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Molecular characterization and antibiotic susceptibility of Shiga toxin- producing Escherichia coli (STEC) isolated from raw milk of dairy bovines in Khyber Pakhtunkhwa, Pakistan
Safir et al
https://orcid.org/0000-0002-9622-5217
Ullah Safir Conceptualization Investigation Writing – original draft 1 *
Hassan Khan Saeed Ul Supervision 1 *
Ali Tariq Resources 2
Zeb Muhammad Tariq Resources 2
https://orcid.org/0000-0002-9622-699X
Riaz Muhammad Hasnain Resources Validation Writing – review & editing 2
Khan Siraj Data curation Writing – review & editing 3
Goyal Sagar M. Conceptualization Investigation 4
1 Department of Zoology, Faculty of Biological Science, Quaid-i-Azam University, Islamabad, Pakistan
2 Directorate of Livestock and Dairy Development Department Khyber Pakhtunkhwa, Veterinary Research Institute Peshawar, Peshawar, Pakistan
3 Department of Pharmacy, Faculty of Biological Science, Quaid-i-Azam University Islamabad, Islamabad, Pakistan
4 College of Veterinary Medicine, University of Minnesota, St. Paul, MN, United States of America
Rouby Sherin Reda Editor
Beni Suef University Faculty of Veterinary Medicine, EGYPT
Competing Interests: The authors have declared that no competing interests exist

* E-mail: drsafibm@yahoo.com (SU); saeedkhan@qau.edu.pk (SUHK)
3 9 2024
2024
19 9 e030783026 12 2023
11 7 2024
https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

This study investigated the virulence potential and antibiotic susceptibility analysis of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups, which are significant cause of food borne diseases. A study collected 800 samples of dairy bovine raw milk through various sources, 500 from milk shops, 200 from dairy farms, 26 from milk collection centers, and 74 from street vendors. Using a standard method, E. coli was detected in 321 out of the 800 samples collected. Out of the 321 E. coli-positive samples isolated, 148 were identified as STEC using selective media, specifically Cefixime Tellurite Sorbitol MacConkey’s Agar (CT-SMA). Out of the 148 positive samples, 40 were confirmed as STEC non-O157 strains using multiplex PCR, indicating a prevalence of 5% (40 out of 800 samples). STEC isolates were subjected to antimicrobial susceptibility testing, and all isolates were resistant to at least one or more antimicrobials tested through the disk diffusion method, revealed high resistance to Amoxicillin 100%, Ceftriaxone 50%, and Penicillin 44.5%, and notably 44% of the strains exhibited Streptomycin resistance, while Enrofloxacin 55%, Florfenicol 50% and Norfloxacin 44%, demonstrated the highest susceptibility. Out of 40 STEC non-O157, twelve were subjected to Multi Locus Sequence Typing (MLST) sequencing through Illumina Inc. MiSeq platform’s next-generation sequencing technology, United States. The genome investigation evidenced the persistence of twelve serotypes H4:O82, H30:O9a, H4:O82, H16:O187, H9:O9, H16:O113, H30:O9, H32:O, H32:O, H32, H32, and H38:O187, linked to the potential infections in humans. Conclusion: STEC isolates showed resistance to multiple antimicrobials, raising concerns for both animal and public health due to widespread use of these drugs in treatment and prevention. The study contributes new insights into monitoring STEC in raw milk, emphasizing the critical role of whole genome sequencing (WGS) for genotyping and sequencing diverse isolates. Still a deficiency in understanding STEC pathogenesis mechanisms, ongoing surveillance is crucial for safeguarding human health and enhancing understanding of STEC genetic characteristics.

The author(s) received no specific funding for this work. Data AvailabilityAll relevant data are within the paper.
Data Availability

All relevant data are within the paper.
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pmc1. Introduction

Milk is an important source of nutrition for humans because it contains a wide range of essential nutrients. Its consumption is deeply rooted in tradition across both urban and rural regions of Pakistan [1]. Although raw milk is nutrient-rich, it is highly perishable and provides an optimal environment for the rapid growth of microorganisms. Consequently, it becomes a potential carrier for the transmission of foodborne illnesses [2]. An impactful zoonotic pathogen worldwide is Shiga toxin-producing Escherichia coli (STEC O157:H7). This Gram-negative bacterium is accountable for inducing severe health complications in those affected, leading to symptoms such as bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura [3, 4]. Escherichia coli commonly inhabits the gastrointestinal tracts of both humans and animals, usually in a non-pathogenic form. It is crucial to recognize, that specific strains of E. coli can pose significant health risks to humans [5]. These pathogenic strains can be classified into various types, encompassing Shiga toxin-producing, enterohemorrhagic (STEC/EHEC), enteropathogenic (EPEC), enteroaggregative (EAEC), and diffusely adherent (DAEC) forms of E. coli [6]. Shiga toxin-producing Escherichia coli (STEC) is distinguished for generating multiple virulence factors, notably stx1, stx2, eae, and ehxA, which hold particular significance due to their cytotoxic effects. These factors contribute to conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Ruminants, comprising cattle, buffalo, sheep, and goats are acknowledged as the primary reservoirs of STEC [7]. It is noteworthy that humans can act as reservoirs, contributing to person-to-person transmission, especially in familial environments, daycare centers, and institutional settings [8]. The primary source of STEC O157:H7 infection in humans is typically contaminated ground beef, and it’s worth mentioning that contaminated food items such as unpasteurized milk and fruits or vegetables contaminated with feces can also be potential sources of infection [9]. Additionally, waterborne transmission of STEC has been documented, particularly after exposure to sewage-contaminated water, whether through swimming or drinking [10]. The occurrence of Shiga toxin-producing E. coli (STEC) in Pakistan has been documented in a variety of food products, with reported prevalence rates ranging from 1.94% to 56.4% [11]. Antibiotic drug resistance poses a significant challenge in global health [12]. The rising incidence of pathogenic multidrug-resistant (MDR) bacteria, notably extended-spectrum beta-lactamase (ESBL) and carbapenem-producing Enterobacteriaceae limits therapeutic options for clinicians and has prompted a renewed focus on older, potentially toxic drugs. [13]. Undoubtedly, the implementation of appropriate hygiene practices across the entire milk production chain, involving individuals such as milk producers, handlers, collectors, transporters, vendors, and consumers, is critical to safeguard the microbial safety of milk. Regrettably, in numerous developing countries, the adherence to hygienic practices is frequently deficient in the handling of milk and its derivatives, making unpasteurized or un-boiled milk potentially unsafe for consumption [14]. A study carried out by the Food and Agriculture Organization (FAO, 2018) disclosed significant annual economic losses across all stages of the dairy industry, spanning from production to consumption. In Pakistan, reports indicate the prevalence of Shiga toxin-producing Escherichia coli (STEC) in milk, ranging from 8% to 20%. [15]. The pathogenic nature of Shiga toxin-producing Escherichia coli (STEC) is complex, necessitating an estimation of virulence beyond serotypes. Additional markers, such as virulence genes and phylogenetic indicators, should be considered. Whole genome sequencing (WGS) offers a comprehensive view of pathogen information, allowing for an assessment of the phylogenetic relationships between strains originating from diverse sources and geographic regions. The rising prevalence of antimicrobial-resistant E. coli poses a significant global public health concern [16]. Transfer of antimicrobial-resistant strains to humans can occur through the consumption of contaminated food. In Khyber Pakhtunkhwa, there is a lack of comprehensive reports on the characterization of Shiga toxin-producing Escherichia coli (STEC) in raw milk and dairy products. To address this gap, we conducted an analysis of isolated E. coli strains obtained from raw milk of dairy bovines from various sources. The study included antibiotic susceptibility testing, serotyping, and multilocus sequence typing (MLST) sequencing analysis. This research aims to provide technical support for investigations into the safety of dairy milk and to monitor the emergence of pathogenic STEC strains in the Khyber Pakhtunkhwa Province, Pakistan

2. Materials and methods

The research investigation took place across multiple institutions, including the Genomic Laboratory of Veterinary Research Institute (VRI), Peshawar (34.0170° N, 71.5699° E); the Laboratory of the Animal Science Institute at the National Agricultural Research Council in Islamabad, Pakistan 33.6995° N, 73.0363° E, and the College of Veterinary Medicine at the University of Minnesota in St. Paul, USA 46.7296° N, 94.6859° W.

