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Characterization of methicillin-resistant and enterotoxins producing Staphylococcus aureus in bovine milk in India
Journal of Agriculture and Food Research 2 (2020) 100017
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Characterization of methicillin-resistant and enterotoxins producing Staphylococcus aureus in bovine milk in India Achintya Mahanti a, Siddhartha Narayan Joardar a, Samiran Bandyopadhyay b, Jaydeep Banerjee b, Sarbaswarup Ghosh c, Kunal Batabyal a, Tapas Kumar Sar d, Tapan Kumar Dutta e, Indranil Samanta a, * a
Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata, 700037, West Bengal, India Eastern Regional Station, Indian Veterinary Research Institute, Belgachia, Kolkata, West Bengal, India c Sasya Shyamala Krishi Vigyan Kendra, Ramakrishna Mission Vivekananda Educational and Research Institute, Arapanch, Sonarpur, West Bengal, India d Department of Veterinary Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Kolkata, 700037, West Bengal, India e Department of Veterinary Microbiology, Central Agricultural University, Selesih, Aizawl, India b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cattle India MRSA Milk
The present study was conducted to detect the occurrence of methicillin-resistant and enterotoxin producing Staphylococcus aureus in bovine milk with SCCmec, spa type, antimicrobial sensitivity and MIC of antistaphylococcal drugs against the isolates. A total of 450 composite milk samples both from cattle with or without mastitis were collected from all agro-climatic zones of West Bengal. Nine (9.6%) S. aureus isolates were phenotypically detected as MRSA and all of them possessed mecA. Whereas, 12 (12.8%) and 4 (4.3%) isolates possessed the staphylococcal enterotoxins sei and seg, respectively. Six and three isolates belonged to SCCmec type Iva and type V, respectively. Spa typing of the isolates showed MRSA isolates belonged to t304 and t6297. All the MRSA isolates were multidrug resistant. The MIC of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates was 8->256 μg ml1, 0.064–0.256 μg ml1, 0.38–2 μg ml1 and 6–8 μg ml1, and 0.1 μg ml1, respectively. The finding is worrying from public health point of view due to detection of MRSA and enterotoxin (seg,sei) producing S. aureus in milk samples which are commonly associated with human infection or food poisoning cases. This is the first comprehensive study with substantial numbers of samples for characterization of MRSA in bovine milk in India.
1. Introduction Staphylococcus aureus is carried asymptomatically by human (20–40%) and animals, and are associated with a range of menaces such as minor skin infections, septicaemia, toxic shock, endocarditis, and pneumonia in human; and mastitis, udder impetigo, wound infections, tick pyaemia, bumblefoot, and botryomycosis in food animals and birds [1,2]. The staphylococcal infections became more complicated with the emergence of methicillin resistant S. aureus (MRSA) in 1961 and its rapid spread among the community (CA-MRSA) and hospitals (HA-MRSA) [3]. The acquisition of large mobile genetic element staphylococcal cassette chromosome mec (SCCmec) in 30 ends of orfX on the bacterial chromosome is associated with the emergence of MRSA [4]. The SCCmec elements possess mec gene complex consisting of mecA gene and their regulators which encodes a low-affinity penicillin binding protein
(PBP2a/PBP20), site-specific recombinases (ccrA, ccrB) that helps in mobility of the element, and joining regions [4,5]. There are currently 11 types of SCCmec (I-XI) and several sub-types based on the variations in ‘J’ regions [6]. SCCmec types I, IV, V, VI, and VII naturally cause β-lactam resistance only, whereas, SCCmec types II and III are associated with resistance against additional antibiotics due to presence of their consecutive resistance genes in comparatively larger SCCmec. The emergence of livestock associated MRSA (CC398 and MRSAmecC) possessing SCCmec types IV and V or mecC were detected originally in pigs and later in veal calves, cattle, chickens, pet animals, horses, wildlife and human in close contact with the animals [7–10] The LA-MRSA clonal complex (CC398) consists of the sequence types (ST) 398, ST752 and ST753. The ST398 MRSA was originated in human as methicillin sensitive strain and later it was found adapted to livestock (pigs and cattle) as MRSA with the acquisition of mecA [11]. The
* Corresponding author. E-mail addresses: [email protected], [email protected] (I. Samanta). https://doi.org/10.1016/j.jafr.2019.100017 Received 26 October 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 2666-1543/© 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Journal of Agriculture and Food Research 2 (2020) 100017
2.2. Isolation and identification of Staphylococcus aureus
contaminated livestock originated food products including bovine milk is considered as another potential means of clonal transmission into the human both for ST398 and MRSAmecC strains [10,12]. European Food Safety Authority (EFSA) also emphasized on characterization of MRSA present in the food animals and the food items, including human and the environment [13]. Characterization of MRSA isolates for epidemiological investigations are based on different typing schemes such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), X region of S. aureus-specific staphylococcal protein A gene (spa) typing and SCCmec typing. The PFGE, considered as ‘gold standard’ typing method has a major drawback in establishing a database based on its results [14]. The spa-typing and MLST were detected as equally discriminatory for MRSA strains and the useful resources (www.spaserver.ridom.de and www .mlst.net) are available to analyze the genotypes [15]. The enterotoxin-producing S. aureus strains are associated with food poisoning due to production of the heat stable toxins in foodstuffs and the toxins are also resistant to protease and environmental conditions [16]. Currently ten staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, ser) and two staphylococcal-like proteins (selj, selp) are described [17]. The enterotoxigenic S. aureus strains are detected in raw milk and unpasteurized dairy products and the contamination occurred either from the infected cattle or during handling of the milk and dairy products [18,19]. In India, the occurrence of MRSA in hospital patients (HA-MRSA) rose from 6.9% during 1988 to 42–47% during late 1990s and among the critical care patients the prevalence rate is 10–14% [20]. Majority of the HA-MRSA strains in India possessed SCCmec type III (ST239) as observed in the earlier studies [21]. Currently MRSA possessing SCCmec type IV/V (ST672, ST772, ST22) are replacing the MRSA strains with SCCmec type III (ST 239) in healthcare settings in India [22]. In livestock, 20–40% of bovine mastitis is associated with S. aureus in India although the LA-MRSA present in food animals and food items is poorly characterized except a few reports indicating their occurrence [23–26]. Most of the cattle in India are reared in backyard system with more direct contact with human than the intensive farming and the possibility of cross-transmission cannot be ruled out. The supply of unpasteurized milk directly from the farmers into the food chain also increases the occurrence of milk-borne staphylococcal infections or intoxications. The present study was undertaken to detect the occurrence of methicillin-resistant and enterotoxin producing S. aureus in raw milk samples collected from the cattle with or without mastitis in different agro-climatic zones of West Bengal, a major cattle rearing state in India. The study also intended to reveal the SCCmec and spa type of the MRSA isolates along with their antimicrobial sensitivity pattern and minimum inhibitory concentration (MIC) of common anti-staphylococcal drugs against the isolates.
