Susceptibility pattern in methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine mastitis
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
RESEARCH ARTICLE
Published Online: January 22, 2022
Susceptibility pattern in methicillin-resistant
Staphylococcus aureus (MRSA) isolated from
bovine mastitis
Mohsina Mushtaqa* | Rajesh Agrawala | Mohammad Altaf Bhata | Nishi Pandea
aSher-e-Kashmir University of Agricultural Sciences and Technology, R S Pura, Jammu, Jammu and Kashmir, 180009, India.
*Corresponding author: mosu2011@gmail.com
Abstract The study was conducted to determine the phenotypic and genotypic antimicrobial resistance pattern of
methicillin-resistant Staphylococcus aureus (MRSA) isolates from bovine mastitis. A total of 160 milk samples collected
aseptically from mastitis-affected cows at organised dairy farms in and around Jammu were subjected to microbial culture
to isolate and identify methicillin-resistant S. aureus. Out of the total 52 Staphylococcus aureus isolates, methicillin
resistance (MRSA) was recorded in only 17 (32.69%) isolates, out of which 7 (41.17%) isolates carried the mecA gene. The
highest sensitivity towards enrofloxacin (100%) and lowest towards penicillin (5.88%) were recorded. Multidrug resistance
(MDR) was observed in 82.35% MRSA isolates. It is concluded that enrofloxacin is the most effective drug against mastitis
in terms of sensitivity. However, judicious use of antibiotics is required to prevent the development of resistance against
this drug in the near future.
Keywords: antimicrobial resistance, mastitis, methicillin-resistant, Staphylococcus aureus
1. Introduction
Bovine mastitis is an inflammatory disease of the mammary gland of dairy cows that results in a huge economic loss to
the dairy industry throughout the world, particularly affecting developing countries like India. It is caused by several infectious
(bacteria, virus, fungus, mycoplasma) and non-infectious (trauma, injury) agents. The severity of the inflammation depends
largely on the nature of the causative agent, age, breed, immunological, health, and lactation state of the animal and is
classified as clinical, subclinical (Ruegg 2017).
Bacterial pathogens are the main cause of infectious bovine mastitis, further categorised into contagious and
environmental types. Udder of the infected cows serves as the major reservoir for the contagious bacteria (Staphylococcus
aureus, Streptococcus agalactiae, Mycoplasma spp. and Corynebacterium bovis) that spread from cow to cow primarily during
milking and mostly lead to chronic sub-clinical infections (Radostits et al 2007).
Subclinical mastitis is asymptomatic, although transient episodes of abnormal milk may appear, accompanied by a
decrease in milk production and an increase in somatic cell count (SCC). Infection is caused mainly by Staphylococcus aureus
and Streptococcus (Zaatout et al 2019). Staphylococcus aureus is the leading cause of bovine mastitis with a major effect on
milk production and bulk tank somatic cell count (SCC) (Keefe 2012).
Methicillin-resistant Staphylococcus aureus (MRSA) is a serious threat to public health, resulting in hospital and
community-associated infections (Olayinka et al 2009). MRSA in livestock has emerged from the indiscriminate use of
antimicrobials as growth promoters and for preventive and therapeutic measures (Cuny et al 2015; Mehndiratta and Bhalla
2012). Methicillin resistance of Staphylococcus aureus is the result of the acquisition of Staphylococcal Cassette Chromosome
(SCC) mecA gene that alters the penicillin-binding protein (PBP2), resulting in the loss of affinity to all β-lactam antibiotics (Cuny
et al 2015; Lee 2003).
The present study was conducted to determine the phenotypic and genotypic antimicrobial resistance of Methicillin-
resistant Staphylococcus aureus (MRSA) isolates from mastitis affected dairy cows.
2. Materials and Methods
2.1. Sample collection
One hundred sixty pooled milk samples were collected from lactating crossbred Holstein Friesian dairy cows affected
with clinical and sub-clinical mastitis. After proper disinfection of the teat surface with 70% ethyl alcohol, a few streams of milk
Appl. Vet. Res. (2022) 1:e2022003 Received: January 2, 2022 | Accepted: January 20, 2022Mushtaq et al. (2022) 2
from each quarter were discarded, and 10 ml of milk was aseptically collected in sterile polyethylene screw-capped bottles
from all four quarters. The samples were kept in an icebox and immediately carried to the laboratory for analysis.
