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. 2022 Dec 16;15(12):2870–2876. doi: 10.14202/vetworld.2022.2870-2876

Isolation of multidrug-resistant Escherichia coli and Salmonella spp. from sulfonamide-treated diarrheic calves

Mohammad Amdadul Haque 1,†, Muhammad Tofazzal Hossain 2,†, Md Shafiqul Islam 1, Md Zahorul Islam 1, Purba Islam 1, Sourendra Nath Shaha 3, Mahmudul Hasan Sikder 1, Kazi Rafiq 1,†,✉
PMCID: PMC9880849  PMID: 36718340

Abstract

Background and Aim:

The bovine industry is threatened by one of the most serious and deadly enteric diseases, calf diarrhea, particularly in developing nations like Bangladesh. In this context, bacterial resistance to antimicrobial drugs and its detrimental consequences have become a critical public health issue that is difficult to address globally. This study aimed to isolate and identify Escherichia coli and Salmonella spp. with their antibiogram and antibiotic resistance gene detection from sulfonamide-treated diarrheic calves.

Materials and Methods:

Twelve diarrheic calves suffering from calf diarrhea in a dairy farm were selected and a total of 36 fecal samples were aseptically collected directly from rectum before, during, and at the end of treatment for each calf to determine the total viable count, total E. coli count and total Salmonella count. A polymerase chain reaction was used for the specific detection of E. coli and Salmonella genus targeting fliC and invA genes, respectively. Antibiotic sensitivity test of the isolated E. coli and Salmonella spp. were performed by the disk diffusion method for eight commonly used antibiotics.

Results:

A total of 36 E. coli (100%) and 12 Salmonella spp. (33%) were isolated from the samples and were confirmed by polymerase chain reaction. Total viable count was found to be ranged from 35 × 107 to 99 × 1010 colony-forming unit (CFU)/g fecal sample before starting sulfonamide treatment, 34 × 105 to 25 × 1010 CFU/g during treatment with sulfonamide, and 48 × 103 to 69 × 1010 CFU/g immediately after completion of sulfonamide treatment. Total E. coli count was found to be ranged from 4 × 104 to 36 × 1010 CFU/g, 24 × 104 to 23 × 108 CFU/g, and 13 × 104 to 85 × 1010 CFU/g, whereas total Salmonella count was found to be ranged from 16 × 106 to 18.5 × 1011 CFU/g, 15 × 104 to 44 × 107 CFU/g, and 13.2 × 105 to 21 × 1010 CFU/g fecal sample before starting sulfonamide treatment, during treatment with sulfonamide immediately after completion of sulfonamide treatment, respectively. The in vitro antibiotic sensitivity test showed that all the E. coli and Salmonella spp. isolated from diarrheic calves (100%) contained multidrug-resistant (MDR) phenotypes. Escherichia coli isolates were found 100% resistant to amoxicillin (AMX), cefuroxime, cephalexin (CN), erythromycin (ERY), and tetracycline (TET); whereas 94.4%, 86.1%, and 77.8% isolates were resistant to doxycycline (DOX), moxifloxacin (MOF), and gentamycin (GEN), respectively. In case of Salmonella isolates, all were found 100% resistant to AMX, CN, and ERY; whereas 91.7% of resistance was observed for DOX, MOF, cefuroxime, GEN, and TET. Based on the molecular screening of the antibiotic resistance genes, tetA gene was present in 83.3% of the isolated E. coli and 75% of the isolated Salmonella strains, whereas 83.3% E. coli and 79.2% Salmonella isolates contained blaTEM gene.

Conclusion:

These findings suggest that MDR E. coli and Salmonella spp. might be responsible for calf scouring, which is challenging to treat with antibiotics or sulfonamide drugs alone. Therefore, it is important to check the antibiotic sensitivity pattern to select a suitable antibiotic for the treatment of calf scoring. A suitable antibiotic or combination of an antibiotic and sulfonamide could be effective against E. coli and Salmonella spp. responsible for calf scouring.