The milk samples were inoculated into nutrient broth (HiMedia, India) for overnight enrichment at 37 C and subsequently it was inoculated into the mannitol salt agar (HiMedia, India) to obtain golden yellow colour colonies. The selected colonies were picked and streaked on nutrient agar (HiMedia, India) slant for further biochemical confirmation. The biochemically confirmed isolates were subjected to PCR for detection of thermonuclease (nuc) gene having species-specific sequences for confirmation of Staphylococcus aureus [28]. 2.3. Detection of phenotypical resistance to oxacillin and cefoxitin All the S. aureus isolates were tested by disc diffusion test with oxacillin (1 μg; HiMedia, India) and cefoxitin (30 μg; HiMedia, India) following the standard guideline [29]. The minimum inhibitory concentration of oxacillin (MICoxa) was measured with MIC Strip (HiComb™, HiMedia, India) coated with oxacillin (0.016–256 μg mL1) following the guidelines of the manufacturer. The MIC study was conducted with the S. aureus isolates that have exhibited phenotypical resistance to cefoxitin or oxacillin. 2.4. PCR based detection of mecA S. aureus isolates which exhibited phenotypic resistance to oxacillin and or cefoxitin were subjected to PCR for detection of mecA [30]. 2.5. SCCmec and spa typing of methicillin-resistant S. aureus isolates SCCmec and spa typing of the confirmed MRSA isolates were performed with the primers as described earlier [31,32]. The spa types were assigned by Ridom SpaServer (http://spaserver.ridom.de). 2.6. PCR based detection of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin (TSST) All the S. aureus isolates both methicillin resistant and sensitive were screened for staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, and sej) and TSST gene by PCR [33]. 2.7. Antimicrobial sensitivity of methicillin-resistant and enterotoxins producing S. aureus isolates All the methicillin-resistant and enterotoxin(s) producing S. aureus isolates were tested for their phenotypical sensitivity to different antimicrobials [29]. The antibiotics used were co-trimoxazole (25 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), ampicillin (10 μg), cefoxitin (30 μg), tetracycline (30 μg), levofloxacin (5 μg), penicillin-G (10 U), ampicillin/sulbactam (10/10 μg), ceftriaxone (30 μg), ceftizoxime (30 μg), erythromycin (15 μg), doxycycline hydrochloride (30 μg), azithromycin (15 μg), and amoxyclav (30 μg) (HiMedia, India).
2. Materials and methods 2.1. Sampling A total of 450 composite milk samples were randomly collected from healthy cattle (n ¼ 168) and cattle with clinical (n ¼ 107) or sub-clinical mastitis (n ¼ 175) as evident by California mastitis test (CMT)/MAST INDEX (Tulip Diagnostics Limited, India) irrespective of their age and breed during 2014-16 from all the six agro-climatic zones of West Bengal, India (Table S1). The farmers reared the cattle by backyard farming system typically consisted of 2–3 cattle per family. Milk samples were collected directly in sterile vials from the teat after discarding two/three primary strips of milk. Milk samples from the cattle with clinical mastitis were collected following the guidelines of national mastitis council [27]. All the four quarter milk samples were mixed and brought into the laboratory maintaining cold chain and were stored at 4 C until further processing.
2.8. MIC of linezolid, vancomycin, ampicillin and ciprofloxacin against MRSA isolates The MIC of linezolid, vancomycin, ampicillin and ciprofloxacin was detected for the MRSA isolates using MIC strip (HiComb™, HiMedia, India) as per the manufacturer guidelines. 3. Results and discussion The present study detected 60 composite milk samples (60/450, 13.3%) to harbour S. aureus isolates. In total, 94 isolates were confirmed as S. aureus based on biochemical tests and PCR based detection of nuc gene (Table S1). The raw milk samples or milk products varied widely (7–69%) in possession of S. aureus in different studies throughout the 2
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Journal of Agriculture and Food Research 2 (2020) 100017
European countries (Austria, Belgium, Denmark, Finland, France, Germany, Iceland, Sweden, Switzerland, and United Kingdom), United States, United Arab Emirates, and South Africa. The single clone of MRSA with spa type t6297 was reported earlier from India (http://spaserver .ridom.de). The spa type t304 was also reported from raw milk cottage cheese and goat cheese in Romania [46]. All the S. aureus isolates were subjected to PCR for detection of different enterotoxin genes and 12 isolates possessed sei (12/94, 12.8%) and four isolates harboured seg (4/94, 4.3%) gene(s) (Table- 2). Similarly occurrence of ‘seg/sei’ genotype was detected with higher frequency in bovine milk and milk products in Italy and Portugal [17,47]. The staphylococcal enterotoxin genotypes (seg/sei or sea/seg/sei) were described to be associated with food poisoning outbreaks which increased the public health concern of the present isolates [48,49]. Moreover, seg/sei enterotoxin genotype was also detected in S. aureus strains isolated from bovine mastitis [45]. The finding makes us cautious about the collection of milk from diseased animals as the seg/sei possessing S. aureus thus can enter the human food chain. None of the MRSA isolates in the present study were detected to possess enterotoxin or TSST gene(s) (Table 2) which is supportive to the hypothesis that LA-MRSA associated with mastitis are devoid of the genes encoding enterotoxins, TSST and haemolysins [50,51]. All the methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) enterotoxins producing S. aureus isolates were tested against 15 antimicrobial agents to study their sensitivity pattern. All the isolates were resistant to penicillin-G (Table 1, Table 2). Most of the isolates were resistant to ceftizoxime (90.5%), ampicillin (71.4%), cefoxitin (42.9%), amoxyclav (38.1%) and erythromycin (38.1%). All the MRSA isolates were multidrug resistant (resistant to at least three antimicrobial agents) (Table 1). Co-resistance to tetracycline, macrolide and aminoglycoside in addition to the β-lactams was earlier reported in MRSA strains isolated from livestock or milk and milk products [17,40,52]. The finding is worrying specially for mastitis isolates as β-lactam, aminoglycosides and macrolides are commonly used to treat mastitis which would be compromised. However, the present isolates were sensitive to chloramphenicol and levofloxacin. Low use of chloramphenicol and levofloxacin in food animals is probably the cause of such sensitivity as observed in our previous study [26]. MIC value of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates (n ¼ 9) was 8->256 μg mL1, 0.064–0.256 μg mL1, 0.38–2 μg mL1, 6–8 μg mL1, and 0.1 μg mL1, respectively (Table 1). All the MRSA isolates were found sensitive to linezolid and vancomycin which are used in the treatment of refractory cases of MRSA in human.
