Isolation, Identification and Antibacterial Resistance Spectrum of Bacterial Pathogens of Respiratory Tract Infection in a Large-Scale Cattle Farm

Yunxia Guo1, Xia Zhou1*, Xin Huang2 and Fagang Zhong2

1College of Animal Science and Technology, Shihezi University, Shihezi 832003, China.

2State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, China.

ABSTRACT

The current study aimed to report the bacterial pathogens and their antibacterial resistance spectrum causing bovine respiratory tract infections in a large-scale cattle farm. Nasopharyngeal swabs were collected from animals (n=122) having clinical manifestations of respiratory tract diseases. Standard culture procedure followed by the Kirby-Bauer disc diffusion assay was applied to calculate the antimicrobial resistance profile of bacterial isolates. The survey results showed that bronchitis and bronchopneumonia were commonly occurring respiratory diseases in cattle. The highest incidence of bacterial organism Staphylococcus aureus (83.6%), followed by Streptococcus pneumoniae (80.3%) and Klebsiella pneumoniae (77.0%) were recorded in the respiratory tract samples. Pasteurella multocida, Bacillus obstructivus and Mycoplasma alkalescens exhibited 100% resistance against penicillin, while Bacillus subtili and M. alkalescens showed 100% resistance against tetracycline. Mycoplasma dispar, B. subtilis, M. alkalescens, B. obstructivus, and Stah. aureus against cefoxitin; and M. alkalescens, and Microccus luteus against cefoperazone exhibited ≥90% resistance. Overall, the majority of bacterial isolates exhibited ≥70% resistance against many antimicrobials. The antimicrobial spectrum profiles whistle an alarming situation for the regulatory bodies to cut the non-judicial use of antimicrobial agents.

Article Information

Received 11 March 2023

Revised 25 April 2023

Accepted 12 May 2023

Available online 21 July 2023

(early access)

Published 01 December 2023

Authors’ Contribution

YG collected the experimental data. XZ conceived the research project. XH and FZ assisted in data analysis, as well as in writing of manuscript.

Key words

Respiratory tract infections, Antimicrobial, Susceptibility, Bovine, Pneumonia

DOI: https://dx.doi.org/10.17582/journal.pjz/20230311140332

* Corresponding author: [email protected]

0030-9923/2024/0001-0033 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

China is contributing about 3.8% of world raw dairy milk production, with an estimated volume of 32 million tons during the year 2019. Dairy cattle are facing many infectious pathogens associated with numerous body systems like respiratory, gastrointestinal, urogenital etc. (Silva and Bittar, 2019). Respiratory tract infections account for 6% of total global infections (Ghimire et al., 2022). An upsurge in respiratory diseases has been reported in winter; while other contributing factors include unhygienic bedding, environmental pollution, malnutrition and managemental issues (Kumar et al., 2014).

A number of infectious agents were recognized as etiological agents of respiratory diseases, including bacteria, virus, yeast, and protozoans. Bacterial infections are frequent as compared to other infections; however, they affect a smaller group of the population (Carvajal and Perez, 2020). It has been estimated that normal microbiomes of respiratory tract like Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae etc., could be involved in respiratory tract infections (Prat and Lacoma, 2016). Mannhemia haemolytica, Pasteurella multocida, and Histophilus somni were isolated as common pathogens of bovine respiratory tract infections (Confer, 2009). Though, these pathogens are the commensals of nostril and nasopharynx (upper respiratory tract) in healthy cattle, and became opportunistic when host defenses are compromised (Timsit et al., 2013). Mycoplasma dispar, Mycoplasma bovis and Mycoplasma bovirhinis were isolated from sick cattle as well (Friis, 1980). Co-infection of many of these bacterial pathogens along with potential viruses may contribute to bovine respiratory disease (BRD), one of the most devastating diseases of the cattle industry globally (Gaudino et al., 2022).

Since the beginning of the 21st century, the problem of antimicrobial resistance is becoming alarming day-by-day. Studies have demonstrated the association between antibiotic use and prevalence of resistance in microbial organisms (Catry et al., 2016; Donaldson et al., 2006). Data from food-producing animal farms have demonstrated that many farmers use a deviated dose of antimicrobials than those labeled on the leaflet (Timmerman et al., 2006), which potentially convert microbiota into bugs (Dewulf et al., 2007).