2.2. Sample processing
0.5 ml of the fresh milk sample was transferred to a test tube containing 5 ml of sterile peptone water (PW) enrichment
broth. After proper mixing, the tubes were incubated at 37 °C for 24 h, followed by inoculation of a loopful of enriched milk
sample onto a Baird-Parker agar (BPA) plate supplemented with egg-yolk tellurite emulsion (HiMedia, Mumbai). Moreover, the
inoculated plates were incubated at 37 °C for 24-48 h.
2.3. Isolation of Staphylococcus aureus
All the presumptive S. aureus isolates were subjected to Gram’s staining followed by biochemical characterisation using
catalase, oxidase, DNase and coagulase tests. Molecular confirmation of the S. aureus isolates was done by targeting the
species-specific thermonuclease gene (nuc gene) using PCR. The DNA was extracted from the isolates by the snap and chill
method.
2.4. In-vitro drug sensitivity of S. aureus isolates and detection of MRSA isolates
All the S. aureus isolates were examined for their antibiogram pattern by disc diffusion method as described by Bauer
et al (1966) against a panel of 13 antibiotics (Table 1). The isolates that exhibited resistance towards methicillin were
categorised as MRSA. Antibiotic discs were obtained from HiMedia Lab, Mumbai.
Table 1 Antibiotics and their concentration used in disc diffusion method.
S. No. Name of the antibiotic Concentration (µg)
1 Methicillin (MET) 5
2 Tetracycline (TE) 30
3 Gentamicin (GEN) 10
4 Enrofloxacin (EX) 5
5 Penicillin G (P) 10 Units
6 Streptomycin (S) 25
7 Amoxyclav (Amoxicillin + clavulanic acid) (Amc) 30
8 Ciprofloxacin (CIP) 5
9 Ceftriaxone (CTR) 30
10 Vancomycin (VA) 10
11 Erythromycin (E) 15
12 Chloramphenicol (C) 30
13 Ampicillin (AMP) 10
2.5. PCR detection of nuc and mecA genes in S. aureus and MRSA isolates
PCR amplification was performed using a 25 μl reaction containing a final concentration of 0.2 mM dNTPs, 2.5 mM
MgCl2, 1× buffer, 25 μM of each forward and reverse primer, 1 U of Taq polymerase, 2 μl template DNA and the sterile
nuclease-free water was added to make up the reaction volume. PCR amplification was performed in DNA thermal cycler. The
cycling conditions included an initial denaturation of DNA at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for
1 min, annealing at 55 °C for 30 sec and extension at 72 °C for 45 sec, followed by a final extension of 7 min at 72 °C and hold
at 4 °C.
3. Results and Discussion
52 (32.5%) Staphylococcus aureus isolates were obtained out of 160 pooled milk samples collected from mastitic cows
based on the morphological, biochemical and molecular characterisation. Sharma and Sindhu (2007), Abera et al (2010) and
Baghel et al. (2018) reported the recovery rate of S. aureus as 38.81%, 42.14% and 35.87%, respectively, which corroborate
with the findings of the present study.
All the presumptive S. aureus isolates (52) subjected to identification of species-specific (nuc) gene revealed an amplicon
size of 270 bp (Figure 1). Kuyma et al (2003) reported similar findings, who detected the PCR product, a single DNA band of 270
bp in all the 29 S. aureus strains (100%) isolated from milk samples.
https://www.malque.pub/ojs/index.php/avrMushtaq et al. (2022) 3
The antibiotic susceptibility pattern of the 52 S. aureus isolates determined by the disc diffusion method revealed the
highest sensitivity towards enrofloxacin (100%) and highest resistance towards penicillin G (88.46%) (Figure 2). One of the
plausible explanations for this is that the newer chemotherapeutic agents like enrofloxacin, vancomycin, ciprofloxacin and
chloramphenicol are less commonly used to treat mastitis resulting in higher efficacy of these drugs. In contrast, the frequent
use of some antibiotics like penicillin, streptomycin, amoxiclav and methicillin in animals results in their ineffectiveness against
bacterial isolates. Kumar et al (2011) reported a similar antibiogram pattern, who observed the highest sensitivity to
vancomycin (100%). Bhat et al (2017) reported a similar antibiotic sensitivity pattern of isolates recovered from clinical mastitis
cases in bovines of the Jammu region with a maximum sensitivity of the isolates towards enrofloxacin, gentamicin, amoxicillin/
sulbactam, ceftriaxone/tazobactam, ceftizoxime, ampicillin/sulbactam and least sensitivity towards oxytetracycline and
penicillin. Chandrasekaran et al (2014) also reported maximum sensitivity of S. aureus isolates towards enrofloxacin (79.8%)
and highest resistance towards penicillin (63.5%).
