Emergence of Pathogenic Strains of Staphylococcus aureus in Goat Milk and Their Comparative Response to Antibiotics

Iqra Muzammil1, Muhammad Ijaz Saleem1, Amjad Islam Aqib2,*, Ambreen Ashar3,

Syed Ashar Mahfooz1, Sajjad ur Rahman4, Muhammad Shoaib4, Muhammad Aamir Naseer1, Imran Khan Sohrani1, Javeed Ahmad1, Razaullah Saqi1, Fizzah Laeeq Lodhi1 and

Qaisar Tanveer5

1Department of Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000

2Department of Medicine, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100

3Government College for Women University, Faisalabad-38000

4Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000

5Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad-38000

ABSTRACT

The nutraceutical milk of goat in agrobased countries is at risk of contamination with pathogenic strains of Staphylococcus aureus. The current study was designed to investigate prevalence of pathogenic strains of S. aureus, assessment of risk factors, and in-vitro antibiogram of non-biofilm producing S. aureus (nbpSA) and biofilm positive S. aureus (bpSA) from mastitic goats. The purposive sampling technique was applied to collect n=200 milk samples from different regions of goat populated areas of district Faisalabad-Pakistan. Using surf field mastitis test, collected milk samples were screened for subclinical mastitis at the spot for subsequent identification of pathogenic strains of S. aureus through microbiological examination in the laboratory. Non-probability statistical tools conferred 42% (84/200, CI=35.37-48.93) prevalence of subclinical mastitis, 38.1% S. aureus (32/84, CI=28.45-48.79), 15.6% MRSA (5/32, CI=6.87-31.76), 46.9% haemolytic S. aureus (15/32, CI=30.87-63.56) and 34.4 % biofilm producing S. aureus (11/32, CI=20.41-51.69). Earthen floor type (OR=1.75, p=0.0996), poor drainage system (OR=7.33, p=0.002), pond as source of drinking water (OR=2.05, p=0.179), stall feeding (OR=7.27, p<0.001), 4-6 years of age of goat (OR=4.2, p=0.0874), and teat injury (OR=13.74, p<0.001) were potential risk factors for subclinical mastitis. The in-vitro findings of current study revealed 100% sensitivity of S. aureus against gentamicin, oxytetracycline, amoxicillin, and linezolid while 80% of biofilm negative S. aureus (nbpSA) showed sensitivity against amoxicillin+clavulanic acid. None of the isolate from bpSA and nbpSA was resistant against linezolid, gentamicin, and oxytetracycline in this study. bpSA were highly resistant against amoxicillin and vancomycin. The study found higher prevalence of pathogenic strains of S. aureus, higher number of potential risk factors, and diversified responses to antibiotic.

Article Information

Received 24 October 2019

Revised 22 May 2020

Accepted 20 August 2020

Available online 16 June 2021

Authors’ Contribution

MIS, AIA and SUR designed the research. AA and SAM supervised the research. IKS, RS and JA did sampling. IM and MAN did lab work and wrote the article. MS analyzed the data statistically. FLL and QT review the article.

Key words

Goat, Mastitis, S. aureus, Biofilm, Risk factors, Antibiogram.

DOI: https://dx.doi.org/10.17582/journal.pjz/20191024181008

* Corresponding author: amjadwaseer@gmail.com

0030-9923/2021/0005-1659 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan

Introduction

Livestock plays an important role in the agriculture sector of Pakistan. The total goat population of Pakistan is up to 74.1 million to produce 0.915 million tons of milk and 0.717 million tons of mutton annually (Anonymous, 2018). Milk production in goats is an active and emergent business in harsh climate areas where large ruminants cannot be reared or are difficult to rear and it largely contributes to the mainstream dairy milk production (Silanikove et al., 2010). Milk of goats has certain properties like better digestibility, alkalinity, buffer capacity and medicinal importance which make goat milk better than human and cow milk (Park, 2001). Mastitis can be illustrated as a result of pathological alterations in mammary glands resulting in elevation in somatic cell count of milk (Contreras et al., 2003). Mastitis occurs as clinical and/or subclinical form (Aqib et al., 2018). In clinical mastitis, signs of inflammation, redness, heat and pain are present, whereas there are no obvious indications of swelling in subclinical mastitis except decrease in milk production and increased somatic cell count (Sarker and Samad, 2011). Sub-clinical mastitis has occurrence of 45% in goats in Punjab whereas 53.3% in Khyber-Pakhtunkhwa (KPK), Pakistan (Najeeb et al., 2013; Ali et al., 2010).