world including India depending upon husbandry practices, hygienic measures adapted in milk processing plants, geographical location and bacteria isolation techniques [17,19,24,34,35]. Nine (9/94, 9.6%) S. aureus isolates were confirmed as MRSA based on phenotypical test and presence of mecA gene (Table 1). The disc diffusion test with breakpoints of 14 mm or less diameter zone surrounding the oxacillin (1 μg) disc and 21 mm or less diameter zone surrounding cefoxitinim (10 μg) disc along with presence of mecA gene are recommended for detection of MRSA isolates [36]. Occurrence of MRSA in milk and milk products varied widely (0.028–9.3%) in different countries such as Italy, The Netherlands, Finland, Japan, Switzerland, Korea and Belgium due to variations in the sampling, methodologies and geographical locations [15,17,19,37–41]. The present study detected seven MRSA isolates (7/94, 7.44%) in the milk samples collected from the cattle with clinical/sub-clinical mastitis (Table 1). Since the first report of MRSA associated with bovine mastitis in 1972, occurrence of MRSA was detected as sporadic and low (1.1–1.6%) in cattle with clinical mastitis in other countries [37,39,41,42]. The higher occurrence of MRSA (9.3%) was detected in the study when sub-clinical mastitis was taken under consideration which might be one of the reason for higher occurrence of MRSA in mastitic cattle in the present study [38]. Detection of MRSA in cattle with sub-clinical mastitis is more worrisome from public health point of view as the cattle appeared healthy and earlier study reported the zoonotic transmission of MRSA from cattle with sub-clinical mastitis [8]. Moreover, earlier studies conducted in India detected higher frequency (5.4–29.41%) of MRSA in cattle and buffaloes with clinical mastitis and human patients (44%) probably due to over usage of antibiotics for therapy [20,24,43]. The SCCmec type IVa and type V were detected in six (MRSA 1–4, 8, 9) and three (MRSA 5–7) MRSA isolates in the present study (Table 1). Our finding is similar with the earlier studies which also reported occurrence of MRSA/VRSA with SCCmec type IV and/or V in bovine milk samples collected from the cattle with mastitis in different countries including India [26,38–41,44]. Possession of SCCmec type IV and V is a typical feature of community associated MRSA (CA-MRSA) and there is recent emergence of MRSA possessing SCCmec type IV/V in healthcare settings in India replacing the old strains [22]. Although there is no evidence of MRSA anthropozoonosis from cattle to human in India, the possibility cannot be ruled out due to close contact with the livestock in backyard husbandry practices. The direct contact with cattle also enhances the probability of zooanthroponosis (human to animal zoonotic transmission) of MRSA as occurrence of typical human MRSA clone (Geraldine clone) was detected in French cattle [45]. In spa typing the MRSA isolates were grouped into t304 and t6297 types. The global occurrence of t304 was estimated as 0.53% mostly in Table 1 Characterization of MRSA isolated from bovine milk and MIC of common drugs. Isolates
Agro-climatic Zone
District
SCCmec Type
Resistance Pattern
Enterotoxin gene
OX (μg/ ml)
LZ (μg/ ml)
VAN (μg/ ml)
CIP (μg/ ml)
AMP (μg/ml)
MRSA 1 MRSA 2 MRSA 3 MRSA 4 MRSA 5 MRSA 6 MRSA 7 MRSA 8 MRSA 9
New Alluvial Zone New Alluvial Zone Red and Laterite zone Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones
Murshidabad
IVa
AMP, CX, P, CZX
NIL
32
0.1
0.064
2
6
Murshidabad
IVa
AMP, CX, P, CZX
NIL
8
0.1
0.064
1
6
Purulia
IVa
CIP, AMP, CX, P, CZX, E, AZM
NIL
>256
0.1
0.064
1.5
8
South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas
IVa
NIL
8
0.1
0.064
2
6
NIL
>256
0.1
0.256
0.75
8
V
COT, AMP, CX, TE, P.A/S, CTR, CZX, E, DO, AMC AMP, CX, TE, P, A/S, CZX, E, DO, AMC AMP, CX, P, CZX, E
NIL
8
0.1
0.128
0.38
6
V
AMP, CX, P, CZX
NIL
8
0.1
0.128
0.38
6
IVa
AMP, CX, P, CZX
NIL
8
0.1
0.128
2
6
IVa
AMP, CX, TE, P, A/S, CTR, E, DO, AMC
NIL
>256
0.1
0.256
2
8
V
3
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Journal of Agriculture and Food Research 2 (2020) 100017
Table 2 Characterization of methicillin sensitive and enterotoxin-producing S. aureus isolated from bovine milk in India. Isolates
Agro-climatic Zone
District
MSSA 17 MSSA 18 MSSA 19 MSSA 20 MSSA 24 MSSA 28 MSSA 34 MSSA 54 MSSA 55 MSSA 62 MSSA 77 MSSA 84
Tarai Zone Tarai Zone Tarai Zone Tarai Zone Tarai Zone Tarai Zone Old Alluvial Zone New Alluvial Zone New Alluvial Zone Red and Laterite zone Coastal and Saline zones Coastal and Saline zones
Darjeeling Darjeeling Darjeeling Darjeeling Darjeeling Coochbehar Burdwan Murshidabad Murshidabad Paschim Medinipur North 24 Parganas North 24 Parganas
Enterotoxins gene sea
seb
sec
sed
see
seg
seh
sei
sej
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
þ – – – þ – – þ – – þ –
– – – – – – – – – – – –
þ þ þ þ þ þ þ þ þ þ þ þ
– – – – – – – – – – – –
TSST
Resistance Pattern
– – – – – – – – – – – –
AMP, P, CZX COT, AMP, TE, P, A/S, E, AZM, AMC P, CZX CIP, AMP, P.A/S, CZX, E, AZM, AMC CIP, AMP, P, CZX P, CZX, E, AZM, AMC AMP, P, CZX, AMC P, CZX P, CZX P, CZX AMP, P, CZX, AMC P, CZX
Dan, L. vanderGraaf van Bloois, [8] E. Juhasz-Kaszanyitzky, S. Janosi, P. Somogyi, A. E. Van Duijkeren, et al., MRSA transmission between cows and humans, Emerg. Infect. Dis. 13 (2007) 630–632. [9] O. Denis, C. Suetens, M. Hallin, B. Catry, I. Ramboer, M. Dispas, et al., Methicillinresistant Staphylococcus aureus ST398 in swine farm personnel, Belgium, Emer, Inf. Disp. 15 (2009) 1098–1101. [10] L. García-Alvarez, M.T. Holden, H. Lindsay, C.R. Webb, D.F. Brown, M.D. Curran, et al., Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study, Lancet Infect. Dis. 11 (2009) 595–603. [11] L.B. Price, M. Stegger, H. Hasman, M. Aziz, J. Larsen, P.S. Andersen, et al., Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock, MBio 3 (2012) e00305–e00311. [12] J.H. Lee, Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans, Appl. Environ. Microbiol. 69 (2003) 6489–6494. [13] EFSA, Assessment of the public health significance of meticillin resistant Staphylococcus aureus (MRSA) in animals and foods - scientific opinion of the panel on biological hazards, EFSA J 993 (2009) 1–73. [14] T.L. Bannerman, G.A. Hancock, F.C. Tenover, J.M. Miller, Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus, J. Clin. Microbiol. 33 (1995) 551–555. [15] E. Hata, K. Katsuda, H. Kobayashi, I. Uchida, K. Tanaka, M. Eguchi, Genetic variation among Staphylococcus aureus strains from bovine milk and their relevance to methicillin-resistant isolates from humans, J. Clin. Microbiol. 48 (2010) 2130–2139. Argudín, M.C. Mendoza, M.R. Rodicio, Food poisoning and Staphylococcus [16] M.A. aureus enterotoxins, Toxins 2 (2010) 1751–1773. [17] V. Carfora, A. Caprioli, N. Marri, D. Sagrafoli, C. Boselli, G. Giacinti, et al., Enterotoxin genes, enterotoxin production, and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy, Int. Dairy J. 42 (2015) 12–15. [18] F.J. Angulo, J.T. LeJeune, P.J. Rajala-Schultz, Unpasteurized milk: a continued public health threat, Clin. Infcet. Dis. 48 (2009) 93–100. [19] A. Riva, E. Borghi, D. Cirasola, S. Colmegna, F. Borgo, E. Amato, et al., Methicillinresistant Staphylococcus aureus in raw milk: prevalence, SCC mec typing, enterotoxin characterization, and antimicrobial resistance patterns, J. Food Prot. 78 (2015) 1142–1146. [20] R. Sunagar, N.R. Hegde, G.J. Archana, A.Y. Sinha, K. Nagamani, S. Isloor, Prevalence and genotype distribution of methicillin-resistant Staphylococcus aureus (MRSA) in India, J. Global Antimicrob. Resist. 7 (2016) 46–52. [21] N. D'Souza, C. Rodrigues, A. Mehta, Molecular characterization of methicillinresistant Staphylococcus aureus with emergence of epidemic clones of sequence type (ST) 22 and ST 772 in Mumbai, India, J. Clin. Microbiol. 48 (2010) 1806–1811. [22] B. Dhawan, C. Rao, E.E. Udo, R. Gadepalli, S. Vishnubhatla, A. Kapil, Dissemination of methicillin-resistant Staphylococcus aureus SCCmec type IV and SCCmec type V epidemic clones in a tertiary hospital: challenge to infection control, Epidemiol. Infect. 143 (2015) 343–353. [23] S. Joshi, S. Gokhale, Status of mastitis as an emerging disease in improved and periurban dairy farms in India, Ann. N. Y. Acad. Sci. 1081 (2006) 74–83. [24] R. Kumar, B.R. Yadav, R.S. Singh, Genetic determinants of antibiotic resistance in Staphylococcus aureus isolates from milk of mastitic crossbred cattle, Curr. Microbiol. 60 (2010) 379–386. [25] S. Bandyopadhyay, I. Samanta, D. Bhattacharyya, P.K. Nanda, D. Kar, J. Chowdhury, et al., Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis–three cases reported from India, Vet. Q. 35 (2015) 56–61. [26] D. Bhattacharyya, J. Banerjee, S. Bandyopadhyay, B. Mondal, P.K. Nanda, I. Samanta, et al., First report on vancomycin-resistant Staphylococcus aureus in bovine and caprine milk, Microb. Drug Resist. 22 (2016) 675–681. [27] National Mastitis Council (NMC), Microbiological Procedures for the Diagnosis of Udder Infection, third ed., National Mastitis Council Inc, Arlington, VA, 1990.