Many variations in antimicrobial resistance profiles of Enterobacteriaceae, Pasteurellaceae and other pathogenic bacteria have been observed between different bovine herds (Tikofsky et al., 2003; Catry et al., 2005; Bokma et al., 2020; Haley et al., 2020). It potentially reflects the antimicrobial use in any particular area, and may alarm the various stakeholders of clinical relevance of antibiotic resistance. The purpose of the current investigation was to isolate, identify and explore the antibacterial resistance spectrum of bacterial isolates of bovine respiratory tract pathogens in a large-scale cattle farm.

MATERIALS AND METHODS

Study approval

The study layout was discussed with the farm manager and formal approval was obtained before the start of sampling collection. The samples were collected with the help of on-farm veterinarians and disease diagnosis was also done by a panel discussion of all veterinarians. The institutional approval was obtained from the Animal Ethics Committee of Shihezi University (Approval No. A2022-14403).

Study design and sampling

The study was carried out in the fall of 2022 (November-December) at a large-scale cattle farm that was owned by a large commercial dairy company in China. The farm has Holstein dairy cattle and is located in Xinjiang Uygur Autonomous Region (XUAR) in Northwest China. The farm has fully managed the conditions of intensive dairy farming and all animals were fed silage rather than left to graze. Immature animals (neonatal calves, and one-month-old calves) have separate sections with special care, management and feeding protocols.

Nasopharyngeal swab samples were collected from animals (n=122) suffering from respiratory tract diseases with numerous clinical signs like coughing, sneezing, fever above 40 ºC, difficulty breathing, etc. The animals were restrained properly in standing position and 90% alcohol was applied to nostrils as a disinfectant before sample collection. Sterile transport swabs (Transystem, Copan, Brescia, Italy) were introduced medioventrally in the nasal cavity and after rotating several times were taken out and placed in a transport medium. Within 12h samples were transported to the laboratory and processed for bacteriological investigation.

Bacterial growth

Swabs were inoculated on various general and selective culture media (Oxoid, Ltd., UK) like blood agar, nutrient agar, MacConkey agar, Brilliant Green agar, de-Man-Rogosa Sharpe agar, Eosin Methylene Blue agar and pleuropneumonia-like organism agar, etc. After inoculation, the Petri dishes were incubated aerobically at 37±1ºC for 24-72 hours. For slow-growing organisms like Mycoplasma or Mycobacterium, the incubation was maintained up to 7 days (Van Driessche et al., 2017; Kabir et al., 2022). The identification of bacterial isolates was done according to Quinn et al. (1992). All analyses were carried out in triplicates and pure cultures were stored for antimicrobial susceptibility testing.

Antibiotic susceptibility testing

The Kirby-Bauer disc diffusion assay was applied to calculate the antibiotic susceptibility patterns of bacterial isolates following the guideline of the Clinical and Laboratory Standard Institute (CLSI, 2021). A single loopful of each pure culture was suspended in Mueller Hinton Broth (Oxoid, Ltd., UK) and cultured on Mueller Hinton agar plates after standardizing to 0.5 McFarland standards. The plates were incubated at 37±1ºC for 24h then zones of inhibition were measured. The breakpoints of CLSI for the tested antimicrobials were used to calculate the susceptibility patterns of bacterial isolates.

Statistical analysis

The data was computed in Excel (Microsoft Inc., USA) Spread Sheets. The prevalence of bacterial organisms in respiratory tract samples and their resistance spectrum were calculated in percentages.

RESULTS and Discussion

In beef and dairy cattle, respiratory diseases result in massive economic losses that demand deep insight into etiological agents like stressors (e.g., transportation, weaning, dietary changes, etc), the interaction between the host and microbiome, intrinsic immunity as well as chronic sub-clinical inflammation in airways that may lead to the occurrence of respiratory diseases. Next-generation sequencing data have shown that there is a great variation in the upper and lower respiratory tract microbiome that potentially convert into pathogens due to stressors (Chai et al., 2022). The genera associated with common respiratory tract infections like Mycoplasma, Pasteurella and Mannheimia were observed in the nostrils of healthy cattle (Nicola et al., 2017), while Pasteurella, Mycoplasma, Histophilus, and Mannheimia were reported in the nasopharyngeal samples of both BRD-affected and healthy cattle (Zeineldin et al., 2017).