Figure 1 PCR amplification of nuc gene at 270 bp. Lane M - 100bp DNA ladder; Lane P - Positive control; Lane N - Negative
control; Lane 1-7 - Positive isolates
Multidrug resistance (MDR), i.e. resistance to 3 or more than 3 antibiotics, was also shown by the S. aureus and MRSA
isolates. MRSA strains show multidrug-resistant (MDR) due to the presence of other resistance genes on the cassette
chromosome harbouring the mecA gene (Holmes and Zadoks 2011). Kozerski et al (2014) reported similar findings of 12
(24.50%) isolates (7 S. aureus and 5 CoNS) that were resistant to more than three classes of antibiotics. On the contrary, Umaru
et al (2016) reported a higher (96.4%) prevalence of MDR in S. aureus isolates. Methicillin-resistant Staphylococci frequently
show multiple resistance to several classes of antimicrobial agents, including aminoglycosides, clindamycin, macrolides,
quinolones, sulfonamides and tetracycline (CLSI 2012), leaving only vancomycin as the drug of choice against these agents.
Figure 2 Antibiotic sensitivity of Staphylococcus aureus (n=52 isolates).
In the present study, methicillin resistance (MRSA) was recorded in only 17 (32.69%) out of the 52 S. aureus isolates.
The antibiogram pattern of MRSA isolates showed the highest sensitivity towards enrofloxacin and vancomycin (100% each)
https://www.malque.pub/ojs/index.php/avrMushtaq et al. (2022) 4
followed by Streptomycin (88.23%), Ciprofloxacin and Chloramphenicol (82.35% each), Tetracycline (70.58%), Ceftriaxone and
Gentamicin (47.05% each), Erythromycin (41.17%) and Amoxyclav (23.52%). MRSA showed the highest resistance towards
Penicillin G and Ampicillin (94.11% each) (Figure 3). The present findings follow those of Joshi et al (2013), who observed 100%
susceptibility of MRSA isolates towards Vancomycin.
Figure 3 Antibiotic Sensitivity of MRSA (n=17 isolates).
Methicillin resistance was detected in only 7 (41.17%) out of the 17 MRSA isolates based on PCR amplification of the
mecA gene at 532 bp. Our findings corroborate those of Koupahi et al (2016), who observed the presence of the mecA gene in
105 (47.72%) out of 220 S. aureus isolates. Similar findings were reported by Havaei et al (2015), who identified 10 (18.52%)
mecA positive out of 54 S. aureus isolates. These results suggest the presence of some other genes like mecC responsible for
methicillin resistance in MRSA isolates, as observed by Paterson et al (2014). Another reason includes the overproduction of
the E-lactamase enzyme (Sancak 2000; Olayinka et al 2009).
4. Conclusions
The present study advocates that enrofloxacin being highly sensitive towards S. aureus and MRSA isolates, is a good
choice of antibiotic for bovine mastitis treatment. However, the drug should not be used indiscriminately to avoid future
resistance. Also, multidrug resistance (MDR) shown by S. aureus and MRSA isolates is a cause of concern, indicating a zoonotic
threat that needs to be addressed as soon as possible to prevent bovine mastitis and for ensuring public safety.
Conflict of Interest
The authors declare that there is no conflict of interest.
Funding
SKUAST-Jammu provided the funds for Ph.D. research.
References
Abera M, Demie B, Aragaw K, Regassa F, Regassa A (2010) Isolation and identification of Staphylococcus aureus from bovine mastitic milk and their drug
resistance patterns in Adama town, Ethiopia. Journal of Veterinary Medicine and Animal Health 2:29–34.
Baghel A, Chhabra D, Sharda R, Shukla S, Audarya S, Sikrodia R, Gangil R (2018) Isolation of Staphylococcus from bovine mastitis and their antibiotic sensitivity
pattern. The Indian Journal of Veterinary Sciences and Biotechnology 13:49-52
Bauer AW, Kirby WMM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardised single disc diffusion method. American Journal of Clinical
Pathology 45:493–96.
Bhat AM, Soodan JS, Singh R, Dhobi IA, Hussain T, Dar MY, Mir M (2017) Incidence of bovine clinical mastitis in Jammu region and antibiogram of isolated
pathogens. Veterinary World 10:2231–916.
Chandrasekaran D, Nambi AP, Thirunavukkarasu PS, Vairamuthu S, Venkatesan P, Tirumurugaan KG (2014) A study on treatment of resistant mastitis in dairy
cows. Journal of Applied Research in Clinical and Experimental Therapeutics 6:786–91.