4. Conclusions The occurrence of MRSA was detected towards higher side in milk samples collected from cattle with clinical or sub-clinical mastitis in comparison to the published literature. The MRSA isolates belonged to SCCmec type IVa and V, and Spa types t304 and t6297. All the MRSA isolates were multidrug resistant. The MIC of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates was detected, and linezolid and vancomycin was fond effective. The finding is worrying from public health point of view due to detection of enterotoxin (seg,sei) producing S. aureus isolates in milk samples which are commonly associated with food poisoning cases. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that no conflict of interest exists. Acknowledgements The authors provide sincere thanks to Honourable Vice Chancellor, West Bengal University of Animal and Fishery Sciences for the infrastructure and facilities. Appendix ASupplementary data Supplementary data to this article can be found online at https://do i.org/10.1016/j.jafr.2019.100017. References [1] F.D. Lowy, Staphylococcus aureus infections, N. Engl. J. Med. 339 (1998) 520–532. [2] A. van Belkum, D.C. Melles, J. Nouwen, W.B. van Leeuwen, W. van Wamel, M.C. Vos, et al., Co-evolutionary aspects of human colonisation and infection by Staphylococcus aureus, Infect. Genet. Evol. 9 (2009) 32–47. [3] M. Barber, Methicillin-resistant staphylococci, J. Clin. Pathol. 14 (1961) 385–393. [4] T. Ito, Y. Katayama, K. Asada, N. Mori, K. Tsutsumimoto, C. Tiensasitorn, et al., Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus, Antimicrob. Agents Chemother. 45 (2001) 1323–1336. [5] Y. Katayama, T. Ito, K. Hiramatsu, A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus, Antimicrob. Agents Chemother. 44 (2000) 1549–1555. [6] International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC), Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements, Antimicrob. Agents Chemother. 53 (2009) 4961–4967. [7] A.J. De Neeling, M.J.M. Van den Broek, E.C. Spalburg, M.G. van Santen-Verheuvel, W.D.C. Dam-Deisz, H.C. Boshuizen, et al., High prevalence of methicillin resistant Staphylococcus aureus in pigs, Vet. Microbiol. 122 (2007) 366–372. 4
A. Mahanti et al.
Journal of Agriculture and Food Research 2 (2020) 100017 [41] V. Gindonis, S. Taponen, A.L. Myllyniemi, S. Py€ or€al€a, S. Nyk€asenoja, S. Salmenlinna, et al., Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland, Acta Vet. Scand. 55 (2013) 61. [42] L.A. Devriese, L.R. Van Damme, L. Fameree, Methicillin (cloxacillin)-resistant Staphylococcus aureus strains isolated from bovine mastitis cases, Zentralblatt für Veterinarmed. - Reihe B 19 (1972) 598–605. [43] A. Tyagi, A. Kapil, P. Singh, Incidence of methicillin resistant Staphylococcus aureus (MRSA) in pus samples at a tertiary care hospital, AIIMS, New Delhi, J. Indian Acad. Clin. Med. 9 (2008) 33–35. [44] A. Feßler, C. Scott, K. Kadlec, R. Ehricht, S. Monecke, S. Schwarz, Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis, J. Antimicrob. Chemother. 65 (2010) 619–625. [45] M. Haenni, L. Galofaro, C. Ponsin, M. Bes, F. Laurent, J.Y. Madec, Staphylococcal bovine mastitis in France: enterotoxins, resistance and the human Geraldine methicillin-resistant Staphylococcus aureus clone, J. Antimicrob. Chemother. 66 (2010) 216–218. [46] L. Ciolacu, B. Stessl, A.S. Bolocan, E.A. Oniciuc, M. Wagner, K. Rychli, A.I. Nicolau, Tracking foodborne pathogenic bacteria in raw and ready-to-eat food illegally sold at the eastern EU border, Foodb. Pathog. Dis. 13 (2016) 148–155. [47] V. Pereira, C. Lopes, A. Castro, J. Silva, P. Gibbs, P. Teixeira, Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal, Food Microbiol. 26 (2009) 278–282. [48] J.O. Cha, J.K. Lee, Y.H. Jung, J.I. Yoo, Y.K. Park, B.S. Kim, et al., Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea, J. Appl. Microbiol. 101 (2006) 864–871. [49] A. Kerouanton, J.A. Hennekinne, C. Letertre, L. Petit, O. Chesneau, A. Brisabois, et al., Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France, Int. J. Food Microbiol. 115 (2007) 369–375. [50] T. Matsunaga, S.I. Kamata, N. Kakiichi, K. Uchida, Characteristics of Staphylococcus aureus isolated from peracute, acute and chronic bovine mastitis, J. Vet. Med. Sci. 55 (1993) 297–300. [51] S. Monecke, P. Kuhnert, H. Hotzel, P. Slickers, R. Ehricht, Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle, Vet. Microbiol. 125 (2007) 128–140. [52] A. Battisti, A. Franco, G. Merialdi, H. Hasman, M. Iurescia, R. Lorenzetti, et al., Heterogeneity among methicillin-resistant Staphylococcus aureus from Italian pig finishing holdings, Vet. Microbiol. 142 (2010) 361–366.
[28] O.G. Brakstad, K. Aasbakk, J.A. Maeland, J. A, Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene, J. Clin. Microbiol. 30 (1992) 1654–1660. [29] CLSI, Performance Standards for Antimicrobial Susceptibility Testing, Clinical and Laboratory Standard Institute. CLSI document, Wayne, PA, 2014. M100-S24. Stegger, P.S. Andersen, A. Kearns, B. Pichon, M.A. Holmes, G. Edwards, et al., [30] A. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251, Clin. Microbiol. Infect. 18 (2012) 395–400. [31] D. Harmsen, H. Claus, W. Witte, J. Rothg€anger, H. Claus, D. Turnwald, et al., Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management, J. Clin. Microbiol. 41 (2003) 5442–5448. [32] K. Zhang, J.A. McClure, S. Elsayed, T. Louie, J.M. Conly, Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus, J. Clin. Microbiol. 43 (2005) 5026–5033. [33] A. Løvseth, S. Loncarevic, K.G. Berdal, Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates, J. Clin. Microbiol. 42 (2004) 3869–3872. [34] D.R. Kalorey, Y. Shanmugam, N.V. Kurkure, K.K. Chousalkar, S.B. Barbuddhe, PCRbased detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases, J. Vet. Sci. 8 (2007) 151–154. [35] V. Sharma, S. Sharma, D.K. Dahiya, A. Khan, M. Mathur, A. Sharma, Coagulase gene polymorphism, enterotoxigenecity, biofilm production, and antibiotic resistance in Staphylococcus aureus isolated from bovine raw milk in North West India, Ann, Clin. Microbiol. Antimicrob. 16 (2017) 65. [36] J.M. Andrews, BSAC standardized disc susceptibility testing method (version 8), J. Antimcrob. Chemother. 64 (2009) 454–489. [37] J.S. Moon, A.R. Lee, H.M. Kang, E.S. Lee, M.N. Kim, Y.H. Paik, et al., Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea, J. Dairy Sci. 90 (2007) 1176–1185. [38] W. Vanderhaeghen, T. Cerpentier, C. Adriaensen, J. Vicca, K. Hermans, P. Butaye, Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows, Vet. Microbiol. 144 (2010) 166–171. [39] H. Huber, S. Koller, N. Giezendanner, R. Stephan, C. Zweifel, Prevalence and characteristics of meticillin-resistant Staphylococcus aureus in humans in contact with farm animals, in livestock, and in food of animal origin, Switzerland, Euro Surveill. 15 (2010) (2009) 19542. [40] M. Tavakol, R.G.O. Riekerink, O.C. Sampimon, W.J. van Wamel, A. van Belkum, T.J. Lam, Bovine-associated MRSA ST398 in The Netherlands, Acta Vet. Scand. 54 (2012) 28.