2.1 Sample collection

In this study, a total of 800 samples were collected, including, 200 from dairy farms, 26 from milk collection points, 74 from street vendors, and 500 from milk shops across various regions of the Khyber Pakhtunkhwa Province during the period of 2020–2021, details mentioned in the (Fig 1). The analysis of E. coli was conducted using techniques that adhere to the standards recommended by the International Organization for Standardization (ISO) specifically designed for E. coli detection and characterization.

10.1371/journal.pone.0307830.g001 Fig 1 Geographical distribution of raw milk samples collected from dairy bovines in various regions of Khyber Pakhtunkhwa, Pakistan.

2.2 Pre-enrichment and bacterial isolation

In the analysis of raw milk samples, each 10 ml portion was individually mixed with 90 ml in a 100 ml sterile conical flask, and pre-enrichment medium (buffered peptone water—BPW) was added in 1:9 ratio, and processed under a laminar flow hood. The flask was sealed with cotton and wrapped in aluminum foil. Following this, samples were incubated overnight in a shaking incubator at 37°C for duration of 24 h. A loopful of culture sample was streaked onto MacConkey agar plates and left to incubate at 37°C for 24 h, resulting in the development of colonies with a pink color on the plates. Subsequently, the pink colonies from the MacConkey agar plates were sub-cultured on Eosin Methylene Blue (EMB) agar (Oxide, UK), where a characteristic metallic sheen was observed. The bacterial identity was confirmed through a series of tests, including Gram staining and biochemical tests such as Oxidase, Catalase, Indol, Methyl red, Vogues Proskauer (VP), and Citrate (IMVC). For the selective isolation of Shiga toxin-producing Escherichia coli (STEC), colonies were streaked on sorbitol MacConkey agar plates supplemented with 2.0μg/ml of cefotaxime (CT-SMAC). These plates were then incubated at 37°C for 24 h, leading to the growth of white, colorless, and pink colonies mentioned in Table 1 and Fig 1.

10.1371/journal.pone.0307830.t001 Table 1 Cross tabulations of milk source with (E.coli, STEC non-O157:H7,) contaminations n = 800.

Source detail	E.coli	STEC non-O157:H7	Total	
Positive	Not Detected	Positive	Not Detected	
Milk Shops	207	293	30	470	500	
Dairy Farms and Individual Farmer	073	127	08	192	200	
Milk Vendors	028	046	01	073	074	
Milk Collection Centre	013	013	01	025	026	
Total	321	479	40	760	800	
Probability Statistics	p-Value = 0.449	p-Value = 0.305		

2.3 DNA extraction

The 148 samples STEC-positive isolates from raw milk were selected for molecular characterization and confirmation of virulence genes through mPCR. Genomic DNA was extracted from E. coli (STEC) culture in brain heart infusion broth using Genomic DNA Extraction Kit (Invitrogen). Genomic DNA extraction was carried out from a 1ml sample of the overnight bacterial culture following the standard protocol provided by the kit. The specific measurement of DNA concentration in the extracted samples was determined using a Qubit 2.0 Fluorometer. The quality and reliability of the DNA extracted samples were loaded onto a 0.8% agarose gel, and electrophoresis was conducted. This gel electrophoresis process is a common method for assessing the reliability and purity of DNA samples.

2.4 Multiplex Polymerase Chain Reaction (mPCR)

The presence of encoding genes, namely stx1, stx2, eae, and ehxA, was determined through multiplex polymerase chain reaction (mPCR) assays. Genomic DNA, extracted from the samples in question, served as the template for these assays, utilizing primers specified in Table 2. The mPCR reaction mixture, with a total volume of 25μl, included 3μl of template DNA, 2.5μl of 10× PCR Buffer (Bio-Rad), 2μl of dNTP, 0.5μL of rTaq each for the forward and reverse primers, and 17.0 μl of nucleotide-free water [17]. The mPCR conditions involved an initial step at 95°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at various temperatures specified in Table 2 for 40 seconds, and extension at 72°C for 1 minute. The process concluded with a final extension at 72°C for 10 minutes before storage at 4°C. Positive and blank controls included DNA from the STEC O157:H7 strain and nucleotide-free water, respectively. Visualization of the mPCR product was achieved through 1% agarose gel electrophoresis under ultraviolet light.

10.1371/journal.pone.0307830.t002 Table 2 Primers, concentration and annealing temperature used for molecular characterization of STEC O157:H7 (applied biosystems, Waltham, USA).

Genes	PCR conditions	PCR reaction volume	
Stx1, Stx2, eae, ehxA	1cycle	2.5 μl of 10x PCR buffer	
96°C-10 min	0.15mM Mgcl2	
35 cycles	0.1mM of each dNTP	
95°C-45 Sec	0.5 μl of each primer	
60°C-45 Sec	one unit of Taq DNA polymerase	
72°C-45 Sec	3 μl of DNA	
1 Cycle final volume of 25 μl with sterile water 72°C -8 min	
0157(rfb gene)	1cycle 2.5 μl of	2.5 μl of 10x PCR buffer 10x PCR	
96°C-5 min	0.15mM Mgcl2 buffer	
25 cycles	0.1mMofeach dNTP (Thermoscientific,Waltham,USA	
95°C-1min	1.0 μl mM of 0157 rfb gene) primers	
56°C, 58°C—1min	one unit of Taq DNA polymerase	
72°C-1 min	5 μl of DNA	
1 Cycle,72°C -10 min held 4°C forever the final volume of 20 μl with sterile water	

2.5 Antibiotic susceptibility test

The antimicrobial susceptibility of the Shiga toxin-producing Escherichia coli (STEC) isolates was assessed using the disc diffusion method, following the protocols outlined by Kirby-Bauer and the Clinical and Laboratory Standards Institute. Each strain was plated on Muller Hinton agar seeded in three fields by cross streak and incubated at 37°C for 24h. In order to verify the accuracy of this test 40 STEC strains were tested, adjusting to 0.5 of the McFarland [18]. It was then spread on Mueller-Hinton agar plates. After waiting for a few minutes, antibiotic discs were applied aseptically. A total of 12 types of antibiotics are available in the market. including Penicillin (p10 iu) Amoxicillin,(Aml30μg) Sulfamethoxazole (smx 50μg), Amoxicillin & Clavulanic acid,(aug30μg) Gentamicin,(cn10μg) Streptomycin,(s10μg) Oxytetracyclin,(ot30μg) Ceftriaxone (cro30μg), Norfloxacin, nor 10 μg Enrofloxacin, (enr 5μg) Florefenical, (ffc 30μg) and Cefotaxime & Clavulanic acid, (ctl 40μg) were employed in Table 3. The plates were then incubated for 24 h at 37°C, and Measured the inhibition zone of diameter expressed the sensitivity of the bacteria, which defines the bacteria as resistant (≤ 9 mm), moderately sensitive (10–11 mm), or sensitive (≥ 12 mm) The results were interpreted by measuring the inhibition zone diameter and comparing those with the standard chart developed by Clinical Laboratory Standards Institute guidelines [19].

