Detection of Altered Pattern of Antibiogram and Biofilm Character in Staphylococcus aureus Isolated From Dairy Milk

1Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan-38000 2Department of Medicine, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan-63100 3Government College Women University, Faisalabad 4Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan-38000 5Department of Zoology, Rawalpindi Women University, Rawalpindi, Pakistan Article Information Received 03 December 2019 Revised 25 January 2020 Accepted 04 February 2020 Available online 04 June 2020


INTRODUCTION
S taphylococcus aureus has emerged as superbug of animal and human that is compromising health and economy (Aqib et al., 2018). S. aureus has various pathogenic attributes major of which are multidrug resistance and biofilm production (Munita et al., 2015). The latter becomes more of concern due to its ability to minimize antibiotics' effect, colonization to epithelial lining, longer persistence, evading immune response, and boosting of pathogenesis (Melchior et al., 2006). Such resistant strains are distinguished by systemic heterogenicity, genetic variety, interactions between complex community and the extracellular matrix of macromolecular substances (Begum et al., 2007). Studies report it to be second most etiology accounting to 17 million annual human deaths, while on the other hands it stands to be pertinent global problem in dairy milk production (Cosandey et al., 2016).
The emergence of resistive S. aureus strain in dairy has tuned to 61% in some of countries with fear to go rise as in case of prevailing risk factors (Aqib et al., 2018). It seems to be mushrooming as a pandemic. Such devastating O n l i n e

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scenario is presumed to be due to be multifactorial (Marques et al., 2007). From which, mainly in concern is biofilm production (Melchior et al., 2006). The ability of biofilm production may be strain specific or genetical trait of strain. Bacteria in biofilms use dense extracellular matrix to protect themselves from antibiotics (Vancraeynest et al., 2004). The resistance to antimicrobials ranges between 100-1000 times in biofilm enclosed pathogenic strains than those of planktonic cells (Begum et al., 2007). Moreover, the strains are responsible for transfer of resistance to the interacting bacteria within biofilm (Munita et al., 2015). Biofilm is reported to be well established even in case of lower number of somatic cell count representing biofilm microbes surviving in udder and contributing in prolong sustainment of pathogen at farm (Melchior et al., 2006). Commonly practiced antibiotics in mammary infections are macrolides, fluoroquinolones, streptogramins, beta-lactam, lincosamides and beta-lactams that are now facing resistance. Usage of these antibiotics at subinhibitory level makes the scene worsen (Kumar et al., 2010). It is reported that the production of biofilm can be enhanced by sub-inhibitory concentration of antibiotics. Also, the recurrence of mastitis has been attributed to the subinhibitory concentrations of antibiotics that induces biofilm character to get established (Vancraeynest et al., 2004).
Strategies to cop this scenario can be implemented more effectively by understanding the prevalence of genetic patterns, availability and susceptibility of genes expression to antibiotics which are facing resistance, addressing the risk factors associated with spread of biofilm producing microbes in routine dairy analysis (Aqib et al., 2018). Similarly, antibiotics must be evaluated against biofilm producing S. aureus. Current study was planned to estimate prevalence and prevailing risk factors of biofilm producing S. aureus of dairy origin, and to find comparative evaluation of antibiotics' efficacy against biofilm S. aureus.

Sampling
The sampling areas included were various small animal holders (having 1-5 animals) and accessible farms located in the jurisdiction of district Nankana Sahib, district Okara and district Faisalabad. These districts were selected based on higher dairy population and accessibility to dairy animals. A Total of n=250 milk samples were collected from dairy animals (n=90 cattle, n=160 buffalo) that were positive for subclinical mastitis using purposive sampling method of non-probability sampling technique. These samples were screened by Surf Field Mastitis Test for subclinical mastitis, as the test has been used in recent studies (Aqib et al., 2017(Aqib et al., , 2018.

Risk factors analysis
Predesigned dichotomous questionnaires having questions of udder condition and consistency. Use of teat dip, teat abnormalities, age, parity number, lactation stage, system of rearing, tick infestation, body condition, feeding, owner knowledge about mastitis, use of therapeutic drug and treatment approach were filled on-spot to access the potential risk factors (Aqib et al., 2017).

Isolation and identification of Staphylococcus aureus
Positive samples were processed for isolation of characteristics yellow pinpoint round colonies of S. aureus. The confirmation was done using gram's staining, selective media growth and biochemical tests (i.e. Catalase test, Coagulase test) (Aqib et al., 2017(Aqib et al., , 2018.

Detection of biofilm producing Staphylococcus aureus
Biofilm production was identified by Tissue culture plate method. Briefly stating, optical density at 570nm of overnight incubated culture (150µl) in tryptic soy broth was determined using tissue culture plate reader. The culture of bacteria was poured in wells and further incubated for 24 h at 37°C. Negative control with only broth and positive control with broth seeded with strong biofilm producing standard strain was also run. Optical density at was measured after washing the wells with PBS thrice, and staining with crystal violet. Optical density < 0.12 indicate None/ Weak, 0.12-0.24 show moderate, while > 0.24 did show high biofilm production (Hassan et al., 2011).