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Characterization of methicillin-resistant and enterotoxins producing Staphylococcus aureus in bovine milk in India Achintya Mahanti a, Siddhartha Narayan Joardar a, Samiran Bandyopadhyay b, Jaydeep Banerjee b, Sarbaswarup Ghosh c, Kunal Batabyal a, Tapas Kumar Sar d, Tapan Kumar Dutta e, Indranil Samanta a, * a
Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata, 700037, West Bengal, India Eastern Regional Station, Indian Veterinary Research Institute, Belgachia, Kolkata, West Bengal, India c Sasya Shyamala Krishi Vigyan Kendra, Ramakrishna Mission Vivekananda Educational and Research Institute, Arapanch, Sonarpur, West Bengal, India d Department of Veterinary Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Kolkata, 700037, West Bengal, India e Department of Veterinary Microbiology, Central Agricultural University, Selesih, Aizawl, India b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cattle India MRSA Milk
The present study was conducted to detect the occurrence of methicillin-resistant and enterotoxin producing Staphylococcus aureus in bovine milk with SCCmec, spa type, antimicrobial sensitivity and MIC of antistaphylococcal drugs against the isolates. A total of 450 composite milk samples both from cattle with or without mastitis were collected from all agro-climatic zones of West Bengal. Nine (9.6%) S. aureus isolates were phenotypically detected as MRSA and all of them possessed mecA. Whereas, 12 (12.8%) and 4 (4.3%) isolates possessed the staphylococcal enterotoxins sei and seg, respectively. Six and three isolates belonged to SCCmec type Iva and type V, respectively. Spa typing of the isolates showed MRSA isolates belonged to t304 and t6297. All the MRSA isolates were multidrug resistant. The MIC of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates was 8->256 μg ml1, 0.064–0.256 μg ml1, 0.38–2 μg ml1 and 6–8 μg ml1, and 0.1 μg ml1, respectively. The finding is worrying from public health point of view due to detection of MRSA and enterotoxin (seg,sei) producing S. aureus in milk samples which are commonly associated with human infection or food poisoning cases. This is the first comprehensive study with substantial numbers of samples for characterization of MRSA in bovine milk in India.
1. Introduction Staphylococcus aureus is carried asymptomatically by human (20–40%) and animals, and are associated with a range of menaces such as minor skin infections, septicaemia, toxic shock, endocarditis, and pneumonia in human; and mastitis, udder impetigo, wound infections, tick pyaemia, bumblefoot, and botryomycosis in food animals and birds [1,2]. The staphylococcal infections became more complicated with the emergence of methicillin resistant S. aureus (MRSA) in 1961 and its rapid spread among the community (CA-MRSA) and hospitals (HA-MRSA) [3]. The acquisition of large mobile genetic element staphylococcal cassette chromosome mec (SCCmec) in 30 ends of orfX on the bacterial chromosome is associated with the emergence of MRSA [4]. The SCCmec elements possess mec gene complex consisting of mecA gene and their regulators which encodes a low-affinity penicillin binding protein
(PBP2a/PBP20), site-specific recombinases (ccrA, ccrB) that helps in mobility of the element, and joining regions [4,5]. There are currently 11 types of SCCmec (I-XI) and several sub-types based on the variations in ‘J’ regions [6]. SCCmec types I, IV, V, VI, and VII naturally cause β-lactam resistance only, whereas, SCCmec types II and III are associated with resistance against additional antibiotics due to presence of their consecutive resistance genes in comparatively larger SCCmec. The emergence of livestock associated MRSA (CC398 and MRSAmecC) possessing SCCmec types IV and V or mecC were detected originally in pigs and later in veal calves, cattle, chickens, pet animals, horses, wildlife and human in close contact with the animals [7–10] The LA-MRSA clonal complex (CC398) consists of the sequence types (ST) 398, ST752 and ST753. The ST398 MRSA was originated in human as methicillin sensitive strain and later it was found adapted to livestock (pigs and cattle) as MRSA with the acquisition of mecA [11]. The
* Corresponding author. E-mail addresses: [email protected], [email protected] (I. Samanta). https://doi.org/10.1016/j.jafr.2019.100017 Received 26 October 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 2666-1543/© 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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2.2. Isolation and identification of Staphylococcus aureus
contaminated livestock originated food products including bovine milk is considered as another potential means of clonal transmission into the human both for ST398 and MRSAmecC strains [10,12]. European Food Safety Authority (EFSA) also emphasized on characterization of MRSA present in the food animals and the food items, including human and the environment [13]. Characterization of MRSA isolates for epidemiological investigations are based on different typing schemes such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), X region of S. aureus-specific staphylococcal protein A gene (spa) typing and SCCmec typing. The PFGE, considered as ‘gold standard’ typing method has a major drawback in establishing a database based on its results [14]. The spa-typing and MLST were detected as equally discriminatory for MRSA strains and the useful resources (www.spaserver.ridom.de and www .mlst.net) are available to analyze the genotypes [15]. The enterotoxin-producing S. aureus strains are associated with food poisoning due to production of the heat stable toxins in foodstuffs and the toxins are also resistant to protease and environmental conditions [16]. Currently ten staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, ser) and two staphylococcal-like proteins (selj, selp) are described [17]. The enterotoxigenic S. aureus strains are detected in raw milk and unpasteurized dairy products and the contamination occurred either from the infected cattle or during handling of the milk and dairy products [18,19]. In India, the occurrence of MRSA in hospital patients (HA-MRSA) rose from 6.9% during 1988 to 42–47% during late 1990s and among the critical care patients the prevalence rate is 10–14% [20]. Majority of the HA-MRSA strains in India possessed SCCmec type III (ST239) as observed in the earlier studies [21]. Currently MRSA possessing SCCmec type IV/V (ST672, ST772, ST22) are replacing the MRSA strains with SCCmec type III (ST 239) in healthcare settings in India [22]. In livestock, 20–40% of bovine mastitis is associated with S. aureus in India although the LA-MRSA present in food animals and food items is poorly characterized except a few reports indicating their occurrence [23–26]. Most of the cattle in India are reared in backyard system with more direct contact with human than the intensive farming and the possibility of cross-transmission cannot be ruled out. The supply of unpasteurized milk directly from the farmers into the food chain also increases the occurrence of milk-borne staphylococcal infections or intoxications. The present study was undertaken to detect the occurrence of methicillin-resistant and enterotoxin producing S. aureus in raw milk samples collected from the cattle with or without mastitis in different agro-climatic zones of West Bengal, a major cattle rearing state in India. The study also intended to reveal the SCCmec and spa type of the MRSA isolates along with their antimicrobial sensitivity pattern and minimum inhibitory concentration (MIC) of common anti-staphylococcal drugs against the isolates.
The milk samples were inoculated into nutrient broth (HiMedia, India) for overnight enrichment at 37 C and subsequently it was inoculated into the mannitol salt agar (HiMedia, India) to obtain golden yellow colour colonies. The selected colonies were picked and streaked on nutrient agar (HiMedia, India) slant for further biochemical confirmation. The biochemically confirmed isolates were subjected to PCR for detection of thermonuclease (nuc) gene having species-specific sequences for confirmation of Staphylococcus aureus [28]. 2.3. Detection of phenotypical resistance to oxacillin and cefoxitin All the S. aureus isolates were tested by disc diffusion test with oxacillin (1 μg; HiMedia, India) and cefoxitin (30 μg; HiMedia, India) following the standard guideline [29]. The minimum inhibitory concentration of oxacillin (MICoxa) was measured with MIC Strip (HiComb™, HiMedia, India) coated with oxacillin (0.016–256 μg mL1) following the guidelines of the manufacturer. The MIC study was conducted with the S. aureus isolates that have exhibited phenotypical resistance to cefoxitin or oxacillin. 2.4. PCR based detection of mecA S. aureus isolates which exhibited phenotypic resistance to oxacillin and or cefoxitin were subjected to PCR for detection of mecA [30]. 2.5. SCCmec and spa typing of methicillin-resistant S. aureus isolates SCCmec and spa typing of the confirmed MRSA isolates were performed with the primers as described earlier [31,32]. The spa types were assigned by Ridom SpaServer (http://spaserver.ridom.de). 2.6. PCR based detection of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin (TSST) All the S. aureus isolates both methicillin resistant and sensitive were screened for staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, and sej) and TSST gene by PCR [33]. 2.7. Antimicrobial sensitivity of methicillin-resistant and enterotoxins producing S. aureus isolates All the methicillin-resistant and enterotoxin(s) producing S. aureus isolates were tested for their phenotypical sensitivity to different antimicrobials [29]. The antibiotics used were co-trimoxazole (25 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), ampicillin (10 μg), cefoxitin (30 μg), tetracycline (30 μg), levofloxacin (5 μg), penicillin-G (10 U), ampicillin/sulbactam (10/10 μg), ceftriaxone (30 μg), ceftizoxime (30 μg), erythromycin (15 μg), doxycycline hydrochloride (30 μg), azithromycin (15 μg), and amoxyclav (30 μg) (HiMedia, India).