Keywords: antimicrobial resistance, calf, diarrhea, Escherichia coli, multidrug-resistant, Salmonella spp

Introduction

Calf diarrhea is one of the most important devastating enteric problems that threaten the bovine industry worldwide [1], with high morbidity and mortality rates, especially in a developing country like Bangladesh [2]. In Bangladesh, calf diarrhea remains the most frequently recorded clinical concern in livestock sector [3]. Bacteria (Salmonella spp., Escherichia coli, and Clostridium perfringes), protozoa (Cryptosporidium parvum), and viruses (coronavirus and rotavirus) may cause diarrhea in calves [4–7] alone or in combination with other associated pathogens [8]. Among these agents, E. coli and Salmonella spp. are the most economically important pathogens [9] and are frequently associated with calf diarrhea in Bangladesh [2]. To treat bacterial diarrhea in calves, a course of antimicrobial therapy is required. However, antimicrobials are used indiscriminately and in low doses for preventive and curative purposes worldwide in calf feed to prevent the major economic loss caused by the bacteria [10]. Sulfonamide has been used widely to treat bacterial and protozoal infections over several decades. In addition, sulfonamides are commonly used alone or in combination with trimethoprim or with other antibiotics for both prophylactic and treatment of calf diarrhea [11]. Although sulfonamides are highly effective against calf diarrhea caused by both E. coli and Salmonella spp. [12]; however, persistent and indiscriminate use of antimicrobials, incomplete course, and lack of maintenance of withdrawal period may lead to the development of a new generation of virulent and resistant bacterial strains that may reduce its efficacy or effectiveness. In this regard, field veterinarian from different parts of Bangladesh is claiming the ineffectiveness of sulfonamide therapy in calf diarrhea. Antimicrobial drug resistance to bacteria and its adverse consequence has become a serious public health concern worldwide [13]. Antimicrobial resistance (AMR) has been frequently observed in Salmonella spp. and E. coli species, especially in pre-weaned dairy calves [4]. In these regards, several studies have been done for the isolation, identification, antimicrobial sensitivity testing, and characterization of the resistant genes from both E. coli and Salmonella spp. in home and abroad [14–16]. However, to the best of our knowledge, there are no data available regarding the isolation and identification of E. coli and Salmonella spp. with their antimicrobial sensitivity pattern from sulfonamide-treated diarrheic calves time-dependently in Bangladesh.

Therefore, this study was carried out to isolate E. coli and Salmonella spp. with their antibiotic sensitivity pattern and antibiotic resistance genes during the course of sulfonamide treatment in diarrheic calves. Our present study findings highlighted the detection of multidrug-resistant (MDR) E. coli and Salmonella spp. from sulfonamide-treated diarrheic calves, which is difficult to treat clinically with sulfonamide or antibiotic singly.

Materials and Methods

Ethical approval

All experimental procedures were performed according to the guidelines for the care and use of animals as described by Animal Welfare and Experimentation Ethics Committee, Bangladesh Agricultural University, Mymensingh-2202 (Approval number: AWEEC/BAU/2018[11]).

Study period and location

The study was conducted from October 2018 to March 2019 in collaboration with the Department of Pharmacology, and Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh.

Collection of samples

Twelve diarrheic calves (1–3 months of age) suffering from calf scours (calf diarrhea) in a dairy farm located at Trishal Upazila, Mymensingh district, Bangladesh were selected and a total of 36 fecal samples were aseptically collected at three different time points directly from rectum basis on their previous history of treatment failure against calf diarrhea treated with sulfonamide, conventional antibiotics, or their combinations. The calves were divided into three groups (C, T and TC), where “C” represents samples (C1–C12) collected from diarrheic calves before treatment with sulfonamide, “T” represents samples (T1–T12) collected during the treatment with sulfonamide and “TC” for samples collected immediately after completion of sulfonamide treatment. The samples were transferred to sterile polythene zip-lock bags after collection and brought to the bacteriological laboratory, Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, in a transport box containing ice.