Statistical analysis
The data obtained was analyzed by descriptive O n l i n e

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statistics for antibiotics while association of risk factors were analyzed by chi-square at 5% probability using SPSS statistical computer program (version 20).

Prevalence of biofilm producing Staphylococcus aureus in cattle and buffalo
The present study showed that amongst the 250 subclinical mastitis samples, 61.60% (154/250) were positive for S. aureus. However, the prevalence of S. aureus was found to be higher in buffalo milk samples (65.62%,105/160) than in cattle milk samples (54.44%,49/90) which was non-significant difference (p<0.05) ( Table I). There were 72.73% of S. aureus isolates positive for biofilm production. Biofilm producing stains of S. aureus isolated from cattle and buffaloes were noted to be 77.55% and 70.48%, respectively.
It was found that all the isolates from biofilm producing S. aureus (bpSA) and non biofilm producing S. aureus (nbpSA) of cattle and buffalo milk were 100% resistant to Cefotaxime, Fusidic acid, and Ampicillin. The general trend of sensitivity fell into Cefoxitin, Gentamicin, Oxytetracycline, and Trimethoprim + Sulphamethoxazole presenting 60-80% range of sensitive isolates in current study.

Comparison of antibiogram between bpSA and nbpSA
The study found overall (cattle and buffalo milk) higher resistant isolates against Vancomycin, Oxacillin, Amoxy clavulanate presenting >70% resistance while against Cefotaxime, Fusidic acid, Ampicillin 100% resistant strains from bpSA and nbpSA were noted (Table  III). The general higher trend of resistance was noted in bpSA isolates at non-significant difference (p>0.0%) against all antibiotics while comparison of bpSA and nbpSA differed significantly (p<0.05) against Trimethoprim + Sulphamethoxazole at resistant, intermediate and sensitive cadre. In case of Trimethoprim + Sulphamethoxazole, significant (p<0.05) higher percentage of resistant bpSA and intermediate bpSA while significantly (p<0.05) lower sensitive bpSA strains were noted.
Higher percentages of cattle milk based resistant isolates were noted from bpSA and nbpSA against Vancomycin, Oxacillin, Amoxy clavulanate, Cefotaxime, Fusidic acid, and Ampicillin. All the isolates from bpSA and nbpSA were resistant against the latter three antibiotics while among former three although higher percentages of bpSA were resistant but difference with nbpSA was non-significant (p>0.05). Linezolid, Cefoxitin, Gentamicin, and Trimethoprim + Sulphamethoxazole were the antibiotics that proved equally effective against both bpSA and nbpSA. Statistical analysis of comparison of bpSA and nbpSA at resistant, intermediate and sensitive cadre of isolates against all the antibiotics were non-significant (p>0.05) except Trimethoprim + Sulphamethoxazole where nbpSA showed significantly (p<0.05) higher percentage of sensitive strains than to that of bpSA isolates. The study noted most of the p values as NA (not applicable) on account of either 100% or 0.00% response at resistant, intermediate and sensitive cadre of strains against various antibiotics. The analysis did reveal that higher resistance to antibiotics existed in those strains that were even not producing biofilm.
The buffalo milk-based study showed higher percentages of sensitive strains of both biofilm producing S. aureus (bpSA) and non-biofilm producing (nbpSA) against Cefoxitin, Gentamicin, Trimethoprim + Sulphamethoxazole, Oxytetracycline in current study. While higher resistance was observed against Vancomycin, Oxacillin, Amoxy clavulanate, Linezolid with percentages to be >90, >60, 60-88, and 44%, respectively. All the isolates from both bpSA and nbpSA were 100% resistant to Ampicillin, Cefotaxime, and Fusidic acid. Statistical comparison of antimicrobial response of biofilm producing S. aureus (bpSA) and nonbiofilm producing (nbpSA) against a list of 11 antibiotics at resistant, intermediate, and sensitive cadre was quite variable depending upon isolates' origin and the kind of antibiotic. The bpSA and nbpSA isolates obtained from buffalo milk did show non-significant difference against Vancomycin, Oxacillin, Amoxy clavulanate, and Linezolid at all three cadre i.e. resistant, intermediate, sensitive. The bpSA did show significant (p<0.05) higher percentage of resistant strains against combination of Trimethoprim + Sulphamethoxazole while significant higher sensitive strains of nbpSA were noted.

Comparison of resistant, intermediate, and sensitive strains of bpSA against antibiotics
Biofilm producing S. aureus did present significant difference among all antibiotic resistant, intermediate and sensitive strains except in case of Trimethoprim + Sulphamethoxazole where these strains did nonsignificantly differ (p>0.05) (Fig. 1)

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Altered Pattern of Antibiogram and Biofilm Character in S. aureus Vancomycin, Cefotaxime, Fusidic acid, and Ampicillin, while Gentamicin and Oxytetracycline sensitive strains were found 100% from bpSA of cattle milk origin. The study noted significant difference of resistant, intermediate, and sensitive isolates of bpSA to different antibiotics except Oxacillin, Amoxy clavulanate, and Linezolid where non-significant difference (p>0.05) was observed. bpSA did show 80 percent resistant isolates against Trimethoprim + Sulphamethoxazole which was very high percentage as compared to those of cattle milkbased isolates. Resistance to Vancomycin and Linezolid was also reduced compared to that of cattle milk bpSA.