2. Materials and methods 2.1. Sampling A total of 450 composite milk samples were randomly collected from healthy cattle (n ¼ 168) and cattle with clinical (n ¼ 107) or sub-clinical mastitis (n ¼ 175) as evident by California mastitis test (CMT)/MAST INDEX (Tulip Diagnostics Limited, India) irrespective of their age and breed during 2014-16 from all the six agro-climatic zones of West Bengal, India (Table S1). The farmers reared the cattle by backyard farming system typically consisted of 2–3 cattle per family. Milk samples were collected directly in sterile vials from the teat after discarding two/three primary strips of milk. Milk samples from the cattle with clinical mastitis were collected following the guidelines of national mastitis council [27]. All the four quarter milk samples were mixed and brought into the laboratory maintaining cold chain and were stored at 4 C until further processing.
2.8. MIC of linezolid, vancomycin, ampicillin and ciprofloxacin against MRSA isolates The MIC of linezolid, vancomycin, ampicillin and ciprofloxacin was detected for the MRSA isolates using MIC strip (HiComb™, HiMedia, India) as per the manufacturer guidelines. 3. Results and discussion The present study detected 60 composite milk samples (60/450, 13.3%) to harbour S. aureus isolates. In total, 94 isolates were confirmed as S. aureus based on biochemical tests and PCR based detection of nuc gene (Table S1). The raw milk samples or milk products varied widely (7–69%) in possession of S. aureus in different studies throughout the 2
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European countries (Austria, Belgium, Denmark, Finland, France, Germany, Iceland, Sweden, Switzerland, and United Kingdom), United States, United Arab Emirates, and South Africa. The single clone of MRSA with spa type t6297 was reported earlier from India (http://spaserver .ridom.de). The spa type t304 was also reported from raw milk cottage cheese and goat cheese in Romania [46]. All the S. aureus isolates were subjected to PCR for detection of different enterotoxin genes and 12 isolates possessed sei (12/94, 12.8%) and four isolates harboured seg (4/94, 4.3%) gene(s) (Table- 2). Similarly occurrence of ‘seg/sei’ genotype was detected with higher frequency in bovine milk and milk products in Italy and Portugal [17,47]. The staphylococcal enterotoxin genotypes (seg/sei or sea/seg/sei) were described to be associated with food poisoning outbreaks which increased the public health concern of the present isolates [48,49]. Moreover, seg/sei enterotoxin genotype was also detected in S. aureus strains isolated from bovine mastitis [45]. The finding makes us cautious about the collection of milk from diseased animals as the seg/sei possessing S. aureus thus can enter the human food chain. None of the MRSA isolates in the present study were detected to possess enterotoxin or TSST gene(s) (Table 2) which is supportive to the hypothesis that LA-MRSA associated with mastitis are devoid of the genes encoding enterotoxins, TSST and haemolysins [50,51]. All the methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) enterotoxins producing S. aureus isolates were tested against 15 antimicrobial agents to study their sensitivity pattern. All the isolates were resistant to penicillin-G (Table 1, Table 2). Most of the isolates were resistant to ceftizoxime (90.5%), ampicillin (71.4%), cefoxitin (42.9%), amoxyclav (38.1%) and erythromycin (38.1%). All the MRSA isolates were multidrug resistant (resistant to at least three antimicrobial agents) (Table 1). Co-resistance to tetracycline, macrolide and aminoglycoside in addition to the β-lactams was earlier reported in MRSA strains isolated from livestock or milk and milk products [17,40,52]. The finding is worrying specially for mastitis isolates as β-lactam, aminoglycosides and macrolides are commonly used to treat mastitis which would be compromised. However, the present isolates were sensitive to chloramphenicol and levofloxacin. Low use of chloramphenicol and levofloxacin in food animals is probably the cause of such sensitivity as observed in our previous study [26]. MIC value of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates (n ¼ 9) was 8->256 μg mL1, 0.064–0.256 μg mL1, 0.38–2 μg mL1, 6–8 μg mL1, and 0.1 μg mL1, respectively (Table 1). All the MRSA isolates were found sensitive to linezolid and vancomycin which are used in the treatment of refractory cases of MRSA in human.
world including India depending upon husbandry practices, hygienic measures adapted in milk processing plants, geographical location and bacteria isolation techniques [17,19,24,34,35]. Nine (9/94, 9.6%) S. aureus isolates were confirmed as MRSA based on phenotypical test and presence of mecA gene (Table 1). The disc diffusion test with breakpoints of 14 mm or less diameter zone surrounding the oxacillin (1 μg) disc and 21 mm or less diameter zone surrounding cefoxitinim (10 μg) disc along with presence of mecA gene are recommended for detection of MRSA isolates [36]. Occurrence of MRSA in milk and milk products varied widely (0.028–9.3%) in different countries such as Italy, The Netherlands, Finland, Japan, Switzerland, Korea and Belgium due to variations in the sampling, methodologies and geographical locations [15,17,19,37–41]. The present study detected seven MRSA isolates (7/94, 7.44%) in the milk samples collected from the cattle with clinical/sub-clinical mastitis (Table 1). Since the first report of MRSA associated with bovine mastitis in 1972, occurrence of MRSA was detected as sporadic and low (1.1–1.6%) in cattle with clinical mastitis in other countries [37,39,41,42]. The higher occurrence of MRSA (9.3%) was detected in the study when sub-clinical mastitis was taken under consideration which might be one of the reason for higher occurrence of MRSA in mastitic cattle in the present study [38]. Detection of MRSA in cattle with sub-clinical mastitis is more worrisome from public health point of view as the cattle appeared healthy and earlier study reported the zoonotic transmission of MRSA from cattle with sub-clinical mastitis [8]. Moreover, earlier studies conducted in India detected higher frequency (5.4–29.41%) of MRSA in cattle and buffaloes with clinical mastitis and human patients (44%) probably due to over usage of antibiotics for therapy [20,24,43]. The SCCmec type IVa and type V were detected in six (MRSA 1–4, 8, 9) and three (MRSA 5–7) MRSA isolates in the present study (Table 1). Our finding is similar with the earlier studies which also reported occurrence of MRSA/VRSA with SCCmec type IV and/or V in bovine milk samples collected from the cattle with mastitis in different countries including India [26,38–41,44]. Possession of SCCmec type IV and V is a typical feature of community associated MRSA (CA-MRSA) and there is recent emergence of MRSA possessing SCCmec type IV/V in healthcare settings in India replacing the old strains [22]. Although there is no evidence of MRSA anthropozoonosis from cattle to human in India, the possibility cannot be ruled out due to close contact with the livestock in backyard husbandry practices. The direct contact with cattle also enhances the probability of zooanthroponosis (human to animal zoonotic transmission) of MRSA as occurrence of typical human MRSA clone (Geraldine clone) was detected in French cattle [45]. In spa typing the MRSA isolates were grouped into t304 and t6297 types. The global occurrence of t304 was estimated as 0.53% mostly in Table 1 Characterization of MRSA isolated from bovine milk and MIC of common drugs. Isolates