Subclinical mastitis in goats is predominantly caused by transmissible bacteria e.g. Staphylococcus spp., Streptococcus spp., Pasteurella spp. and E. coli (Persson and Olofsson, 2011; Contreras et al., 2007). Staphylococcus aureus is the major causative agent where its frequency of isolation and identification vary from 4-40% of the entire isolated pathogens (Leitner et al., 2007). Antimicrobial resistance is reported in S. aureus probably due to excessive administration of antimicrobials (over-prescription, suboptimal termination of treatment regimen and/or insufficient dose administration of antimicrobials) resulting in lateral gene transfer (transformation, transduction and conjugation) of DNA from resistant strain for survivability (Castro-Sánchez et al., 2016). S. aureus can produce biofilm which act as a protective layer for the pathogen and provides continuous persistence via development of resistant genes e.g. mecA, vanA, icaA, icaB etc. (Jyothi et al., 2018). Production ability of coagulase enzyme is considered a significant phenotypic determinant in S. aureus linked with pathogenicity (Moreillon et al., 1995). Multiple drug resistance (MDR) is one of additional challenges in bacterial mastitis (Hameed et al., 2007). In 1972, first MRSA was isolated from dairy mastitic milk (Devriese et al., 1972). MRSA is now becoming major bacterial etiology of mastitis in addition to its isolation from vaginal and nasal swabs of animals (Cortimiglia et al., 2015).

Treatment with broad spectrum antibiotics along with anti-inflammatory drugs is used to treat mastitis in goats. Mechanism of development of drug resistance in bacteria associated with goat mastitis is very important to understand transmission frequency, better management strategies and developing valuable remedial interference (Aqib et al., 2018a; Merz et al., 2016). Therefore, epidemiological studies of pathogenic strains of S. aureus along with their response to antibiotics are necessary for prevention and treatment protocols. Subclinical mastitis in goats remained as neglected issue despite of its increasing prevalence and antimicrobial resistance of bacterial etiologies. Current study was thus designed to investigate the occurrence of different pathogenic strains of S. aureus, associated risk factors, and in-vitro antibiogram of biofilm positive and biofilm negative S. aureus in mastitic goats from Faisalabad, Pakistan.

Materials and methods

Sampling plan and screening for subclinical mastitis

Faisalabad is the second biggest city of province Punjab and the third most populated city of Pakistan having a total area of 5,856km². Purposive sampling technique (Thrusfield, 2007) was applied to collect milk samples (n= 200) from dairy farms located in district Faisalabad (Samundri, n= 62; Rasoolpur, n= 60; Livestock Farm of University of Agriculture Faisalabad, n= 36; Jhapal, n= 42) depending upon the willingness of the farmers to participate in the study and accessibility to Mastitis Research Laboratory, University of Agriculture Faisalabad, Pakistan. Milk samples were collected by strictly following the guidelines of National Mastitis Council of the USA (Reyher and Dohoo, 2011). Samples were screened by using Surf Field Mastitis test (SFMT) proposed by Muhammad et al. (2010). The SFMT positive milk samples, maintained in cold chain (4°C), were transferred to Mastitis Research Laboratory, University of Agriculture Faisalabad, Pakistan and preserved at -20°C till further process (Cengiz et al., 2015).

Risk factor analysis

A questionnaire comprising information like age of animal, type of housing, type of drainage system, type of floor, condition of floor, farm hygiene, source of drinking water, feeding system, vaccination against diseases, deworming, mastitis control program, parity number, stage of lactation, body condition score, milk consistency, milk yield, and teat injury was filled at the time of sampling to assess risk factors associated with mastitis. The risk factors were assumed based on the previous studies conducted by Amin et al. (2011) and Megersa et al. (2010).