10.1371/journal.pone.0307830.t003 Table 3 Antimicrobial susceptibility of shiga toxin-producing E. coli (STECnon-O157:H7) isolated from raw milk in Khyber Pakhtunkhwa.

(CLSI 2020).

Antibiotics								
Discs	Resistance	%	Intermediate	%	Susceptible	%	
Penicillin	G p 10 iu	8	44	6	33	4	22	
Amoxicillin	Aml 30 μg	18	100	0	0	0	0	
Amoxicillin clavulanic acid	Aug 30 μg	7	38	5	27	6	33	
Sulphathiazole	Smx 50 μg	6	33	8	44	4	33	
Gentamicin	Cn 10 μg	7	38	4	22	7	38	
Streptomycin	S 10 μg	8	44	5	27	5	27	
Oxytetracycline	Ot 30 μg	6	33	8	44	4	22	
Ceftriaxone	Cro 30 μg	9	50	6	33	3	16	
Norfloxacin	Nor 10 μg	6	33	4	22	8	44	
Enrofloxacin	Enr 5 μg	5	27	3	16	10	55	
Florfenicol	Ffc 30 μg	4	22	5	27	9	50	
Cefotaxime& clavulanic acid	Ctl 40 μg	8	44	4	22	6	33	
Chi-Square = 37.01	P-Value = 0.023	
*RXC Method by using EPI-info software

2.6 Whole genome sequencing assembly

The genomic DNA were extracted from 12 STEC strains and were processed for the sequencing using Illumina MiSeq platform’s next-generation sequencing technology, following library preparation procedures provided by Illumina Inc., based at the University of Minnesota Genomic Center in the United States. Raw sequence data were analyzed using FASTAQ software, and subsequently, the reads underwent de novo assembly using the PATRIC (Pathosystems Resource Integration Center) web tool (https://www.patricbrc.org/). A filtration step was applied, excluding reads shorter than 150 base pairs. The resulting contigs were then annotated using Prokka, employing the default e value cutoff (version 1.13). It is important that for previously assembled genomes incorporated into the TORMS analysis, the same parameters were applied.

2.7 In silico serotyping and Multi-locus Sequence Typing (MLST) analysis

The EC Typer tool for Escherichia coli Sero-typing, Version was used to confirm the serotype of isolates as O157 using default parameters O antigen minimum >90% identity coverage and H antigens minimum >90% identity and 60% coverage. Genetic relatedness of raw milk from dairy bovine strains were determined using an in-silico E.coli MLST Finder 2.0 scheme seven housekeeping genes loci adenylate kinase (adk), fumarate hydratase (fumC), DNA gyrase (gyrB), isocitrate/isopropylmalate dehydrogenase (icd), malate dehydrogenase (mdh), purA (adenylosuccinate dehydrogenase), and recA (ATP/GTP binding motif) previously described for E. coli were used in MLST. The E.coli MLST database was used to assign a number to each locus and sequence type (ST) for each unique combination of loci [20]. As indicated in the Table 4.

10.1371/journal.pone.0307830.t004 Table 4 Serotyping status for STEC non-O157:H7 isolates from raw milk of dairy bovine In Khyber-Pakhtunkhwa.

 Samples ID	H antigen	Fim H typing	O antigen	
ST-202	H4	FimH type—E.coli	O82	
ST-203	H30	FimH type: fimH54	O9	
ST-223-A	H4	FimH type: fimH54	O82	
ST-223-B	H4	FimH type: fimH30	O82	
ST-224	H16	FimH type: Unknown or presumptive new variant *	O187	
ST-228	H9	FimH type: fimH54	O9a	
ST 237	H16	FimH type: fimH53	O113	
ST275	H30	FimH type: fimH54	O9	
ST-289	H9	FimH type: fimH54	O9a	
ST-315	H32	FimH type—E.coli	 -	
ST-369	H32	FimH type—E.coli	 -	
ST-621	H38	FimH type: fimH54	O187	

3. Results

A total of (n = 800) raw milk samples were collected from dairy farms (200) milk collection, (74) street vendors (26) and milk shops (500) in different regions of Khyber Pakhtunkhwa, Province in 2020–21 (Fig 1) for PCR detection of Shiga toxin encoding genes (stx). Of the 800 samples (n = 800) tested, 5% (40 samples) were positive for stx1, stx2, eae, and ehxA virulence genes. Details were provided in Table 5 and Fig 2. The isolation rates of STEC strains were 4.0% (8/200) from dairy farms, 6% (30/500) from milk shops, 1.4% (1/74) from street vendors, and 3% (1/26) from milk collection centers. Detection of selected virulence genes (stx1, stx2, eae, ehxA) revealed that more than 5% (40/800) of the isolates carried more than two types of virulence-associated genes, specifically both stx1 and stx2 simultaneously (see Fig 3 and Table 6). In our study, analysis of raw milk isolates revealed that 38% of the isolates carried the stx1 virulence gene, while 25% harbored the stx2 gene (Fig 1). Additionally, the hlyA gene was detected in 19% of the raw milk isolates, Furthermore, all raw milk strains examined in this study demonstrated the presence of the eae gene, accounting for 18% of the isolates (Fig 3). Additionally, all the STEC isolates were further subjected to serotyping via latex agglutination tests, using latex beads coated with specific antibodies STEC O157:H7 from Pro-Lax in the UK. Our data presented in the (Table 3) and result indicated that several antibiotics showed varying levels of effectiveness against the isolates. Enrofloxacin demonstrated the highest effectiveness, with 55% of isolates being susceptible. Similarly, Norfloxacin showed a high susceptibility rate of 50%, followed by Florfenicol at 44.5% and Gentamicin at 38%. In contrast, the isolates displayed low sensitivity to Ceftriaxone, with only 16% showing susceptibility. Amoxicillin on the other hand, exhibited complete resistance across all isolates, with 100% resistance observed. In our study, it was observed that all isolates exhibited resistance to gentamicin, Oxytetracyline, Amoxicillin and Clavulanic acid, and Cefotaxime. Antibiotic susceptibility profiles were comprehensively established for these isolates using twelve commonly used antibiotics from various drug classes (Table 3). Specifically, the isolates showed varying levels of resistance and susceptibility across the antibiotics tested. The results provide insight into the effectiveness of different antibiotic treatments against the studied isolates

10.1371/journal.pone.0307830.g002 Fig 2 Regional wise E.coli status n = 800.

10.1371/journal.pone.0307830.g003 Fig 3 Multiplex polymerase chain reaction (PCR) results for the presence of virulence genes (eae 100bp, Stx1 150bp, Stx2, ehxA 534bp) in Shiga toxin-producing Escherichia coli (STEC) strains isolated from raw bovine milk samples in Khyber Pakhtunkhwa, Pakistan.

The figure includes the following lanes: M Lane shows the 100bp ladder, Lane 1 is the positive control, Lane 2 is the negative control, and Lanes 3–6 represent the test samples.

10.1371/journal.pone.0307830.t005 Table 5 Chi-square and fisher’s exact test (statistics) of E.coli with (STEC non-O157:H7) n = 800.

Escherichia coli (E.coli)	Shiga Toxin producing Escherichia coli (STEC non-O157:H7)	Total	
Positive	Not Detected	
Positive	40	281	321	
Not Detected	00	479	479	
Total	40	760	800	
Chi-square = 62.830	P-Value = 0.000	Fisher’s Exact Test = 0.000	
a. 0 cells (.0%) have expected count less than 5. The minimum expected count is 16.05.

b. Computed only for a 2x2 table

10.1371/journal.pone.0307830.t006 Table 6 Prevalence of STEC O157:H7 virulence genes (Stx1, Stx2, eae, ehxA) from bovine raw milk.