Agro-climatic Zone
District
SCCmec Type
Resistance Pattern
Enterotoxin gene
OX (μg/ ml)
LZ (μg/ ml)
VAN (μg/ ml)
CIP (μg/ ml)
AMP (μg/ml)
MRSA 1 MRSA 2 MRSA 3 MRSA 4 MRSA 5 MRSA 6 MRSA 7 MRSA 8 MRSA 9
New Alluvial Zone New Alluvial Zone Red and Laterite zone Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones Coastal and Saline zones
Murshidabad
IVa
AMP, CX, P, CZX
NIL
32
0.1
0.064
2
6
Murshidabad
IVa
AMP, CX, P, CZX
NIL
8
0.1
0.064
1
6
Purulia
IVa
CIP, AMP, CX, P, CZX, E, AZM
NIL
>256
0.1
0.064
1.5
8
South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas South 24 Parganas
IVa
NIL
8
0.1
0.064
2
6
NIL
>256
0.1
0.256
0.75
8
V
COT, AMP, CX, TE, P.A/S, CTR, CZX, E, DO, AMC AMP, CX, TE, P, A/S, CZX, E, DO, AMC AMP, CX, P, CZX, E
NIL
8
0.1
0.128
0.38
6
V
AMP, CX, P, CZX
NIL
8
0.1
0.128
0.38
6
IVa
AMP, CX, P, CZX
NIL
8
0.1
0.128
2
6
IVa
AMP, CX, TE, P, A/S, CTR, E, DO, AMC
NIL
>256
0.1
0.256
2
8
V
3
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Journal of Agriculture and Food Research 2 (2020) 100017
Table 2 Characterization of methicillin sensitive and enterotoxin-producing S. aureus isolated from bovine milk in India. Isolates
Agro-climatic Zone
District
MSSA 17 MSSA 18 MSSA 19 MSSA 20 MSSA 24 MSSA 28 MSSA 34 MSSA 54 MSSA 55 MSSA 62 MSSA 77 MSSA 84
Tarai Zone Tarai Zone Tarai Zone Tarai Zone Tarai Zone Tarai Zone Old Alluvial Zone New Alluvial Zone New Alluvial Zone Red and Laterite zone Coastal and Saline zones Coastal and Saline zones
Darjeeling Darjeeling Darjeeling Darjeeling Darjeeling Coochbehar Burdwan Murshidabad Murshidabad Paschim Medinipur North 24 Parganas North 24 Parganas
Enterotoxins gene sea
seb
sec
sed
see
seg
seh
sei
sej
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
– – – – – – – – – – – –
þ – – – þ – – þ – – þ –
– – – – – – – – – – – –
þ þ þ þ þ þ þ þ þ þ þ þ
– – – – – – – – – – – –
TSST
Resistance Pattern
– – – – – – – – – – – –
AMP, P, CZX COT, AMP, TE, P, A/S, E, AZM, AMC P, CZX CIP, AMP, P.A/S, CZX, E, AZM, AMC CIP, AMP, P, CZX P, CZX, E, AZM, AMC AMP, P, CZX, AMC P, CZX P, CZX P, CZX AMP, P, CZX, AMC P, CZX
Dan, L. vanderGraaf van Bloois, [8] E. Juhasz-Kaszanyitzky, S. Janosi, P. Somogyi, A. E. Van Duijkeren, et al., MRSA transmission between cows and humans, Emerg. Infect. Dis. 13 (2007) 630–632. [9] O. Denis, C. Suetens, M. Hallin, B. Catry, I. Ramboer, M. Dispas, et al., Methicillinresistant Staphylococcus aureus ST398 in swine farm personnel, Belgium, Emer, Inf. Disp. 15 (2009) 1098–1101. [10] L. García-Alvarez, M.T. Holden, H. Lindsay, C.R. Webb, D.F. Brown, M.D. Curran, et al., Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study, Lancet Infect. Dis. 11 (2009) 595–603. [11] L.B. Price, M. Stegger, H. Hasman, M. Aziz, J. Larsen, P.S. Andersen, et al., Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock, MBio 3 (2012) e00305–e00311. [12] J.H. Lee, Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans, Appl. Environ. Microbiol. 69 (2003) 6489–6494. [13] EFSA, Assessment of the public health significance of meticillin resistant Staphylococcus aureus (MRSA) in animals and foods - scientific opinion of the panel on biological hazards, EFSA J 993 (2009) 1–73. [14] T.L. Bannerman, G.A. Hancock, F.C. Tenover, J.M. Miller, Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus, J. Clin. Microbiol. 33 (1995) 551–555. [15] E. Hata, K. Katsuda, H. Kobayashi, I. Uchida, K. Tanaka, M. Eguchi, Genetic variation among Staphylococcus aureus strains from bovine milk and their relevance to methicillin-resistant isolates from humans, J. Clin. Microbiol. 48 (2010) 2130–2139. Argudín, M.C. Mendoza, M.R. Rodicio, Food poisoning and Staphylococcus [16] M.A. aureus enterotoxins, Toxins 2 (2010) 1751–1773. [17] V. Carfora, A. Caprioli, N. Marri, D. Sagrafoli, C. Boselli, G. Giacinti, et al., Enterotoxin genes, enterotoxin production, and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy, Int. Dairy J. 42 (2015) 12–15. [18] F.J. Angulo, J.T. LeJeune, P.J. Rajala-Schultz, Unpasteurized milk: a continued public health threat, Clin. Infcet. Dis. 48 (2009) 93–100. [19] A. Riva, E. Borghi, D. Cirasola, S. Colmegna, F. Borgo, E. Amato, et al., Methicillinresistant Staphylococcus aureus in raw milk: prevalence, SCC mec typing, enterotoxin characterization, and antimicrobial resistance patterns, J. Food Prot. 78 (2015) 1142–1146. [20] R. Sunagar, N.R. Hegde, G.J. Archana, A.Y. Sinha, K. Nagamani, S. Isloor, Prevalence and genotype distribution of methicillin-resistant Staphylococcus aureus (MRSA) in India, J. Global Antimicrob. Resist. 7 (2016) 46–52. [21] N. D'Souza, C. Rodrigues, A. Mehta, Molecular characterization of methicillinresistant Staphylococcus aureus with emergence of epidemic clones of sequence type (ST) 22 and ST 772 in Mumbai, India, J. Clin. Microbiol. 48 (2010) 1806–1811. [22] B. Dhawan, C. Rao, E.E. Udo, R. Gadepalli, S. Vishnubhatla, A. Kapil, Dissemination of methicillin-resistant Staphylococcus aureus SCCmec type IV and SCCmec type V epidemic clones in a tertiary hospital: challenge to infection control, Epidemiol. Infect. 143 (2015) 343–353. [23] S. Joshi, S. Gokhale, Status of mastitis as an emerging disease in improved and periurban dairy farms in India, Ann. N. Y. Acad. Sci. 1081 (2006) 74–83. [24] R. Kumar, B.R. Yadav, R.S. Singh, Genetic determinants of antibiotic resistance in Staphylococcus aureus isolates from milk of mastitic crossbred cattle, Curr. Microbiol. 60 (2010) 379–386. [25] S. Bandyopadhyay, I. Samanta, D. Bhattacharyya, P.K. Nanda, D. Kar, J. Chowdhury, et al., Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis–three cases reported from India, Vet. Q. 35 (2015) 56–61. [26] D. Bhattacharyya, J. Banerjee, S. Bandyopadhyay, B. Mondal, P.K. Nanda, I. Samanta, et al., First report on vancomycin-resistant Staphylococcus aureus in bovine and caprine milk, Microb. Drug Resist. 22 (2016) 675–681. [27] National Mastitis Council (NMC), Microbiological Procedures for the Diagnosis of Udder Infection, third ed., National Mastitis Council Inc, Arlington, VA, 1990.