Identification of pathogenic strains of S. aureus

SFMT positive samples were cultured on blood agar at 37°C for 24 h. Characteristic pinpoint colonies were further cultured on Mannitol Salt Agar, selective and differential medium for S. aureus, following the same incubation conditions. Series of biochemical tests were performed following guidelines of Bergey’s Manual of Determinative Bacteriology (Bergey and Holt, 1994).

Isolates were identified for their expression as α, β and γ haemolysis on blood agar by inoculating S. aureus on blood agar at 37°C for 24 h. Methicillin resistant S. aureus were identified by their resistance against oxacillin disc following standard protocol described in clinical and laboratory standard institute. Biofilm was identified by Congo Red Agar (CRA) method, a previously established method (Freeman et al., 1989). For biofilm identification, fresh culture of S. aureus was grown on CRA and incubated for 24 h at 37°C. After incubation, colour of colonies indicated strength of biofilm i.e. pinkish red colonies - no biofilm production; slight blackish -weak production; black sheeting - moderate production; and jet black dry sheeting - strong biofilm production (França et al., 2012; Mathur et al., 2006).

In-vitro drug response against bpSA and nbpSA

In-vitro drug response was evaluated using the Kirby Bauer disc diffusion test. The positive isolates were subjected to testing against various antibiotics such as vancomycin (30µg), chloramphenicol (10µg), oxytetracycline (30µg), trimethoprim+sulphamethoxazole (25µg), gentamicin (10µg), linezolid (30µg), amoxicillin-clavulanic acid (20µg), amoxicillin (10µg), and oxacillin (1µg). Fresh cultures adjusted at 1.5×108 CFU were swabbed on Muller Hinton Agar whereas antibiotic discs were aseptically placed at equal distances from each other following the guidelines of CLSI (2015). Incubation was given at 37°C for 18-20 h and zone of inhibitions were measured by Vernier Callipers in millimetres and compared with provided standards.

Statistical analysis

The obtained data were analysed by descriptive statistics for occurrence of S. aureus and antibacterial activity of antibiotics whereas risk factor analysis was assessed by odd’s ratio at 5% probability using IBM SPSS (version 20).

Results

Prevalence of subclinical mastitis and pathogenic strains of S. aureus

The present study found overall 42.0% (84/200) prevalence of subclinical mastitis from goats based on Surf Field Mastitis Test (SFMT). The prevalence of subclinical mastitis was found higher in Jhapal (59.5%) followed by Samundri (38.7%), Rasoolpur (38.3%) and UAF Livestock Farm (33.3%) while there was non-significant association (p> 0.05) among different areas. The overall prevalence of S. aureus was found 38.1% while among S. aureus there was 15.6% MRSA, 46.9% haemolytic S. aureus, 34.4% biofilm producing S. aureus during current study. The higher prevalence of S. aureus and MRSA was noted from livestock farm (50.0% and 33.3%) followed by Samundri (33.3% and 12.5%), Rasoolpur (39.1% and 11.1%) and Jhapal (36.0% and 11.1%). The percentage of haemolytic S. aureus and biofilm producing S. aureus was 83.3% and 83.3% from UAF livestock farm, 44.4% and 33.3% from Rasoolpur, 37.5% and 12.5% from Samundri, and 33.3% and 22.2% from Jhapal, respectively. The study found non-significant difference (p>0.05) for S. aureus, MRSA and haemolytic S. aureus while significant difference was noted for biofilm producing S. aureus among different areas of study (Table I; Fig. 1).

 

Table I.- Prevalence of subclinical mastitis, Staphylococcus aureus, methicillin resistant S. aureus, hemolytic and biofilm producing Staphylococci from different areas of Faisalabad.