STEC Virulence genes	N (%)	Subtypes	Number of positive samples	
Stx1, eae	4 (10.0)	Not detected	108(72.64%)	
Stx1, stx2, ehxA	5 12.5)	STEC non-O157	40 (5.0%)	
eae	5 (12.5)	
Stx1, ehxA	6 (15.0)	
ehxA	4 (10.0)	
Stx1, eae, ehxA	3 (7.5)	
Stx2, eae, ehxA	2 (5.0)	
Stx1, stx2	6 (15.0)	
Stx2	3(7.5)	
Stx1	2 (5.0)	
Total	40 (5.0%)		148 (18%)	
*Percentage W.R.T Total Number of Cases of 40 and Genes W.R.T STEC non-O157 Sub-Divisions.

Serotypes remain a crucial tool for epidemiological purposes as they allow for the characterization of lineages and the composition of populations. In this particular study, a total of 12 serogroups were identified among the STEC isolates, as shown in the Table 4.

3.1 In Silico sero-typing and multi-locus sequence typing

Forty samples bacterial isolates from raw milk of dairy bovine (n = 40) were checked and the quality of twelve samples DNA concentration found correct and were sequenced. In the in-silico serotyping revealed that all STEC non-O157 isolates possessed the H antigens determinant, and these serogroups, including H4:O82, H30:O9a, H4:O82, H16:O187, H9:O9, H16:O113, H30:O9, H32:O, H32-O, H32, H32 and H38:O187 were found respectively in Table 4, to be associated with the potential to cause infections in humans. MLST analysis revealed six different sequence types (ST). Most raw milk samples isolates belonged to a total of 12 categories of sequence types (ST) were identified within the 40 STEC isolates through the utilization of the MLST methodology (Fig 4). Phylogenetic analysis revealed that the MLST genotype ST 275, ST 224, and ST 223A, 203 exhibited a relationship and displayed a close affinity to epidemic MLST genotypes. Similarly, ST-223B, ST,-202, and ST-369, ST-315 showed a close relationship, among themselves. In contrast, ST-621 and ST237 exhibited distinct characteristics differing from the rest. In spirit, MLST has provided valuable insights into the genetic diversity and relationships among E. coli strains in a complex environment, shedding light on potential novel types and emphasizing the need for continued research in this field.

10.1371/journal.pone.0307830.g004 Fig 4 MLST phylogenetic analysis.

4. Discussion

Shiga Toxin-producing E.coli (STEC) are most infectious diarrheagenic E coli affecting the public health worldwide, STEC infections are also a leading cause of frequent foodborne illnesses [21]. Usually the most threatening STEC are those of O157:H7 serotype commonly result in hemolytic uremic syndrome (HUS), which is a life-threatening condition characterized by hemolytic anemia, and renal failure [22]. This study investigated the molecular characterization of STEC strains by determining the virulence genes and antibiotic resistance profiles. These molecular factors play important roles in determining the pathogenicity of STEC, thus impacting public health. Out of the 800 raw milk samples, 321 (40.1%) were found to be positive for E. coli. following culturing on a specific medium, namely sorbitol MacConkey agar, 148 (18.5%) of the positive samples were confirmed. Subsequently, when subjected to multiplex Polymerase Chain Reaction (mPCR) reaction protocol (Table 2). 40 (5.0%) of the isolates were identified as Shiga toxin-producing E. coli. These findings align with previous studies, such as the one [15], where 8.75% of milk samples from the city of Peshawar were positive for E. coli O157:H7, corresponding to our present results. Additionally, [23] reported an STEC prevalence of 5.7% in raw milk samples from the Apulia region. A study conducted [24]. found a prevalence of E. coli STEC O157:H7 at 10% in raw milk. Moreover, the serotype O157:H7 was identified in 6.21% of raw milk samples collected from cows, goats, and buffalos [25]. In the studied dairy bovine population, non-O157 STEC had a prevalence of 5.0% in raw milk. In contrast, the prevalence of this subgroup was 24.7% in Germany, 18.0% in Egypt, 58.1% in California, and 35.9% in Spain. In concordance to zero prevalence of serogroup O157 in this study, the low values were reported from other countries, zero % in Egypt and 3.8% in Spain. So, the dominance of non-O157 STEC subgroup over O157 is in agreement with [26], who reported the ranges of prevalence of nonO157 and O157 STEC from (0.42 to 74%) and (0.2 to 48.8%), respectively, around the globe. The variations in prevalence values might be due to the differences in epidemiological determinants like stocking density, age, season, spatial distribution, sampling time, strategy, handling and laboratory practices [27]. Another study conducted in Madurai, India, found E. coli in 65% of raw milk samples. The variation in the occurrence of E. coli in milk and milk products across different regions may be attributed to factors such as seasonal variations, farm size, feed type, hygiene practices, farm management, and differences in sampling procedures. The pathogenicity of STEC O157:H7 is linked to the production of Shiga toxin genes; including stx1, stx2, eae, and ehxA, or a combination of these toxins. To confirm the presence of Shiga toxin genes in the isolates, mPCR was carried out on all positive E. coli isolates as shown in the Fig 3. The results showed that 40 out of 148 (5%) isolates were positive for Shiga toxin genes (stx1, stx2, eae, and ehxA). These findings are consistent with [11], who reported 73% with stx1 and 62% with stx2 in raw milk. In addition to stx1 and stx2, the prevalence of ehxA (19%) was also very high, showing a good agreement with previously studies. It is worthy of note that ehxA is generally used as a diagnostic indicator because the presence of ehxA is frequently correlated with the Shiga toxin [28]. In the present study, all eae-positive STEC strains isolated in Khyber Pakhtunkhwa, province was detected to be positive for stx2 in Table 6. The determination of such strains represents a high risk on public health in this region. However, it’s important to note that results from various studies may not always align. For instance, some studies have reported a higher occurrence of stx2 compared to stx1 in bulk milk samples, and the presence of the stx2 gene can vary in different regions, as indicated by a study on STEC O157 in beef cattle in Quetta, Pakistan [29]. The antimicrobial resistance (AMR) of STEC is also a serious problem that the world is now facing. It’s worth noting that antibiotics such as Enrofloxacin (55%), Florfenicol (50%), Gentamicin (38%), and Norfloxacin (44%) demonstrated a notably high level of effectiveness in combating all the isolates. In contrast, the isolates exhibited a considerably low sensitivity to Ceftriaxone 16% and highly resistant Amoxicillin 100%. Additionally, the isolates displayed elevated levels of resistance against Penicillin, Amoxicillin, Clavulanic acid, Cefotaxime, Streptomycin, Oxytetracycline, and Sulfamethoxazole. In agreement with these studies, a low proportion of STEC isolates from the present study was susceptible to those types of antimicrobials mentioned in Table 3. These findings suggest a serious profile of AMR in STEC in food-producing animals. It is worthy of note that all STEC isolates were sensitive to Ciprofloxacin, Norfloxacin and Florfenicol in the present study. This raises concerns about their role as potential reservoirs for multidrug-resistant STEC, which could potentially be transmitted to humans through the consumption of raw milk. This concern has also been reported in Lahore, Pakistan by [30]. The virulence potential of STEC, several factors should be considered, including serotype, stx subtype, virulence genes, phylogroups, and sequence type (ST). These strains belonged to different serotypes with various combinations of H and O antigens, including H4:O82, H9:O9a, H11:O160, H16:O113, H16:O156, H16:O187, H30:O9, H32, H32 and O187:H14 and H32:O mentioned in Table 4. They exhibited the ability to encourage infections in humans, substantiated by facts from the phylogenomic database of enteric bacteria. Certain subtypes of the stx2 gene (such as stx2a, stx2c, and stx2d) have been associated with severe human diseases. In this study, twelve isolates were found to possess these stx2 subtypes, raising concerns about their potential to pose a health threat. Stx2 producing STEC strains, linked to patients with acute diarrhea and Hemolytic Uremic Syndrome (HUS), were also identified in this study, underscoring the clinical significance of the four isolates carrying stx2. In this study, 12 types of STs were determined for the 40 STEC isolates. In particularly, many isolates belonging to different STs possessed the same serogroups (Table 2). These findings are consistent with the findings. Suggesting that STEC isolates with the same serogroups might have genotypical diversity. Various STEC serotypes were identified in the raw milk samples, with O82:H4 and O9a:H9 being the most frequently detected serotypes. In another study conducted in American countries, STEC strains were found in cattle, beef products, and other food items [31]. Additionally, STEC O113:H21 strains were isolated from beef and cattle samples in Chile. Various sequence types (STs) of STEC strains have been isolated globally from both human and animal hosts, causing illnesses. For instance, MLST analysis of ST 203 and ST 223 revealed that H4-082 is associated with severe hemolytic uremic syndrome (HUS) infections. Similarly, non-O157 STEC lineages of H16: O187 have been linked to human infections in South American countries, with isolated cases reported in England. Different STEC strains have been reported in various countries, originating from environmental sources, food, clinical infections, and diverse animals [32]. Remarkably, several STs were identified, including, ST- 202, ST 203, ST 223, ST 224, ST 228, ST 237, ST 275, ST 369, ST 315, and ST 612 The ST prediction in this study identified the presence of the ST224 (O187:H14) clone in raw milk samples from Pakistan. Previous research has also reported the presence of STEC strains in cattle and among children. Additionally, STEC strains have been detected in food samples in South Korea, and they have been a predominant lineage in cattle samples in Sweden, as well as in pigs, cattle, milk, and water samples from dairy farms in China.