Enumeration of bacterial load

One gram of each feces sample was used to determine the total viable count (TVC), total E. coli count (TEC), and total Salmonella count (TSC) according to the previously published methods [17, 18]. Briefly, a total of 900 μL of phosphate buffer solution was taken in eight Eppendorf tubes, and 100 μL suspension was used to prepare ten serial-fold dilution of each content. Then, 10 μL from each dilution was dropped on plate count agar (HiMedia, India) for TVC, on Eosin-Methylene-Blue (EMB) agar (HiMedia) for TEC, and on Salmonella-Shigella (SS) agar (HiMedia) for TSC and were overnight incubated in a bacteriological incubator at 37°C. Colonies for suitable dilution were counted, and TVC, TEC, and TSC were calculated.

Isolation and identification of E. coli and Salmonella spp.

All collected feces samples were enriched in nutrient broth followed by overnight incubation at 37°C. The enriched culture of each sample was then streaked onto EMB and SS agar media for the isolation of E. coli and Salmonella spp., respectively. A suspected single colony was further streaked onto same media to obtain pure cultures [19]. In addition, Gram’s staining was also performed for morphological identification of E. coli and Salmonella spp. isolated from fecal samples [19, 20].

Molecular detection

Primers and polymerase chain reaction (PCR) conditions used for the specific detection of E. coli and Salmonella genus targeting 16S rRNA and invA genes, respectively, are presented in Table-1 [20–22]. For PCR, genomic DNA was extracted from E. coli and Salmonella spp. by simple boiling method as described previously by Hossain et al. [23]. Briefly, a pure colony of each isolate was inoculated into the broth. After overnight incubation, 1 mL of cultural broth was centrifuged at 10,000 rpm for 3 min. The supernatant was discarded and suspended with 100 μL distilled water, boiling for 20 min followed by cold shock for about 7 min and then centrifuged at 10,000 rpm for 10 min. Finally, supernatant was collected, stored and used as DNA template for PCR. The PCR was performed in an applied Biosystem Thermocycler (Thermo Fisher Scientific, USA) in a total volume of 25 μL reaction mixture with 12.5 μL master mixture 2× (Promega, USA), 3 μL (50 ng) genomic DNA, 1 μL of each primer, and 7.5 μL nuclease-free water. Amplified products were analyzed by electrophoresis in 1.5% of agarose gel, stained in ethidium bromide, and finally visualized under an ultraviolet transilluminator (Biometra, Germany). The size of PCR amplicons was assessed using a 100 bp DNA ladder (Promega).

Table-1.

Primer sequences and PCR conditions.

Target gene Primer sequences Amplified segment (bp) Primary denaturation Amplification (30–34 cycles) Final extension Reference
Secondary denaturation Annealing Extension
16SrRNA for E. coli F: CCCCCTGGACGAA GACTGAC
R: ACCGCTGGCAACA AAGGATA		 401 95°C 5 min 94°C 30 s 57°C 90 s 72°C 90 s 72°C 10 min [21]
invA for Salmonella F: ATCAGTACCAGTC GTCTTATCTTGAT
R: TCTGTTTACCGGG CATACCAT		 211 94°C 5 min 94°C 30 s 52°C 1 min 72°C 45 s 72°C 5 min [20]
blaTEM F: CATTTCCGTGTCG CCCTTAT
R: TCCATAGTTGCCT GACTCCC		 793 95°C 5 min 95°C 1 min 52°C 1 min 72°C 1 min 72°C 7 min [22]
tetA F: GGTTCACTCGAAC GACGTCA
R: CTGTCCGACAAGT TGCATGA		 577 95°C 5 min 95°C 1 min 52°C 1 min 72°C 1 min 72°C 10 min [20]
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PCR=Polymerase chain reaction, E. coli=Escherichia coli

Antibiotic sensitivity test

Antibiotic sensitivity test of the isolated E. coli and Salmonella spp. was performed by disk diffusion method [24]. Freshly grown isolates having a concentration equivalent to 0.5 McFarland standards were spread on Mueller-Hinton agar media (HiMedia) using a sterile cotton swab and eight commonly used antibiotics of HiMedia, namely, amoxicillin (AMX, 30 μg/disc), gentamycin (GEN, 10 μg/disc), tetracycline (TET, 30 μg/disc), erythromycin (ERY, 15 μg/disc), doxycycline (DOX, 30 μg/disc), moxifloxacin (MOF, 5 μg/disc), cephalexin (CN, 30 μg/disc), and cefixime (5 μg/disc) were placed on the media. All results of antibiotic susceptibility for E. coli and Salmonella spp. were interpreted according to the guidelines provided by Clinical and Laboratory Standards Institute [25].