Area		Subclinical mastitis (SM) on SFMT basis*	Staphylococcus aureus#	Methicillin resistant S. aureus within S. aureus‡	Haemolytic S. aureus@	Biofilm producing S. aureus†
Samundri	No. observed	24/62	8/24	1/8	3/8	1/8
	Prevalence (%)	38.7	33.3	12.5	37.5	12.5
	CI (95%)	27.58-51.15	17.97-53.29	2.24-47.09	13.68-69.43	2.24-47.09
Rasoolpur	No. observed	23/60	9/23	1/9	4/9	3/9
	Prevalence (%)	38.3	39.1	11.1	44.4	33.3
	CI (95%)	27.09-50.98	22.16-59.21	1.99-43.50	18.87-73.33	12.06-64.58
UAF livestock farm	No. observed	12/36	6/12	2/6	5/6	5/6
	Prevalence (%)	33.3	50.0	33.3	83.3	83.3
	CI (95%)	20.21-49.66	25.38-74.62	9.68-70.00	43.65-96.99	43.65-96.99
Jhapal	No. observed	25/42	9/25	1/9	3/9	2/9
	Prevalence (%)	59.5	36.0	11.1	33.3	22.2
	CI (95%)	44.49-72.95	20.25-55.48	2.24-47.09	12.06-64.58	6.32-54.74
Total	No. observed	84/200	32/84	5/32	15/32	11/32
	Prevalence (%)	42.0	38.1	15.6	46.9	34.4
	CI (95%)	35.37-48.93	28.45-48.79	6.87-31.76	30.87-63.56	20.41-51.69

 

p<0.05 indicate significant difference. Among different areas subclinical mastitis, *, p=0.072; #, p=0.799 and ‡, p=0.623; @, p=0.244; †, p=0.034.

 

Table II.- Risk factors associated with spread of mastitis in dairy goats.

Factor	Variables		No. positive	Percentage (%)		Odds ratio		C.I (95%)		p-value
Housing type	Open		29/80	36.25		1.093		0.49-2.46		0.829
	Street		13/38	34.21		1		-		-
	Backyard		42/82	51.21		2.019		0.91-4.48		0.084
Floor type	Earthen		40/80	50.00		1.75		0.90-3.41		0.099
	Bricks		20/54	37.03		1.029		0.49-2.17		0.939
	Cemented		24/66	36.36		1		-		-
Condition of floor	Even		56/120	46.67		1.625		0.91-2.91		0.102
	Uneven		28/80	35.00		1		-		-
Drainage system	Poor		16/24	66.67		7.333		2.54-21.21		0.0002
	Partially controlled		56/120	46.67		3.208		1.54-6.67		0.002
	Well formed		12/56	21.42		1		-		-
Farm hygiene	Very poor		9/32	28.13		0.671		0.24-1.85		0.44
	Poor		17/40	42.5		1.267		0.51-3.15		0.61
	Normal		44/90	48.89		1.64		0.75-3.57		0.213
	Good		14/38	36.84		1		-		-
Source of drinking water	Pond		9/16	56.25		2.047		0.72-5.82		0.179
	Underground		21/44	47.72		1.454		0.73-2.88		0.282
	Bucket		54/140	38.57		1		-		-
Feeding	Grazing		27/100	27.00		1		-		-
	Stall feeding		35/48	72.92		7.279		3.35-15.80		<0.0001
	Mixed		22/52	42.31		1.983		0.98-4.01		0.057
Vaccinated against diseases	Yes		25/124	20.16		2.467		1.23-4.95		0.011
	No		59/76	77.63		1		-		-
Deworming	Yes		53/128	41.41		1		-		-
	No		31/72	43.05		1.069		0.60-1.92		0.82
Mastitis control measures	Yes		30/76	39.47		1		-		-
	No		54/124	43.55		1.183		0.66-2.11		0.571
Age	Up to 2 years	13/42		31.0	3.138		0.62-15.85		0.166
	2-4 years	29/102		28.4	2.781		0.59-13.01		0.194
	4-6 years	12/32		37.5	4.2		0.81-21.77		0.087
	6-8 years	2/16		12.5	1		-		-
	Above 8 years	1/8		12.5	1		0.08-13.02		1
Parity	1-2 kidding	27/49		55.10	0.859		0.28-2.63		0.79
	2-4 kidding	20/34		58.82	1		0.31-3.26		1
	>5 kidding	10/17		58.82	1		-		-
Stage of lactation	Early	23/42		54.76	1		-		-
	Mid	12/23		52.17	0.901		0.33-2.50		0.841
	Late	22/35		62.86	1.398		0.56-3.49		0.473
Body condition score (BCS)	Poor	29/47		61.70	1.381		0.40-4.77		0.609
	Normal	20/40		50.00	0.857		0.24-3.00		0.809
	Good	7/13		53.84	1		-		-
Milk consistency	Thin	70/165		42.42	1.228		0.51-2.97		0.648
	Thick	9/24		37.50	1		-		-
	Purulent	5/11		45.45	1.389		0.33-5.90		0.656
Milk yield	Decreased	27/45		60.00	1.25		0.56-2.78		0.584
	Not Decreased	30/55		54.54	1		-		-
Teat injury	Yes	59/76		77.63	13.743		6.86-27.55		<0.0001
	No	25/124		20.16	1		-		-