5. Conclusions

The non-O157 STEC serogroups with variable virulence characteristics were quite prevalent at the studied raw milk of dairy bovine in Khyber Pakhtunkhwa, Pakistan and because of its zoonotic potential may create public health hazards. It is crucial to monitor and regulate antibiotic usage in both animals and humans to curb the emergence of antibiotic resistance. Moreover, this data holds significance in gaining insights into the health implications associated with raw milk consumption and guiding future interventions to ensure the safety of dairy products in the region. Emphasizing milk pasteurization is recommended, along with the implementation of hygiene practices during milk production. Additionally, adopting appropriate herd management practices, eliminating high-shedders, and considering interventions such as vaccination, dietary adjustments, or the use of probiotics in feed are advisable measures. Finally, detailed studies are essential foe evaluating the Stx1 as a complete diagnostic tool for non-O157 serogroups across the globe.

The authors thanks’ the Veterinary Research Institute Peshawar, Animal Science Institute, National Agriculture Research Council Islamabad and University of Minnesota United States of America for providing research facilities and support for this study.

Abbreviations

STEC Shiga toxin-producing Escherichia coli

mPCR Multiplex polymerase chain reaction

ESBL Extended-spectrum beta-lactamase

MLST Multi Locus Sequence Typing

HUS Hemolytic uremic syndrome

STEC/EHEC Shiga toxin-producing, enterohemorrhagic

EPEC Enteropathogenic

EAggEC Enteroaggregative E.coli

DAEC Diffusely adherent

HC Hemorrhagic colitis

FAO Food and agriculture organization

WGS Whole genome sequencing

BPW Buffered peptone water

EMB Eosin Methylene Blue

VP Vogues Proskuer

NSF Non-sorbitol fermenting

10.1371/journal.pone.0307830.r001
Decision Letter 0
Rouby Sherin Reda Academic Editor
© 2024 Sherin Reda Rouby
2024
Sherin Reda Rouby
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
1 Feb 2024

PONE-D-23-43641Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, PakistanPLOS ONE

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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: Line 33 Carried the virulence genes bla CTXM and bla TEM - These are antibiotic resistance genes

Line 36 - 37 Multi Locus Sequence Typing (MLST) was performed on twelve (12) - why only 12? why not all 40?

Line 55, 58, 61, 64, 70 - , as documented by (delete)

Line 63 - it's important to note

Line 78 - prevalence of STEC in Pakistan has been reported to vary significantly in various food products...

Line 79 - Antimicrobial resistance is seriously challenging the global health (rephrase this sentence)

Line 82 - Enterobacteriaceae (remove italics) rephrase sentence. It's not making sense.

Line 90 - as documented (FAO 2007) Use more recent refs.

Line 91 -Therefore forty isolated of these bacteria from raw milk from... (rephrase this sentence)

Line 109 - United States of America Materials and Methods

Line 119 - Indole

Line 120 - Voges Proskauer (VP)

Line 130 - Genomic DNA was extracted using the boiling method (17)

Line 146 - to perform an in silico molecular characterization of the sequenced strains. Please indicate which strains were sequenced and on which platform. Also, be clear on the bioinformatics tools that where used for analysis.

Line 163-164 - Unnecessary statement. Delete.

Line 167 - Kirby Bauer CLSI 2013. Why would you use guidelines from over 10 years ago? Please use more recent guidelines.

Line 172 - Antibiogram of the ESBL positive E. coli isolates was performed using antimicrobials (rephrase sentence)

Line 173, 178/179 - Put antibiotic disc concentrations

Line 184 - In Fig. 4

Line 184 - The study identified an E. coli prevalence rate of 40.5% among these samples are described in Table 1, and Fig:1 (rephrase this sentence)

Fig 2 - Very poor fig which is poorly annotated.

Fig 3 - poorly annotated figure. Say more about the presented isolates

Fig 4 - Poor figure. Put a key.

Table 2 - rfb gene mentioned for the first time in this table. The table needs fixing. PCR is a molecular technique, reagents are mixed at microlitre quantities not milliliters.

Table 3 - frequency of positive genes not adding up to 40.

Table 4 - Some antibiotic spellings are wrong e.g Sulphamathoxole, Oxytetracyclin, Ceftriexone. Put antibiotic concentrations. Why use Amoxy & AMC?

Line 257 - Define multi-drug resistance.

Line 267 - However, despite this constraint, the discovery of both bla TM and bla CTXM genes parallel is remarkable (rephrase the sentence). Why is the presence of these genes remarkable?

Reviewer #2: I regret to disappoint authors, but due to numerous methodological errors and ambiguities in the experiment described in the manuscript, I do not recommend this manuscript for publication.

- the authors' goal was to isolate and identify E. coli strains producing shiga toxin, i.e. STEC, from raw milk, because these are strains that may be responsible for various serious infections in humans.

I suggest changing the style in which the introduction was written a bit and removing sentence endings such as: as observed by, as highlighted, as discussed by, according to. They are unnecessary and make understanding difficult. Moreover, authors in such cases should refer to the names of the cited authors, not reference numbers

Lines: 99-102: goal description should be simplified; the sentence is too long and incomprehensible

Line 112: the authors performed selective pre-isolation in the presence of 0.1 mg/ml cefotaxime. According to the CLSI standard, the screening confirmatory test for ESBLs in E. coli uses a 100x lower concentration of cefotaxime. Therefore, with such selective multiplication, most E. coli strains will not grow - a serious methodological error that excludes further results obtained by the authors

Line 146: authors write about molecular characterization based on sequencing. No description of the sequencing procedure (methodology, which strains?)

Line 114: how to check turbidity in a cloudy, even diluted, milk sample?

Lines 157-163: no description of MLST procedure (only genes listed)

Antimicrobial susceptibility profiles:

The procedure for assessing drug susceptibility using the disc diffusion method is a standardized procedure, similar to the screening and confirmatory test for ESBL (the authors described a completely different methodology).