Molecular detection of TET and beta-lactams resistant genes

The presence of TET-resistant tetA and beta-lactam-resistant blaTEM genes in the isolated E. coli and Salmonella spp. was screened by PCR using the mixture conditions as described by Tawyabur et al. [20] and Walker et al. [22], respectively. Primers with PCR conditions used for the specific detection of tetA and blaTEM genes are presented in Table-1.

Statistical analysis

All the collected data were analyzed with the help of GraphPad Prism 6 (2365 Northside Dr. Suite 560. San Diego, CA 92108). The mean differences between before, during, and after the treatments were determined by a one-way analysis of variance followed by Bonfferoni post hoc test [26].

Results and Discussion

Enumeration of TVC, TEC, and TSC from diarrheic calves

The total viable count was found to be ranged from 35 × 107 colony-forming unit (CFU)/g to 99 × 1010 CFU/g fecal sample before starting sulfonamide treatment, 34 × 105 CFU/g to 25 × 1010 CFU/g during the treatment with sulfonamide, and 48 × 103 CFU/g to 69 × 1010 CFU/g immediately after completion of sulfonamide treatment. The lowest and highest TVC was found in calf of sample C-9 and C-2, sample T-12 and T-3, and sample TC-11 to TC-9, respectively (Table-2). Total E. coli count was found 4 × 104 CFU/g to 36 × 1010 CFU/g, 24 × 104 CFU/g to 23 × 108 CFU/g, and 13 × 104 CFU/g to 85 × 1010 CFU/g in samples before starting sulfonamide treatment, during treatment with sulfonamide, and immediately after completion of sulfonamide treatment, respectively (Table-2). total Salmonella count was also found 16 × 106 CFU/g to 18.5 × 1011 CFU/g, 15 × 104 CFU/g to 44 × 107CFU/g, and 13.2 × 105 CFU/g to 21 × 1010 CFU/g in samples before starting sulfonamide treatment, during the treatment with sulfonamide, and immediately after completion of sulfonamide treatment, respectively (Table-2). Variation in results of TVC, TEC, and TSC indicates that sulfonamide is not always effective for diarrheic calves. In this regard, Klaus et al. [27] reported that sulfonamides used for the treatment of neonatal calves with diarrhea were effective in their clinical improvement, but systemic therapy with sulfonamide plus antibiotics provided better performance, with better weight gain and body development.

Table-2.

Results of bacterial load in feces collected from diarrheic calf at three different time points (before starting, during, and immediately after completion of sulfonamide treatment).

Calf ID Bacterial load (CFU/g)
TVC TEC TSC
C: Before T: During TC: After C: Before T: During TC: After C: Before T: During TC: After
1 60 × 1010 14 × 108 26 × 106 42 × 106 41 × 107 16 × 105 16 × 106 44 × 107 13.2 × 105
2 99 × 1010 21 × 106 11 × 106 22 × 105 22 × 105 22 × 105 - - -
3 81 × 109 25 × 1010 33 × 108 22 × 107 22 × 107 17 × 108 21 × 107 18 × 107 82 × 106
4 41 × 109 29 × 109 36 × 1010 35 × 106 35 × 106 22 × 107 - - -
5 85 × 108 25 × 106 18 × 108 10 × 105 10 × 105 16 × 107 - - -
6 20 × 109 27 × 109 53 × 109 4 × 104 24 × 105 13 × 104 - - -
7 38 × 1010 72 × 109 17 × 1010 22 × 1010 24 × 104 21 × 1010 18.5 × 1011 15 × 104 21 × 1010
8 13.9 × 1010 22 × 109 72 × 109 61 × 109 70 × 104 37 × 109 45 × 1010 20 × 104 78 × 108
9 35 × 107 12 × 108 69 × 1010 8 × 108 5 × 106 57 × 1010 - - -
10 39 × 1010 25 × 108 29 × 106 36 × 1010 10 × 108 11 × 105 - - -
11 20 × 108 33 × 109 48 × 103 14 × 108 23 × 108 33 × 1010 - - -
12 22 × 1010 34 × 105 11.3 × 1011 18 × 107 7 × 105 85 × 1010 - - -
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TVC=Total viable cell count, TEC=Total E. coli count, TSC=Total salmonella count, CFU=Colony-forming unit