4. Conclusions The occurrence of MRSA was detected towards higher side in milk samples collected from cattle with clinical or sub-clinical mastitis in comparison to the published literature. The MRSA isolates belonged to SCCmec type IVa and V, and Spa types t304 and t6297. All the MRSA isolates were multidrug resistant. The MIC of oxacillin, vancomycin, ampicillin, ciprofloxacin and linezolid against MRSA isolates was detected, and linezolid and vancomycin was fond effective. The finding is worrying from public health point of view due to detection of enterotoxin (seg,sei) producing S. aureus isolates in milk samples which are commonly associated with food poisoning cases. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that no conflict of interest exists. Acknowledgements The authors provide sincere thanks to Honourable Vice Chancellor, West Bengal University of Animal and Fishery Sciences for the infrastructure and facilities. Appendix ASupplementary data Supplementary data to this article can be found online at https://do i.org/10.1016/j.jafr.2019.100017. References [1] F.D. Lowy, Staphylococcus aureus infections, N. Engl. J. Med. 339 (1998) 520–532. [2] A. van Belkum, D.C. Melles, J. Nouwen, W.B. van Leeuwen, W. van Wamel, M.C. Vos, et al., Co-evolutionary aspects of human colonisation and infection by Staphylococcus aureus, Infect. Genet. Evol. 9 (2009) 32–47. [3] M. Barber, Methicillin-resistant staphylococci, J. Clin. Pathol. 14 (1961) 385–393. [4] T. Ito, Y. Katayama, K. Asada, N. Mori, K. Tsutsumimoto, C. Tiensasitorn, et al., Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus, Antimicrob. Agents Chemother. 45 (2001) 1323–1336. [5] Y. Katayama, T. Ito, K. Hiramatsu, A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus, Antimicrob. Agents Chemother. 44 (2000) 1549–1555. [6] International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC), Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements, Antimicrob. Agents Chemother. 53 (2009) 4961–4967. [7] A.J. De Neeling, M.J.M. Van den Broek, E.C. Spalburg, M.G. van Santen-Verheuvel, W.D.C. Dam-Deisz, H.C. Boshuizen, et al., High prevalence of methicillin resistant Staphylococcus aureus in pigs, Vet. Microbiol. 122 (2007) 366–372. 4
A. Mahanti et al.
Journal of Agriculture and Food Research 2 (2020) 100017 [41] V. Gindonis, S. Taponen, A.L. Myllyniemi, S. Py€ or€al€a, S. Nyk€asenoja, S. Salmenlinna, et al., Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland, Acta Vet. Scand. 55 (2013) 61. [42] L.A. Devriese, L.R. Van Damme, L. Fameree, Methicillin (cloxacillin)-resistant Staphylococcus aureus strains isolated from bovine mastitis cases, Zentralblatt für Veterinarmed. - Reihe B 19 (1972) 598–605. [43] A. Tyagi, A. Kapil, P. Singh, Incidence of methicillin resistant Staphylococcus aureus (MRSA) in pus samples at a tertiary care hospital, AIIMS, New Delhi, J. Indian Acad. Clin. Med. 9 (2008) 33–35. [44] A. Feßler, C. Scott, K. Kadlec, R. Ehricht, S. Monecke, S. Schwarz, Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis, J. Antimicrob. Chemother. 65 (2010) 619–625. [45] M. Haenni, L. Galofaro, C. Ponsin, M. Bes, F. Laurent, J.Y. Madec, Staphylococcal bovine mastitis in France: enterotoxins, resistance and the human Geraldine methicillin-resistant Staphylococcus aureus clone, J. Antimicrob. Chemother. 66 (2010) 216–218. [46] L. Ciolacu, B. Stessl, A.S. Bolocan, E.A. Oniciuc, M. Wagner, K. Rychli, A.I. Nicolau, Tracking foodborne pathogenic bacteria in raw and ready-to-eat food illegally sold at the eastern EU border, Foodb. Pathog. Dis. 13 (2016) 148–155. [47] V. Pereira, C. Lopes, A. Castro, J. Silva, P. Gibbs, P. Teixeira, Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal, Food Microbiol. 26 (2009) 278–282. [48] J.O. Cha, J.K. Lee, Y.H. Jung, J.I. Yoo, Y.K. Park, B.S. Kim, et al., Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea, J. Appl. Microbiol. 101 (2006) 864–871. [49] A. Kerouanton, J.A. Hennekinne, C. Letertre, L. Petit, O. Chesneau, A. Brisabois, et al., Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France, Int. J. Food Microbiol. 115 (2007) 369–375. [50] T. Matsunaga, S.I. Kamata, N. Kakiichi, K. Uchida, Characteristics of Staphylococcus aureus isolated from peracute, acute and chronic bovine mastitis, J. Vet. Med. Sci. 55 (1993) 297–300. [51] S. Monecke, P. Kuhnert, H. Hotzel, P. Slickers, R. Ehricht, Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle, Vet. Microbiol. 125 (2007) 128–140. [52] A. Battisti, A. Franco, G. Merialdi, H. Hasman, M. Iurescia, R. Lorenzetti, et al., Heterogeneity among methicillin-resistant Staphylococcus aureus from Italian pig finishing holdings, Vet. Microbiol. 142 (2010) 361–366.
[28] O.G. Brakstad, K. Aasbakk, J.A. Maeland, J. A, Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene, J. Clin. Microbiol. 30 (1992) 1654–1660. [29] CLSI, Performance Standards for Antimicrobial Susceptibility Testing, Clinical and Laboratory Standard Institute. CLSI document, Wayne, PA, 2014. M100-S24. Stegger, P.S. Andersen, A. Kearns, B. Pichon, M.A. Holmes, G. Edwards, et al., [30] A. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251, Clin. Microbiol. Infect. 18 (2012) 395–400. [31] D. Harmsen, H. Claus, W. Witte, J. Rothg€anger, H. Claus, D. Turnwald, et al., Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management, J. Clin. Microbiol. 41 (2003) 5442–5448. [32] K. Zhang, J.A. McClure, S. Elsayed, T. Louie, J.M. Conly, Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus, J. Clin. Microbiol. 43 (2005) 5026–5033. [33] A. Løvseth, S. Loncarevic, K.G. Berdal, Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates, J. Clin. Microbiol. 42 (2004) 3869–3872. [34] D.R. Kalorey, Y. Shanmugam, N.V. Kurkure, K.K. Chousalkar, S.B. Barbuddhe, PCRbased detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases, J. Vet. Sci. 8 (2007) 151–154. [35] V. Sharma, S. Sharma, D.K. Dahiya, A. Khan, M. Mathur, A. Sharma, Coagulase gene polymorphism, enterotoxigenecity, biofilm production, and antibiotic resistance in Staphylococcus aureus isolated from bovine raw milk in North West India, Ann, Clin. Microbiol. Antimicrob. 16 (2017) 65. [36] J.M. Andrews, BSAC standardized disc susceptibility testing method (version 8), J. Antimcrob. Chemother. 64 (2009) 454–489. [37] J.S. Moon, A.R. Lee, H.M. Kang, E.S. Lee, M.N. Kim, Y.H. Paik, et al., Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea, J. Dairy Sci. 90 (2007) 1176–1185. [38] W. Vanderhaeghen, T. Cerpentier, C. Adriaensen, J. Vicca, K. Hermans, P. Butaye, Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows, Vet. Microbiol. 144 (2010) 166–171. [39] H. Huber, S. Koller, N. Giezendanner, R. Stephan, C. Zweifel, Prevalence and characteristics of meticillin-resistant Staphylococcus aureus in humans in contact with farm animals, in livestock, and in food of animal origin, Switzerland, Euro Surveill. 15 (2010) (2009) 19542. [40] M. Tavakol, R.G.O. Riekerink, O.C. Sampimon, W.J. van Wamel, A. van Belkum, T.J. Lam, Bovine-associated MRSA ST398 in The Netherlands, Acta Vet. Scand. 54 (2012) 28.
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