- antimicrobials used only in veterinary medicine were used, for which there are no criteria in the M100S standard. For some antibiotics used in human medicine there are no criteria in this standard either. So what criteria were used to assess drug susceptibility?

Lines 211-212: whether classical serotyping or molecular methods were used to determine serotypes and serogroups? The authors mix the two versions in “materials and methods” vs “results”

Lines217-221: please provide the specific STs obtained.

Due to the presented inconsistencies, the discussion and conclusion do not seem credible.

Minor comments:

Please use susceptible instead of sensitive and animicrobials instead of antibiotics.

Lines 93-95: I suggest deleting these sentences, the authors did not perform WGS

Lines 152-155: I suggest deleting it

**********

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Reviewer #1: Yes: Joshua Mbanga

Reviewer #2: Yes: Aneta Nowakiewicz

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10.1371/journal.pone.0307830.r002
Author response to Decision Letter 0
Submission Version1
2 May 2024

Thank you for sending reviewers’ comments on our manuscript on,

ONE-D-23-43641

Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, Pakistan

PLOS ONE “

We have revised our manuscript according to these comments. Answers to each of the comments are given below. I hope that this revised manuscript is now found suitable for publication,

Reviewer 1 Comments highlighted in a (Red color)

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Answer. The manuscript has been completely revised in a proper PLOS one Style. The text presentation has been refined, inconsistencies have been eliminated, unity has been improved, and overall readability has been enhanced.

2. Please provide a complete Data Availability Statement in the submission form,

Answer: Data availability statement submission form.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures

Answer: original uncroped gel results figures has been attached.

4. PLOS requires an ORCID ID for the corresponding

Answer: ORCID iD is enlisted (0000-0002-96225217)

5. Please amend the manuscript submission data (via Edit Submission) to include author "Muhammad Hasnain Riaz".

Answer: author name is included as per your suggestion.

6. We note that Figure 4 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online

Answer: Figure 4 has been changed and new non map images attached..

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)

Answer: needful done as per your suggestion.

Reviewer #1: Line 33 Carried the virulence genes bla CTXM and bla TEM - These are antibiotic resistance genes

Answer: needful done as per your suggestion.

Line 36 - 37 Multi Locus Sequence Typing (MLST) was performed on twelve (12) - why only 12? why not all 40?

Answer: MLST was performed on twelve isolates, for genome analysis. While the funding issue for genomic analysis.

Line 55, 58, 61, 64, 70 - , as documented by (delete)

Answer: as per suggestion needful done.

Line 63 - it's important to note

Line 78 - prevalence of STEC in Pakistan has been reported to vary significantly in various food products..

Answer: needful done.

Line 79 - Antimicrobial resistance is seriously challenging the global health (rephrase this sentence)

Answer: rephrase has been done the sentences,

Line 82 - Enterobacteriaceae (remove italics) rephrase sentence. It's not making sense.

Line 90 - as documented (FAO 2007) Use more recent refs.

Line 91 -Therefore forty isolated of these bacteria from raw milk from... (Rephrase this sentence)

Line 109 - United States of America Materials and Methods

Line 119 - Indole

Line 120 - Voges Proskauer (VP)

Line 130 - Genomic DNA was extracted using the boiling method (17)

Line 146 - to perform an in silico molecular characterization of the sequenced strains. Please indicate which strains were sequenced and on which platform. Also, be clear on the bioinformatics tools that where used for analysis.

Line 163-164 - Unnecessary statement. Delete.

Line 167 - Kirby Bauer CLSI 2013. Why would you use guidelines from over 10 years ago? Please use more recent guidelines.

Line 172 - Antibiogram of the ESBL positive E. coli isolates was performed using antimicrobials (rephrase sentence)

Line 173, 178/179 - Put antibiotic disc concentrations

Line 184 - In Fig. 4

Line 184 - The study identified an E. coli prevalence rate of 40.5% among these samples are described in Table 1, and Fig:1 (rephrase this sentence)

Fig 2 - Very poor fig which is poorly annotated.

Fig 3 - poorly annotated figure. Say more about the presented isolates

Fig 4 - Poor figure. Put a key.

Table 2 - rfb gene mentioned for the first time in this table. The table needs fixing. PCR is a molecular technique, reagents are mixed at microlitre quantities not milliliters.

Table 3 - frequency of positive genes not adding up to 40.

Table 4 - Some antibiotic spellings are wrong e.g Sulphamathoxole, Oxytetracyclin, Ceftriexone. Put antibiotic concentrations. Why use Amoxy & AMC?

Line 257 - Define multi-drug resistance.

Line 267 - However, despite this constraint, the discovery of both bla TM and bla CTXM genes parallel is remarkable (rephrase the sentence). Why is the presence of these genes remarkable?

Answer: Your comments have been duly addressed and highlighted, and appropriately relocated to the material methods and results chapters after thorough revision.

Reviewer 2 Comments highlighted in a (purple color)

I regret to disappoint authors, but due to numerous methodological errors and ambiguities in the experiment described in the manuscript, I do not recommend this manuscript for publication.

- the authors' goal was to isolate and identify E. coli strains producing shiga toxin, i.e. STEC, from raw milk, because these are strains that may be responsible for various serious infections in humans.

I suggest changing the style in which the introduction was written a bit and removing sentence endings such as: as observed by, as highlighted, as discussed by, according to. They are unnecessary and make understanding difficult. Moreover, authors in such cases should refer to the names of the cited authors, not reference numbers

Lines: 99-102: goal description should be simplified; the sentence is too long and incomprehensible

Answer: Both chapters have been revised and repetition has been removed, and strong links among sentences have been developed as per your comments.

Line 112: the authors performed selective pre-isolation in the presence of 0.1 mg/ml cefotaxime. According to the CLSI standard, the screening confirmatory test for ESBLs in E. coli uses a 100x lower concentration of cefotaxime. Therefore, with such selective multiplication, most E. coli strains will not grow - a serious methodological error that excludes further results obtained by the authors.

Line 146: authors write about molecular characterization based on sequencing. No description of the sequencing procedure (methodology, which strains?)

Line 114: how to check turbidity in a cloudy, even diluted, milk sample?

Lines 157-163: no description of MLST procedure (only genes listed)

Antimicrobial susceptibility profiles:

The procedure for assessing drug susceptibility using the disc diffusion method is a standardized procedure, similar to the screening and confirmatory test for ESBL (the authors described a completely different methodology).

- antimicrobials used only in veterinary medicine were used, for which there are no criteria in the M100S standard. For some antibiotics used in human medicine there are no criteria in this standard either. So what criteria were used to assess drug susceptibility?

Lines 211-212: whether classical serotyping or molecular methods were used to determine serotypes and serogroups? The authors mix the two versions in “materials and methods” vs “results”

Lines217-221: please provide the specific STs obtained.

Due to the presented inconsistencies, the discussion and conclusion do not seem credible.

Answer. Changes have been made and followed the suggestion.

Minor comments:

Please use susceptible instead of sensitive and antimicrobials instead of antibiotics.

Lines 93-95: I suggest deleting these sentences, the authors did not perform WGS

Lines 152-155: I suggest deleting it

________________________________________

Answer. Changes has been made according to suggestion

Attachment Submitted filename: Response to Reviewers..docx

10.1371/journal.pone.0307830.r003
Decision Letter 1
Rouby Sherin Reda Academic Editor
© 2024 Sherin Reda Rouby
2024
Sherin Reda Rouby
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
14 Jun 2024

PONE-D-23-43641R1Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, PakistanPLOS ONE

Dear Dr. ullah,

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.

==============================

ACADEMIC EDITOR:Please make an effort to address all comments by the reviewers as they are meant to improve the quality of your work. />==============================

Please submit your revised manuscript by Jul 29 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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We look forward to receiving your revised manuscript.