Isolation and identification of E. coli and Salmonella spp. from diarrheic calves

Several enteric pathogens are responsible for causing neonatal diarrhea [27]. In this study, 36 E. coli (100%) and 12 Salmonella spp. (33%) were isolated and detected in 36 collected feces samples regardless of the collecting time. Moreover, 12 Salmonella spp. were isolated from 36 fecal samples collected at three different times (before, during, and after) of treatment with sulfonamide and identified by cultural and staining properties followed by PCR for confirmation (Figures-1 and 2). Isolation rate of E. coli from diarrheic calves in this study has a similarity with the findings of Gupta et al. [28] and Diwakar et al. [6]. Dark blue-black colonies of E. coli with metallic green sheen were found on EMB agar media, and raised, pinhead, round, or circular, black-centered colonies of Salmonella spp. were found on SS agar media. Gram-negative, pink-colored, single, or paired short plump rod-shaped appearance was observed in Gram’s staining both for suspected E. coli and Salmonella. All the culture-positive E. coli and Salmonella spp. were confirmed by PCR and positive bands appeared at 401 bp and 211 bp for E. coli and Salmonella spp., respectively (Figures-1 and 2).

Figure-1.

Figure-1

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Polymerase chain reaction assay for the amplification of species-specific 16S rRNA (401 bp) gene from Escherichia coli. Lanes 1 and 16: 100 bp ladder, L14: positive control, L15: negative control, and Lanes 2–13: E. coli isolates from a diarrheic calf.

Figure-2.

Figure-2

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Polymerase chain reaction assay for the amplification of genus-specific invA (2101 bp) gene from Salmonella spp. Lane L: 100 bp ladder, Lanes 1–9: Salmonella isolates from a diarrheic calf.

Occurrence of MDR E. coli and Salmonella spp. in diarrheic calves

The results of the antibiotic sensitivity test showed that all the E. coli and Salmonella spp. isolated from diarrheic calves (100%) showed MDR pattern (Table-3). It was found that 100% E. coli isolates were resistant to AMX, cefuroxime, CN, ERY, and TET; whereas 94.4%, 86.1%, and 77.8% isolates were resistant to DOX, MOF, and GEN, respectively (Table-3). In case of Salmonella isolates, all were found 100% resistant to AMX, CN, and ERY, whereas 91.7% of resistance was observed for DOX, MOF, cefuroxime, GEN, and TET (Table-3). Based on previously published evidence for the oral administration of these antimicrobial agents, Constable [29] recommended only AMX for the treatment of calf diarrhea. However, in this present study, all the isolates were found resistant to AMX that means it was not effective for these sick calves. Ansari et al. [14] also reported similar type of findings where 100% resistance was also observed against AMX. Gupta et al. [28] found that 83.33% E. coli isolates in their study were MDR, whereas all the E. coli and Salmonella spp. isolates of this study were MDR. Gentamicin was found to be relatively sensitive to the isolates compared to other antibiotics used in this study. Diwakar et al. [6] reported that GEN was the most effective antibiotic in case of calf diarrhea and highly sensitive for E. coli, Shigella, Edwardsiella, Salmonella, and Klebsiella as well as Proteus isolates recovered from cases of calf diarrhea. The presence of MDR E. coli and Salmonella spp. is documented as important public health hazards worldwide. Consequently, hospital costs for the treatment of both humans and livestock become expensive and would definitely prolong treatment duration time.

Table-3.

Results of antimicrobial susceptibility testing for multidrug resistance of E. coli and Salmonella spp. isolated from diarrheic calves.