 

C.I, confidence interval set at 95%; *p< 0.05 indicate significant difference.

Risk factor analysis

The findings of the current study presented type of drainage system, type of feeding, vaccination against diseases, and teats injury as potential risk factors (p< 0.05) of subclinical mastitis. Poor type of drainage system (p= 0.0002) and stall feeding (p< 0.0001) showed higher odds of getting mastitis compared to partially controlled drainage system (p= 0.0018) and mixed feeding (p= 0.0572), respectively. The risk factor analysis revealed backyard type of housing showing higher odds of getting mastitis (p= 0.0843) as compared to open type of housing (p= 0.8289). Similar findings were found in case of earthen type of floor (p= 0.0996) as compared to brick floor (p= 0.9393). Mid stage of lactation (p= 0.8414), 1-2 kidding (p= 0.7900), normal body condition scoring (p= 0.8096) were not proved to be potential risk factors of subclinical mastitis (Table II).

Antibiotic Susceptibility testing against S. aureus and biofilm producing S. aureus

The in-vitro findings of current study revealed 100% sensitivity of S. aureus against gentamicin, oxytetracycline, amoxicillin, and linezolid while 80% of biofilm negative S. aureus (nbpSA) showed sensitivity against amoxicillin + clavulanic acid (Table III; Fig. 1). Higher percentages of goat milk based resistant isolates were noted from bpSA and nbpSA against vancomycin, chloramphenicol, oxacillin, amoxicillin+clavulanic acid and amoxicillin. None of the isolate from bpSA and nbpSA was resistant against linezolid, gentamicin, and oxytetracycline in this study. In case of oxacillin, amoxicillin clavulanate and trimethoprim+sulphamethoxazole there was significant (p<0.05) difference at intermediate cadre, and same was observed at sensitive cadre of isolates. The antibiotics did not differ significantly in efficacies between sensitive bpSA strains and sensitive nbpSA strains (Table III).

 

Discussion

Prevalence of subclinical mastitis, S. aureus, MRSA, hemolytic and biofilm producing S. aureus

The prevalence of subclinical mastitis in current study was in line with findings of Najeeb et al. (2013) who reported 45% subclinical mastitis from goats. On the other hands, 37.5% and 53% subclinical mastitis in goats was also noted in previous studies by Abo-Shama (2014) and Ali et al. (2010). S. aureus has been formerly described

 

Table III.- Comparative response of antibiogram of biofilm producing and non-biofilm producing S. aureus isolated from mastitic dairy goats.

Antibiotic	Resistant %			Intermediate %			Sensitive %
	NBPSA	BPSA	p-value	NBPSA	BPSA	p-value	NBPSA	BPSA	p-value
Vancomycin	20	57.14	0.021	40	42.86	0.014	40	0	0.064
Oxacillin	40	0.000	0.383	40	14.29	0.036	20	85.71	0.057
Amoxicillin+Clavulanic acid	0	42.86	0.322	20	0	0.024	80	57.14	0.026
Linezolid	0	0	N/A	0	28.57	0.787	100	71.43	0.035
Gentamicin	0	0	N/A	0	0	N/A	100	100	N/A
Trimethoprim + Sulphamethoxazole	20	0	0.689	40	0	0.044	40	100	0.056
Oxytetracycline	0	0	N/A	0	0	N/A	100	100	0.457
Chloramphenicol	40	14.29	0.047	40	14.29	0.047	2	71.43	0.037
Amoxicillin	0	71.43	0.057	0	0	N/A	100	28.57	0.479

 

NBPSA, non-biofilm producing S. aureus; BPSA, biofilm producing S. aureus; NA, not applicable.

as one of the most significant causative agent in caprine mastitis (Ali et al., 2010; Najeeb et al., 2013). Higher biofilm positive S. aureus in current study was in contradiction with findings of França et al. (2012) who reported 7.6% bpSA based on CRA from caprine milk.

