasingly severe problems with drug resistance in cattle.”has been moved to the Introduction section. Line 63-64: “ The abuse of antibiotics has worsened the resistance issue in cattle populations.”

 

Comments 9: Lines 179-180: comma symbols are unusual

Response 9: Agree. The sentence has been detected.

 

Comments 10: Line 204: the (no capital T needed in this case)

Response 10: Agree. “The”has been instead of “the”. Line 330: “ the clinical manifestation of strong .”

 

Comments 11: Line 236: no capital V needed in Veterinarians

Response 11: Agree. “Veterinarians”has been instead of “veterinarians”. Line 363: “Secondly, veterinarians should identify the specific pathogen before deciding on the use of antibiotics to ensure targeted and effective treatment. .”

 

Comments 12: Line 247: no need to underline the author name Tianshi Xiao

Response 12: Agree. The underline of the Tianshi Xiao has been removed. Line 375: “ Tianshi Xiao et al.”

 

Comments 13: Line 259: "mL" (not "ml") is the internationally recognized symbol for milliliter

Response 13: Agree. “ml”has been instead of “mL”. Line 177: “ mL”

 

Comments 14: Line 258: How many hearts, livers, spleens etc. were harvested and analysed? What was the sample size in each case and for each organ? These are important details for the statistical analyses you report in a later section (section 5.5).

Response 14: Agree. The number of dead animal bodies and samples were added in the Materials and methods section. Line113-114: “We collected 66 clinical samples from 11 dead calves using aseptic procedures, including the heart, liver, spleen, lungs, kidneys, and intestines from each calf.”

 

Comments 15: Experimental section: For all chemicals and preparations used in all experimental protocols, suppliers must be  indicated as well as purity or at least reagent grade (e.g. PBS, agar, CO₂ gas, formaldehyde, crystal violet, Zoleti, all antibiotics mentioned etc.). Similar information must be provided for all biological supplies acquired commercially (e.g Hep-2 cells, LB broth etc.)

Response 15: Agree. The sources of all chemicals and preparations used in the experimental protocol have been indicated.

Line 116: “ buffered saline (PBS) (Beijing solarbio science﹠technology co.,ltd.).”

Line 117: “ MacConkey agar plate (Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd)”

Line 119: “ LB broth (MDBio,Inc)” 

Line 133: “ 24-well plates (Beijing solarbio science﹠technology co.,ltd.)”

Line 136: “ Dulbecco's Modified Eagle Medium (DMEM) (Beijing solarbio science﹠technology co.,ltd.)”

Line 139: “ formaldehyde (Beijing solarbio science﹠technology co.,ltd.)”

Line 140: “ 0.1% crystal violet (Beijing solarbio science﹠technology co.,ltd.)”

Line 142: “ Fluorescent inverted microscope (Leica, DMIL, Germany)”

Line 145: “ agar plates (Beijing Coolaber)”

Line 150-156: “including fluoroquinolones (norfloxacin at 10 μg, ofloxacin at 5 μg, and enrofloxacin at 10 μg), sulfonamides (sulfamethoxazole containing 23.75 μg of sulfonamide and 1.25 μg of trimethoprim), chloramphenicols (chloramphenicol at 30 μg), aminoglycosides (gentamicin at 10 μg and kanamycin at 30 μg), tetracyclines (doxycycline at 30 μg), cephalosporins (ceftriaxone at 30 μg, ceftazidime at 30 μg, cefalexin at 30 μg, and cefoxitin at 30 μg), and penicillins (ampicillin at 10 μg and amoxicillin at 20 μg), all provided by Hangzhou Microbial Reagent Co., Ltd.”

Line 167-168: “ Zoleti (75 mg/kg) (Virbac Trading Co., Ltd.)”

 

Comments 16: Line 279: Provide all technical details regarding the microscope used (type, maker, place, country, typical settings used)

Response 16: Agree. all technical details regarding the microscope used have been provided. Line 142: “ Fluorescent inverted microscope (Leica, DMIL, Germany)”

 

Comments 17: Line 300: Cite a reference where details regarding this method can be found

Response 17: Agree. References in “Materials and Methods” section have been supplementary.

Line 118-120: “Several typical colonies were selected and enriched in LB broth (MDBio,Inc) at 37℃ overnight and stored at 4℃ for Gram staining and further detection[12].”

Line 126-128: “The results were compared with the National Center for Biotechnology Information (NCBI) database through BLAST [13].”

Line 141-142: “The adhesion phenotype was observed and recorded under a Fluorescent inverted microscope (Leica, DMIL, Germany) [14].”

Line 156-159: “The antibiotic susceptibility of the tested bacteria was determined and interpreted in accordance with the Clinical & Laboratory Standards Institute (CLSI) guidelines (M100 - Ed35) [15-23].”

Line 170-172: “The histopathological sections were sent to Wuhan Servicebio Technology Co., Ltd. The LD₅₀ was calculated using the Reed-Muench method [24].”

 

Comments 18: Add DOIs to all references in the reference list

Response 18: Agree. All DOIs to all references in the reference list have been added.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

After a revised Manuscript version 2, the paper could be considered for publication in the journal Microbiology Research.

Reviewer 3 Report

Comments and Suggestions for Authors

The author responded all raised comments