The incidence of Staph. aureus was highest (83.6%) in the respiratory tract samples, followed by Strep. pneumoniae (80.3%), Klebsiella pneumoniae (77.0%). Other bacteria that were found in the respiratory tract were Mycoplasma bovis (39.344), Enterococcus faecalis (31.148), Klebsiella pneumoniae (77.049), Pasteurella multocida (49.180), Bacteroides pyogenes (36.066), Enterococcus faecium (45.902), Clostridium perfringens (60.656), Mycoplasma dispar (26.230), Bacillus subtilis (62.295), Pseudomonas taetrolens (42.623), Mycoplasma alkalescens (29.508), Bacillus obstructivus (24.590), Strep. pneumoniae (80.328), Staph. aureus (83.607), Microccus luteus (60.656). While Mannheimia haemolytica (19.6%), followed by H. somni (14.7%) were recorded with the least incidences in respiratory tract infection samples of cattle. H. somni and P. multocida are common pathogens of bovine respiratory tract infections and their incidence is usually expected less than M. haemolytica (Anholt et al., 2017; Welsh et al., 2004). Bacillus and Acinetobacter are two bacterial genera that are regarded as microbiota of the nasopharynx and lower respiratory tract of feedlot cattle (Zaheer et al., 2013; Zeineldin et al., 2017). The high incidence of these genera in the respiratory tract infections could reflect microbial seeding of lungs as a result of regurgitation of feedstuffs during rumination and the formation of aerosols during eructation in ruminants. Bacterial species like Enterococcus faecium, S. pneumoniae, Enterococcus faecalis, and Bacillus spp., had a relatively high abundance in dairy cattle (Klima et al., 2019). S. pneumoniae is associated with pneumonia in animals and humans (Borsa et al., 2019), whereas, Enterococcus faecium, Enterococcus faecalis, and Bacillus spp. were regarded as commensal organisms of animal and human gut (Makarov et al., 2022).

Studies have shown that respiratory diseases are the 2nd leading cause of death in bovines after gastrointestinal diseases. The prevalence of these diseases was reported 4 to 80% in cattle in recent literature (Pratelli et al., 2021; Gaudino et al., 2022). The study of Klima et al. (2019) dealt with BRD and found that bronchopneumonia was the most prevalent infection among bovine respiratory tract diseases followed by fibrinous bronchopneumonia. Out of 18 observed cases, only one was diagnosed with Mycoplasma pneumoniae. During the current investigation, bronchitis followed by bronchopneumonia was recorded as the most common respiratory disease of cattle; while fibrinous bronchopneumonia and Mycoplasma pneumoniae were recorded as the least prevalent respiratory diseases of dairy cattle (Table I). M. haemolytica was observed as the most prevalent organism in numerous respiratory tract diseases. This finding is in agreement with previous studies that reported that M. haemolytica is a common isolate of all types of respiratory tract infections in bovines (Panciera et al., 2010; Anholt et al., 2017).

 

Table I. Veterinary diagnosis, number of cases and isolated bacterial species from respiratory tract infections.

Diagnosis	No. of cases	Isolated bacterial species
Chronic bronchopneumonia	14	Mannheimia haemolytica, Enterococcus faecalis, Klebsiella pneumoniae, Mycoplasma dispar, Bacillus obstructivus, Streptococcus pneumoniae, Staphylococcus aureus
Bronchopneumonia	22	M. haemolytica, Mycobacterium bovis, K. pneumoniae, E. faecalis, Pseudomonas taetrolens, B. obstructivus, S. pneumoniae, S. aureus, Micrococcus luteus
Bronchitis	28	M. haemolytica, P. multocida, H. somni, E. faecium, C. perfringens, B. subtilis, P. taetrolens, B. obstructivus, S. aureus, M. luteus
Rhinitis	10	E. faecalis, E. faecium, B. subtilis, S. aureus, M. luteus
Fibrinous pneumonia	16	M. haemolytica, K. pneumoniae, H. somni, M. dispar, P. taetrolens, S. pneumoniae, M. luteus
Acute suppurative bronchopneumonia	9	K. pneumoniae, B. pyogenes, C. perfringens, M. alkalescens, S. pneumoniae, S. aureus
Tracheitis	13	M. haemolytica, P. multocida, H. somni, E. faecium, B. subtilis, S. aureus, M. luteus
Fibrinous bronchopneumonia	4	M. haemolytica, P. multocida, B. pyogenes, E. faecalis, B. obstructivus, S. pneumoniae
Mycoplasma pneumonia	6	M. bovis, E. faecium, M. alkalescens, M. luteus

 

Table II. Antibacterial resistance percentages of bacterial isolates of respiratory tract infection.