Kind regards,

Sherin Reda Rouby, PhD

Academic Editor

PLOS ONE

Additional Editor Comments :

Please make an effort to address all comments by the reviewer as they are meant to improve the quality of your work.

[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: (No Response)

**********

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: Partly

**********

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

Reviewer #1: 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: No

**********

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: No

**********

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: Check paper for gramma especially abstract, but the whole paper needs to be checked and corrected.

In the initial manuscript there was mention of the ESBL genes bla CTX-M and bla-TEM but they’ve been removed in this revised version. Why? Was the detection of antibiotic resistance genes not done at all?

There was also mention of extracting DNA using the boiling method which has also been removed. Does this mean extraction was only done using the DNA kit mentioned in the revised version of the manuscript?

Always make an effort to address all comments by the reviewers as they are meant to improve the quality of your work. Wrong spellings e.g antibiotics, poorly annotated diagrams and weak grammar make the work less appealing. There’s need to improve on the technical aspects of the manuscript. The presentation of results needs improvement.

Line 28 Rephrase sentence. A total of 800 bovine raw milk samples from milk shops (500), ...

Line 30 write CT-SMA in full when writing for the first time.

Line 30 the positibve isolates were subjected to

Line 31 in silico

Line 32 rephrase sentence. 158 isolates were checked why are the percentages out of 800?

Line 34 say something about the listed serogroups

Line 37 ceftriaxone (correct spelling). Put percentages in brackets. Put a full stop after penicillin (44.5%).

Line 41 list the identified sequence types

Line 43 ...had the potential ability to transfer antibiotic resistance and virulence genes. This assertion is based on what exactly?

Line 132 the 0.5 MacFarland’s standard not 0.05

Line 138 indole, Voges Proskauer (VP)

Line 140 justify why you used the concentration of antibiotic you used. 0.1 gm of cefotaxime (CT-SMAC). Was it based on previous studies or was developed in this study.

Lines 144, 145 A subset of 40 samples matching to the same sampling years as the dairy bovine raw milk isolates was selected and subjected to sequencing. What do you mean by same sampling years?? Wasn’t selection based on the isolates that were positive in the STEC mPCR?

Lines 156 – 158 17µl Nuclease free water + 3µl DNA + 2.5µl PCR buffer + 1µl primers + 2µl dNTPs + 0.5µl Taq = 26µl in total. Please cross check and correct. Which enzyme (manufacturer, city) was used in the PCR?

Line 168 Put antibiotic disc concentrations

Line 171 and the zones... complete the sentence.

172 The CLSI document is an official document that can be cited independently. This must be done here.

Line 174 What was the selection criterion used to select the 12 isolates. This should be made clear.

Line 178 You need to put the web addresses of the tools you used for analysis e.g PATRIC (http://www.patricbrc.org). However PATRIC is now housed under BV-BRC (https://www.bv-brc.org/). Specify the specific tool/s you used for assembly, did you use SPAdes? Please provide all more detail on the bioinformatics analysis so that others may follow what you did.

Line 199 stx1, stx2, eae, ehxA. genes in table 2 and figure 2. Please correct this sentence

Line 200 and street vendors were and... Please correct this sentence

Line 202 100% (40/800) – this is not 100%

Line 204 All raw milk samples showed the presence of ... All means 100%. You can’t say all and then put a % which lower than 100%. Please correct this.

Lines 210 & 211 are the percentages susceptibility or resistance?

Line 212 ceftriaxone (16%) and were highly resistant to amoxycillin (100%)

Line 214 You’ve already mentioned the high resistance to amoxicillin or you meant to write amoxicillin and clavulanic acid (AMC)

Figure 3 – Poorly annotated. Lane 3 has pcr amplicons yet its said to be the negative control. Lanes 9 and 10 have the DNA ladder and can not be test samples. The figure casts doubts over the mPCR results presented in text.

Figure 4. Say something about the isolates in the legend. Multi locus sequence types typically are denoted by ST not SF.

Table 1 - rfb gene mentioned for the first time in this table.

Table 5 - Some antibiotic spellings are wrong e.g Sulphamathoxole, Oxytetracyclin, Ceftriexone.

**********

7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Joshua Mbanga

**********

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10.1371/journal.pone.0307830.r004
Author response to Decision Letter 1
Submission Version2
8 Jul 2024

Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, Pakistan PLOS ONE manuscript on, ONE-D-23-43641

Thank you for sending reviewers’ comments on our manuscript.

We have revised our manuscript according to these comments. Answers to each of the comments are given below. I hope that this revised manuscript is now found suitable for publication.

S. # Comments Reply

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 (No response) Not Applicable (N/A)

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.

Response to Reviewer: Thank you for the critical review and suggestions, which have been incorporated to enhance the quality of the manuscript to meet the esteemed journal's criteria. The experiments were conducted in various institutes as required, ensuring that all references are included in the manuscript. Adequate sample sizes and appropriate control measures were applied, and the results support the conclusions, making a significant contribution to the research database for further study. The revised version hopefully fulfills all criteria.

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

Reviewer #1: N/A 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.

Response to Reviewer: Thank you for your review and suggestion. We have thoroughly revised the manuscript to adhere to the proper PLOS One style. The text has been refined for better presentation, inconsistencies have been addressed, unity has been improved, and overall readability has been enhanced. Additionally, the Data Availability Statement is now fully presented in the supplementary file, ensuring that all information is readily accessible without restrictions.

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.

Response to Reviewer: Thank you for your critical review and comment. The revised manuscript has been carefully crafted to remain to the standard English format required by the journal. We have addressed language clarity and corrected grammatical errors during revision. We are confident that the manuscript now meets the publication criteria of the journal.

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)

Response to Reviewer: Thank you for your critical review and comment. Regarding the research study, we assure you of its genuineness and the dual publication of its data. All ethical considerations have been meticulously addressed in accordance with the journal's criteria. Additionally, we have fulfilled all necessary requirements.

Reviewer #1: Check paper for gramma especially abstract, but the whole paper needs to be checked and corrected.

In the initial manuscript there was mention of the ESBL genes bla CTX-M and bla-TEM but they’ve been removed in this revised version. Why? Was the detection of antibiotic resistance genes not done at all?

There was also mention of extracting DNA using the boiling method which has also been removed. Does this mean extraction was only done using the DNA kit mentioned in the revised version of the manuscript?

Always make an effort to address all comments by the reviewers as they are meant to improve the quality of your work. Wrong spellings e.g antibiotics, poorly annotated diagrams and weak grammar make the work less appealing. There’s need to improve on the technical aspects of the manuscript. The presentation of results needs improvement.

Response to Reviewer: Thank you very much for the comments made in the manuscripts. I really appreciate your critical feedback. The ESBL genes blaCTX-M and blaTEM were initially mentioned, but due to lack of supportive data, we have removed them from the manuscript. Antibiotic susceptibility testing was performed instead of genotyping the resistant genes.

For isolation of DNA extraction for normal PCR is mostly done through the boiling process. However, for MLST, genomic DNA extraction was carried out using extraction kits. And DNA kit mentioned in the revised version of the manuscript.

We acknowledge the importance of addressing all reviewer comments to enhance the quality of our work. We addressed and corrected the spelling errors, tried to improve diagram annotations, and strengthen the grammar to make the manuscript more appealing. Additionally, we focused on enhancing the technical aspects and improving the presentation of our results.

Reviewer comments

Line 28 Rephrase sentence. A total of 800 bovine raw milk samples from milk shops (500), ...

Line 30 write CT-SMA in full when writing for the first time.