Clinical Laboratory Standards Institute (CLSI) (2012) Performance Standards for Antimicrobial Susceptibility Testing. 15th Informational Supplement.
CLSI/NCCLS document. Pennsylvania, USA.
Cuny C, Wieler LH, Wolfgang W (2015) Livestock-associated MRSA: the impact on humans. Antibiotics (Basel) 4:521-543.
Havaei SA, Assadbeigi B, Esfahani BN, Hoseini NS, Rezaei N, Havaei SR (2015) Detection of mecA and enterotoxin genes in Staphylococcus aureus isolates
associated with bovine mastitis and characterization of Staphylococcal cassette chromosome mec (SCCmec) in MRSA strains. Iranian Journal of Microbiology
https://www.malque.pub/ojs/index.php/avrMushtaq et al. (2022) 5 7:161–67. Holmes MA, Zadoks RN (2011) Methicillin resistant S. aureus in human and bovine mastitis. Journal of Mammary Gland Biology and Neoplasia 16:373–82. Joshi S, Ray P, Manchanda V, Bajaj J, Chitnis DS, Gautam V, Goswami P, Gupta V, Harish BN, Kagal A, Kapil A, Rao R, Rodrigues C, Sardana R, Devi KS, Sharma A, Balaji V (2013) Methicillin resistant Staphylococcus aureus (MRSA) in India: Prevalence and susceptibility pattern. Indian Journal of Medical Research 137:363–69. Keefe G (2012) Update on control of Staphylococcus aureus and Streptococcus agalactiae for management of mastitis. Veterinary Clinics of North America: Small Animal Practice 28:203–16. Koupahi H, Jahromy SH, Rahbar M (2016) Evaluation of different phenotypic and genotypic methods for detection of methicillin resistant Staphylococcus aureus (MRSA). Iranian Journal of Pathology 11:370–66. Kozerski ND, Oliveira JLPD, Moura RA, Silva DRD, Oliveira AFD, Mello PL, Agostinis R O, Goncalves DD, Otutumi LK, Martins LDA (2014) Antimicrobial profile of multidrug resistant Staphylococcus spp. isolated from bovine mastitis cases in the northwest region of Parana state, Brazil. African Journal of Microbiology Research 8:3392–97. Kumar R, Yadav BR, Singh RS (2011) Antibiotic resistance and pathogenicity factors in Staphylococcus aureus isolated from mastitic Sahiwal cattle. Journal of Biological Sciences 36:175–88. Kuyma K, Malinowski E, Lassa H, Kossowska A (2003) Specific detection of Staphylococcus aureus by PCR in intramammary infection. Bulletin of the Veterinary Institute in Pulawy 47:183–90. Lee JH (2003) Methicillin (oxacillin) resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans. Applied and Environmental Microbiology 69:6489-6494. Mehndiratta PL, Bhalla P (2012) Typing of Methicillin resistant Staphylococcus aureus: a technical review. Indian Journal of Medical Microbiology 30:16-23. Olayinka BO, Olayinka AT, Obajuluwa AF, Onaolapo JA, Olurinola PF (2009) Absence of mecA gene in methicillin-resistant Staphylococcus aureus isolates. African Journal of Infectious Diseases 3:49-56. Paterson GK, Harrison EM, Holmes MA (2014) The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends in Microbiology 22:42–47. Radostits OM, Gay CC, Hinchcliff KW, Constable PD (2007) Veterinary Medicine: A text book of the disease of cattle, horses, sheep, pigs and goats. 10th ed. London: Elsevier Ltd. Ruegg PL (2017) A 100-year review: mastitis detection, management, and prevention. Journal of Dairy Science 100:10381–10397. Sancak B (2000) S. aureus dametisilindirençmekanizmalari. Mikrobiyoloji Bulteni 34:381–89. Sharma A, Sindhu N (2007) Occurrence of clinical and subclinical mastitis in buffaloes in the state of Haryana (India). Itali an Journal of Animal Science 6:965- 967. Umaru GA, Kwaga JKP, Bello M, Raji MA, Maitala YS (2016) Antibiotic resistance of Staphylococcus aureus isolated from fresh cow milk in settled Fulani herds in Kaduna state, Nigeria. Bulletin of Animal Health and Production in Africa 64:173–82. Zaatout N, Ayachi A, Kecha, Kaldec K (2019) Identification of Staphylococci causing mastitis in dairy cattle from Algeria and characterization of Staphylococcus aureus. Journal of Applied Microbiology 127:1305-1314. https://www.malque.pub/ojs/index.php/avr
You can also read