Bacterial isolates (No.)	Antibacterial discs (potency, µg)
	Gen (10)	Sp (100)	Ch (30)	Cip (5)	Nor (10)	Ery (15)	Lin (2)	Tet (30)	Pen (10)	Cef (30)	Ce (75)	Am (10)
Mannheimia haemolytica (24)	12.5	4.16	29.16	33.33	37.5	37.5	45.83	70.83	75	70.83	58.33	50
Mycoplasma bovis (48)	20.83	20.83	29.16	29.16	41.66	50	50	89.58	91.66	87.5	68.75	50
Enterococcus faecalis (38)	26.31	18.42	31.57	34.21	34.21	42.10	55.26	86.84	86.84	65.78	76.31	63.15
Klebsiella pneumoniae (94)	26.59	21.27	26.59	27.65	29.78	42.55	35.10	57.44	62.76	67.02	61.70	55.31
Pasteurella multocida (60)	26.66	16.66	31.66	40	35	48.33	60	95	100	80	78.33	66.66
Histophilus somni (18)	11.11	11.11	22.22	22.22	27.77	38.88	38.88	66.66	61.11	55.55	55.55	38.88
Bacteroides pyogenes (44)	22.72	15.90	27.27	36.36	36.36	38.63	47.72	75	84.09	63.63	65.90	50
Enterococcus faecium (56)	17.87	8.92	26.78	25	32.14	41.07	53.57	71.42	80.35	78.57	69.64	46.42
Clostridium perfringens (74)	29.72	24.32	25.67	28.37	28.37	43.24	55.40	83.78	86.48	82.43	75.67	54.05
Mycoplasma dispar (32)	40.62	28.12	34.37	46.87	40.62	56.25	62.50	93.75	93.75	93.75	87.50	68.75
Bacillus subtilis (76)	30.26	17.10	25	50	42.10	64.47	67.10	100	98.68	93.42	89.47	67.10
Pseudomonas taetrolens (52)	23.07	15.38	34.61	44.23	30.76	57.69	61.53	80.76	86.53	86.53	76.92	44.23
Mycoplasma alkalescens (36)	36.11	25	36.11	41.66	38.88	72.22	72.22	100	100	97.22	91.66	63.88
Bacillus obstructivus (30)	30	20	33.33	36.66	26.66	60	63.33	90	100	93.33	83.33	50
Streptococcus pneumoniae (98)	26.54	9.19	33.68	31.64	27.56	50	65.31	96.94	98.98	83.68	74.49	50
Staphylococcus aureus (102)	33.33	13.72	41.17	37.25	19.60	56.86	70.58	96.07	95.09	92.15	76.47	57.84
Microccus luteus (74)	25.67	17.56	31.08	43.24	27.02	45.94	68.91	82.43	90.54	89.18	90.54	74.32

 

Gen, Gentamicin; Sp, Spectinomycin; Ch, Chloramphenicol; Cip, Ciprofloxacin; Nor, Norfloxacin; Ery, Erythromycin; Lin, Lincomycin; Tet, Tetracycline; Pen, Penicillin; Cef, Cefoxitin; Ce, Cefoperazone; Am, amoxicillin.

 

As shown in Table II, M. haemolytica isolates exhibited >70% resistance against tetracycline, penicillin, and cefoxitin. However, these isolates showed the least resistance (4.16%) against spectinomycin. The isolates of Mycoplasma bovis were found highly resistant against penicillin (91.6%), while the least resistance (20.8%) was recorded against gentamycin and spectinomycin. The isolates of Enterococcus faecalis were found highly (86.8%) resistant against tetracycline and penicillin, followed by cefoperazone (76.3%). K. pneumoniae showed >60% resistance against penicillin, cefoxitin and cefoperazone. The isolates of Pasteurella multocida exhibited 100% and 95% resistance against penicillin and tetracycline, respectively. The isolates of H. somni were found highly resistant against tetracycline (66.6%), while the least resistance (11.1%) was recorded against gentamicin and spectinomycin. The isolates of Bacteroides pyogenes exhibited >60% resistance against four antimicrobials i.e., penicillin (84.1%), tetracycline (63.6%), cefoperazone (65.9%) and cefoxitin (63.6%); while least resistance was recorded against spectinomycin (15.9%). Enterococcus faecium isolates showed the highest resistance against penicillin (80.3%), followed by cefoxitin (78.5%), tetracycline (71.4%) and cefoperazone (69.6%). Clostridium perfringens isolates exhibited >80% resistance against tetracycline, penicillin and cefoxitin; while Mycoplasma dispar exhibited 93.7% resistance against these three antimicrobials. The isolates of Bacillus subtilis and Mycoplasma alkalescens were found fully resistant (100%) against tetracycline; while Bacillus obstructivus and Mycoplasma alkalescens were observed to 100% resistant against penicillin. S. pneumoniae exhibited 96.9% and 98.9% resistance against tetracycline and penicillin, respectively. The isolates of Staphylococcus aureus exhibited >90% resistance against tetracycline, penicillin and cefoxitin, while least resistance was recorded against spectinomycin (13.7%). The isolates of Microccus luteus showed 90.5% resistance against penicillin and cefoperazone; while 89.1% and 82.4% resistance were exhibited against cefoxitin and tetracycline, respectively.