Line 30 the positive isolates were subjected to

Line 31 in silico

Line 32 rephrase sentence. 158 isolates were checked why are the percentages out of 800?

Line 34 say something about the listed serogroups

Line 37 ceftriaxone (correct spelling). Put percentages in brackets. Put a full stop after penicillin (44.5%).

Line 41 list the identified sequence types

Line 43 ...had the potential ability to transfer antibiotic resistance and virulence genes. This assertion is based on what exactly?

Line 132 the 0.5 MacFarland’s standard not 0.05

Line 138 indole, Voges Proskauer (VP)

Line 140 justify why you used the concentration of antibiotic you used. 0.1 gm of cefotaxime (CT-SMAC). Was it based on previous studies or was developed in this study.

Lines 144, 145 A subset of 40 samples matching to the same sampling years as the dairy bovine raw milk isolates was selected and subjected to sequencing. What do you mean by same sampling years?? Wasn’t selection based on the isolates that were positive in the STEC mPCR?

Lines 156 – 158 17µl Nuclease free water + 3µl DNA + 2.5µl PCR buffer + 1µl primers + 2µl dNTPs + 0.5µl Taq = 26µl in total. Please cross check and correct. Which enzyme (manufacturer, city) was used in the PCR?

Line 168 Put antibiotic disc concentrations

Line 171 and the zones... complete the sentence.

172 The CLSI document is an official document that can be cited independently. This must be done here.

Line 174 What was the selection criterion used to select the 12 isolates. This should be made clear.

Line 178 You need to put the web addresses of the tools you used for analysis e.g PATRIC (http://www.patricbrc.org). However PATRIC is now housed under BV-BRC (https://www.bv-brc.org/). Specify the specific tool/s you used for assembly, did you use SPAdes? Please provide all more detail on the bioinformatics analysis so that others may follow what you did.

Line 199 stx1, stx2, eae, ehxA. genes in table 2 and figure 2. Please correct this sentence

Line 200 and street vendors were and... Please correct this sentence

Line 202 100% (40/800) – this is not 100%

Line 204 All raw milk samples showed the presence of ... All means 100%. You can’t say all and then put a % which lower than 100%. Please correct this.

Lines 210 & 211 are the percentages susceptibility or resistance? Line 212 ceftriaxone (16%) and were highly resistant to amoxicillin (100%)

Line 214 You’ve already mentioned the high resistance to amoxicillin or you meant to write amoxicillin and clavulanic acid (AMC)

Figure 3 – Poorly annotated. Lane 3 has pcr amplicons yet its said to be the negative control. Lanes 9 and 10 have the DNA ladder and can not be test samples. The figure casts doubts over the mPCR results presented in text.

Figure 4. Say something about the isolates in the legend. Multi locus sequence types typically are denoted by ST not SF.

Table 1 - rfb gene mentioned for the first time in this table

Table 5 - Some antibiotic spellings are wrong e.g Sulphamathoxole

7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Response to reviewers:

The sentence has been rephrased. Please see line No. 28- 41 edited in red colored texts for ready reference please.

Line 43 .The capacity to potentially transfer antibiotic resistance and virulence genes, examined through comprehensive genome sequencing and analysis.

The sentence from line 132 has been corrected in lines 201-203 and the changes are marked in red for your quick reference.

The sentence from line 138 has been revised and corrected in lines 168-169. The changes are marked in red for your convenience."

The sentences from lines 144-145 have been changed, corrected, and highlighted in red. Please refer to lines 199-203 for the revised text.

The lines 156-158 have been revised. The concentration of PCR buffer and primers used has been corrected and highlighted in red text on lines 210-213 for easy reference.

The antibiotic disc concentrations have been added to line 168 of the manuscript. Please refer to lines 230-232 for the revised text.

Line 171 has been revised with corrections, and the description of the zones of inhibition is now included in the sentence. Please refer to lines 204-214 for details. The CLSI document references for line 172 have been documented. Please see line 234 for details.

Line 174 has been changed, and the criteria for antibiotics include a total of 12 types available in the market. Please refer to lines 231-232 for the text highlighted in red.

Line 178 has been updated with corrections and includes detailed information on next-generation sequencing (NGS) technology using the Illumina MiSeq platform by Illumina Inc., conducted at the University of Minnesota Genomic Center in the United States. Raw sequence data was processed using FASTAQ software, followed by de novo assembly using the PATRIC web tool and annotation with Prokka (version 1.13). Please refer to lines 220-221 for more information.

Line 199 has been revised with corrections. Please refer to lines 240-241 for the text highlighted in red for your reference.

The sentence at line 200 concerning street vendors has been corrected. Please refer to lines 242-244 for highlighted text for your convenience.

The lines 202 to 204 have been changed, sentences have been revised and corrected, and please refer to lines 246-252 for highlighted text for ready reference.

Lines 210 & 211 have been revised and edited. Please refer to lines 259-270 for ready reference

Line 214 have been revised and changed amoxicillin and Clavulanic acid (AMC) see line 266 for ready reference.

, Oxytetracyclines, Ceftriexone. Thank you very much for the comment.

Figure 3 has been updated with properly marked of PCR amplicons. Please see figure no 3.

Figure 4: MLST designated the sample sequence type as ST, but the samples were marked as SF during Samples library preparation and laboratory identification for whole genome sequencing.

Table 1 includes the rfb gene, which was part of our research for identifying STEC O157 strains, but no findings were made. Therefore, its inclusion was necessary.

Table 5 The spelling of antibiotics has been corrected. Please refer to it for references,

Thank you for your option to publish the peer review history of this article. I opt to publish the peer review history of my article. Please proceed accordingly.

Attachment Submitted filename: Rebuttal Response to reviewers.docx

10.1371/journal.pone.0307830.r005
Decision Letter 2
Rouby Sherin Reda Academic Editor
© 2024 Sherin Reda Rouby
2024
Sherin Reda Rouby
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
12 Jul 2024

Molecular Characterization and Antibiotic Susceptibility of Shiga Toxin- Producing Escherichia Coli (STEC) Isolated from Raw Milk of Dairy Bovines in Khyber Pakhtunkhwa, Pakistan

PONE-D-23-43641R2

Dear Dr. ullah,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sherin Reda Rouby, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10.1371/journal.pone.0307830.r006
Acceptance letter
Rouby Sherin Reda Academic Editor
© 2024 Sherin Reda Rouby
2024
Sherin Reda Rouby
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
22 Jul 2024

PONE-D-23-43641R2

PLOS ONE

Dear Dr. ullah,

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

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Sherin Reda Rouby

Academic Editor

PLOS ONE
==== Refs
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17 Sharma VK , Dean-Nystrom EA (2003) Detection of enterohemorrhagic Escherichia coli O157: H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Veterinary microbiology 93 : 247–260. doi: 10.1016/s0378-1135(03)00039-7 12695048
18 McFarland J (1907) The nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. Journal of the American Medical Association 49 : 1176–1178.
19 CLSI.M10S2020.http://www.facm.ucl.ac.be/intranet/CLSI/CLSI-M100S23-susceptibility-testing-2020.
20 Wick LM , Qi W , Lacher DW , Whittam TS (2005) Evolution of genomic content in the stepwise emergence of Escherichia coli O157: H7. Journal of bacteriology 187 : 1783–1791. doi: 10.1128/JB.187.5.1783-1791.2005 15716450
21 Karmali MA (2004) Prospects for preventing serious systemic toxemic complications of Shiga toxin–producing Escherichia coli infections using Shiga toxin receptor analogues. The Journal of infectious diseases 189 : 355–359. doi: 10.1086/381130 14745691
22 Tarr PI , Gordon CA , Chandler WL (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. The lancet 365 : 1073–1086. doi: 10.1016/S0140-6736(05)71144-2 15781103
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