Hemolysins are involved in various pathological processes. Kenny et al. (1992) reported that haemolytic toxins can develop clinical signs in mastitis cases, and Ebrahimi et al. (2007) reported that the udder of mastitic goats contain hemolytic Staphylococci. In the current study, 15.6% of S. aureus were found to be resistant to methicillin which was in line with the previous results of 9.2% as discussed by El-Deeb et al. (2018), 20% by Bochev and Russenova (2005), and 28.57% by Ebrahimi et al. (2007). The methicillin-resistant Staphylococci cannot be successfully treated with beta-lactam antibiotics as discussed by previous studies (Aqib et al., 2018b; Dar et al., 2006).

Risk factors

Potential risk factors of current study were in line with findings of previous studies conducted in Pakistan. Feeding system is significant factor for subclinical mastitis. Poor drainage system or farm hygiene can lead to occurrence of mastitis (Ali et al., 2010; Aqib et al., 2019; Najeeb et al., 2013). Teat injury is also strongly associated with mastitis (Ferdous et al., 2018). Wound on the teats and udder facilitates the entry of microbes into the glands, leading to mastitis (Gebrewahid et al., 2012). The findings of current study were in line with those of previous trials conducted on prevalence of subclinical mastitis in goats by Ali et al. (2010) and Najeeb et al. (2013).

Antibiogram

The results of current study were in line with those reported by Ali et al. (2010) and Saleem et al. (2018) who found 80-100% of S. aureus sensitive against these antibiotics. The decreased use of gentamicin in the late 1990’s and obvious shift in strains of clinical isolates of S. aureus were major factors for increased gentamicin sensitivity (Klevens et al., 2006). Oxytetracycline is used as first line treatment by field workers. Oppliger et al. (2012) also suggested that S. aureus isolated from farm workers were 100% sensitive to oxytetracycline.

Vancomycin resistance is a rising problem in S. aureus isolates and their number is increasing day by day which may be due to the acquired resistance as occurred in case of methicillin (Marques et al., 2013). Glycopeptide antibiotics such as vancomycin are last choice for the severe clinical infections of MRSA throughout the world. But the continuous use of vancomycin for handling of MDR S. aureus infections has caused a decrease in vancomycin sensitivity in many countries (Hiramatsu et al., 1997; Rağbetli et al., 2016). Vancomycin resistance in S. aureus when studied at genomic level shows that the development of vanA gene is associated with this behaviour (Akpaka et al., 2017). Mastitis is well known for its deterioration and lack of response to treatment chiefly due to resistance by bacteria against antibiotics (Shamila-Syuhada et al., 2016).

Conclusion

The present study found overall higher prevalence of subclinical mastitis (42%) in goats with increased percentage of S. aureus (3.8.1%) and pathogenic strains of S. aureus (MRSA 15.6%, hemolytic S. aureus 46.9%, and biofilm producing S. aureus 34.4%). Risk factor analysis revealed type of drainage system, type of feeding, and teats injury as potential risk factors of mastitis. The in-vitro drug trial indicated higher sensitivity of S. aureus against oxytetracycline, trimethoprim + sulphamethoxazole, gentamicin and linezolid against S. aureus and biofilm producing S. aureus. Biofilm producing S. aureus were highly resistant against amoxicillin and vancomycin. Current study reports higher prevalence of pathogenic strains of S. aureus, larger number of potential risk factors, and diversified response of antibiotic susceptibilities which suggest extensive molecular studies and development of effective preventive measures.

Statement of conflict of interest

The authors have declared no conflict of interest.

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