Number of isolates (n = 48)
Antibiotics used E. coli (n = 36) Salmonella spp. (n = 12) Overall resistance (%)
Amoxicillin 36 12 48 (100)
Cefuroxime 36 11 47 (97.9)
Cephalexin 36 12 48 (100)
Doxycycline 34 11 45 (93.8)
Erythromycin 36 12 48 (100)
Gentamycin 28 11 29 (60.4)
Moxifloxacin 31 11 42 (87.5)
Tetracycline 36 11 47 (97.9)
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E. coli=Escherichia coli

Detection of tetA and blaTEM genes in the isolates

Based on the molecular screening of the antibiotic resistance genes, tetA gene was present in 83.3% of the isolated E. coli and 75% of the isolated Salmonella strains (Figure-3), whereas 83.3% E. coli and 79.2% Salmonella isolates contained blaTEM gene (Figure-4). The finding on tetA gene in this present study is compatible with the results of Hafez [30] and Liao et al. [31]; the active efflux is still the primary mechanism underlying E. coli resistance to TET. The findings on blaTEM also have similarities with the results of Hafez [30] and Rahman et al. [32].

Figure-3.

Figure-3

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Amplification of tetA (577 bp) gene in isolated Escherichia coli and Salmonella spp. Lane 10: 100-bp DNA ladder, Lanes 1–9: Amplified product of DNA sample of Escherichia coli, and Lane 11–19: Amplified product of DNA sample of Salmonella spp.

Figure-4.

Figure-4

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Amplification of blaTEM (793 bp) gene in isolated Escherichia coli and Salmonella spp. Lane 1: 100-bp DNA ladder, Lanes 2–5: Amplified product of DNA sample of Escherichia coli, and Lanes 6–10: Amplified product of DNA sample of Salmonella spp.

Therefore, it is important to check the antibiotic sensitivity pattern to select a suitable antibiotic for the treatment of calf scoring. A suitable antibiotic or combination of an antibiotic and sulfonamide could be effective against E. coli and Salmonella spp. responsible for calf scouring. The anticipated data suggested the judicial use of antimicrobials, measurement to preserve antimicrobials’ effectiveness and suitable antimicrobials treatment strategies are necessary to control calf scouring which will definitely help to prevent antibiotic resistance.

Limitations of the study

This study has several limitations, such as the sampling area that was limited to a dairy farm. Further details study with larger samples size from various dairy farms in Bangladesh is needed. Details of further phenotypic and genotypic analysis in a wider range with 16S rRNA sequence profiling of these isolates would definitely help the scientists in this field to combat AMR as well as to stop the spreading of MDR foodborne pathogens to humans.

Conclusion

This study findings indicate a high frequency of AMR among E. coli and Salmonella spp. isolated from sulfonamide-treated diarrheic calves. Escherichia coli and Salmonella spp. are the important causes of calf diarrhea which cannot be managed by the use of sulfonamide drugs or antibiotics alone. For the quick recovery of the diarrheal calves, sulfonamide drugs in combination with antibiotics such as GEN may be beneficial. The results of this study will undoubtedly assist veterinarians in choosing the best treatment strategies against calf diarrhea that will help reduce MDR bacteria and fight against AMR.

Authors’ Contributions

MAH and MTH: Performed all the experiments and prepared the draft of the manuscript; KR and MTH: Conceptualization, designed and supervised the research, revised, and finalized the draft of the manuscript; KR, MSI, MZI, PI, and MHS: Did the statistical analysis and prepared the graphs. MAH, SNS, and MTH: Revised the manuscript and prepared the graphs and tables. All authors have read and approved the final manuscript.

Acknowledgments

The authors acknowledge the Project Based Research Grant (PBRG) support from National Agricultural Technology Project-2 (NATP-2), Bangladesh Agricultural Research Council (BARC), Dhaka, Bangladesh, to Professor Dr. Kazi Rafiqul Islam (Project ID-138). The authors are grateful to the Dairy farm authority for allowing us to do the experiment and sample collection.

Competing Interests

The authors declare that they have no competing interests.

Publisher’s Note

Veterinary World remains neutral with regard to jurisdictional claims in published institutional affiliation.

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