The study of Anholt et al. (2017), reported that P. multocida, T. pyogenes and M. haemolytica were observed highly resistant to antimicrobials. The majority of the isolates exhibited resistance as high as 90.2%. Timsit et al. (2017) reported that M. haemolytica and P. multocida isolates of BRD exhibited > 70% resistance against oxytetracycline. These reports are in accordance with our current study, as we also recorded more than 70% resistance for P. multocida, and M. haemolytica against the antibiotics tetracycline, penicillin and cefoxitin. De novo mutation is suspected of the high occurrence of resistance in bacterial isolates, which is consistent with the selection of multidrug-resistant mobile genetic elements or resistant pathogens that have them (DeDonder and Apley, 2015).

Welsh et al. (2004) published an 8-year study from 1994-2002 and reported a variable resistance (26-77%) for M. haemolytica, H. somni and P. multocida against tetracycline. Tetracycline was adopted for commercial use in 1978 and was recognized as a drug of choice till the end of the 20th century due to its broad antimicrobial spectrum, high availability and low cost (Gasparrini et al., 2020). At present, its’ synergistic action and efficacy in localized infections is acceptable, however for systemic infections particularly those caused by Gram-negative organisms efficacy is highly compromised due to significant antimicrobial resistance developed in bacterial pathogens, human and animal commensals and environmental microbes (Chopra and Roberts, 2001). Ribosome protection and efflux are the main resistance mechanisms of bacterial organisms against tetracyclines (Gasparrini et al., 2020).

Streptococcus pneumoniae is known to colonize the nasopharynx of humans and animals, however, they were isolated commonly as a pathogenic isolate in fibrinous pneumonia and bronchopneumonia. Due to the high occurrence of Streptococcus in the environment and commensal microbiome, and their exposure to over-use of antimicrobials, critical antimicrobial resistance is been reported in animal and human studies (Hayes et al., 2020). In the current study Streptococcus pneumoniae exhibited ≥ 50% resistance against seven (out of twelve) tested antimicrobials which is also an alarming sign for clinicians to treat clinical cases of respiratory infections.

Mycoplasma is wall-less bacteria that are reported as a part of normal microflora in the respiratory and urogenital tract. They also cause mastitis frequently in dairy animals (Saif et al., 2022). In the current study, three species of Mycoplasma viz., M. bovis, M. dispar and M. alkalescens were isolated from respiratory tract samples. All these were observed highly resistant (50-100%) against erythromycin, lincomycin, tetracycline, penicillin, cefoxitin, cefoperazone, and amoxicillin. Being a wall-less bacteria, Mycoplasma is intrinsically resistant to cell-wall-targeting antimicrobials. In addition, recent literature has shown that they have established resistance against several other antibiotics like sulfonamide, nalidixic acid, rifampicin, polymixins, trimethoprim and rifampicin (Gautier-Bouchardon, 2018). Moreover, some other antibiotics like macrolides, tetracyclines, lincosamides, tiamulin and spectinomycin were observed as mycoplasmastatic instead of mycoplasmacidal action (Kleven and Anderson, 1971).

Conclusion

From the results, it could be concluded that Staph. aureus, followed by Streptococcus pneumoniae and Klebsiella pneumoniae were the most prevalent bacterial pathogens in the respiratory tract samples of cattle. Numerous bacterial isolates were observed more than 70% resistant against many antimicrobials. The antimicrobial spectrum results whistle an alarming situation to regulate the non-judicial use of antimicrobials.

Acknowledgements

Authors are grateful to farm manager, staff and colleague veterinarians for their help and support in the sampling process.

Funding

This research was supported by the Xinjiang corps key areas of science and technology project (2021AB012, 2019AB029), Xinjiang corps international science and technology cooperation program (2019BC004) and the XPCC regional innovation guidance program (2018BB036).

IRB approval

The Institutional Review Board (IRB) Approval was granted by the Animal Ethics Committee of Shihezi University (Approval No. A2022-14403).

Ethical statement

Animal handling during sampling was carried out in line with international ethical standard and IRB guidelines.

Statement of conflict of interest

The authors declared no conflict of interest.

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