Comparison of resistant, intermediate, and sensitive strains of nbpSA against antibiotics
Statistical analysis of overall (cattle and buffalo milk) nbpSA resistant, intermediate and sensitive strains to different antibiotics showed significant difference (p<0.05) presenting >70% Vancomycin, Oxacillin, Amoxy clavulanate while 100% resistant strains to Cefotaxime, Fusidic acid, Ampicillin were noted (Fig. 2). Linezolid resistant, intermediate, and sensitive strains of nbpSA did show non-significant difference (p>0.05) presenting 5, 25, and 75% of strains, respectively. Gentamicin, Trimethoprim + Sulphamethoxazole, and Oxytetracycline sensitive strains of nbpSA were found to be 100% in current study. Cefoxitin as exceptional to that of oxacillin presented 70 and 25% sensitive and intermediate strains of nbpSA.
Cattle milk based nbpSA resistant, intermediate, and sensitive strains to various antibiotics significantly differed (p<0.05). Relative to those of buffalo milk based nbpSA isolates, the ones from cattle were lower in percentages of resistant cadre. Vancomycin resistant nbpSA were 20 unites while those of Amoxy clavulanate, Linezolid and Cefoxitin were 10 units lower in percentages compared to nbpSA of buffalo milk. Rest of resistant and sensitive cadre were like that exhibited by buffalo milk based nbpSA.
Comparison of resistant, intermediate, and sensitive strains of nbpSA to different antibiotics showed significant difference (p <0.05) in buffalo milk except that of Linezolid where non-significantly (p >0.05) higher percentage of resistant isolates was noted. In addition to 100% resistant O n l i n e

Risk factor analysis
Statistical analysis of assumed risk factors showed significant (p<0.05) association of teat dipping, tick infestation, body condition, and therapeutic drug use in causing mastitis with spread of S. aureus in dairy milk. On other hands, age, lactation stage, system of rearing, and feeding management did not show significant (p>o.05) association with the spread of S. aureus isolated from mastitis milk. All fibrosed udders presented 100% involvement of S. aureus while the normal udder presented 57.66% of cases associated with bacterial spread. Animals having weak body condition, greater number of parities, had higher percentages of S. aureus involvement (Table  II).

DISCUSSION
Staphylococcus aureus continues to pose major public health challenges in many areas because of antibiotic resistance problems. Findings of higher rate of Staphylococcus prevalence in subclinical mastitis was in line with recent studies (Aqib et al., 2017(Aqib et al., , 2018.

Prevalence of biofilm character
Higher prevalence of S. aureus in current study could be related to higher number of significant risk factors in field condition. The salient of contributing factors included previous mastitis disease history, lack of knowledge about disease, breed, lactation stage, udder anomalies, tick infestation, and lack of teat dipping which prone the animal to infection and aggravate the pathogen persistence in the udder (Aqib et al., 2017). Significant rise in biofilm characters has been in notice of (Marques et al., 2007) who do report that buffalo is more likely to get heaped biofilm character in milk as reported in study where icaA and icaD genes were found in 100% of tested animals. Rising biofilm was justifiable by microbial resistance, longer stay of organism in environment, lack of professional approach to deal infection, irrational antibiotics use against resistant micro-organism (Begum et al., 2007). Higher resistance to penicillin, and ampicillin by S. aureus in current study is line with reports by (Kong et al., 2016) who found 85% and 77% of isolates resistant to antibiotics. Both the biofilm production and beta lactamase coding genes group has been reported to enhance resistance against antibiotics (Marques et al., 2007). Continuous genetic variations and exotic genes uptake by S. aureus results in new phylogenetic categories in some of pathogens like those belonging to agr allele groups which encode increasing resistive pattern of this pathogen against different classes of antibiotics (Jarraud et al., 2002).

Response to antibiotics
Higher percentages of intermediate or sensitive strains to trimethoprim + sulfamethoxazole, amoxy O n l i n e

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Altered Pattern of Antibiogram and Biofilm Character in S. aureus clavulanate and oxacillin was also reported by (Jarraud et al., 2002). Some studies reported very lower percentages of resistant isolates as conducted by (Carfora et al., 2015) found 1.3% of resistant isolates. The higher percentage of resistance strains may also appear even in the absence of biofilm due to various factors inclusive of which are the high frequency gene islands like sec-seg-sei (Cosandey et al., 2016). Multiple pathogenic factors when combine simultaneously may predispose higher resistance. Zhang et al. (2018) reported that a higher portion (83.8%) of S. aureus isolates from animals show biofilm character positive for agr alleles. Most biofilm-producing isolates were positive for microbial surface component recognizing adhesive matrix molecule (MSCRAMM), variant capsule type and ica group genes. The results illustrate a significant association between the prevalence rate of MSCRAMM, capsule type and ica group genes among isolates producing weak, moderate and strong biofilms. Deceasing multidrug resistance in community clinical isolates especially in MRSA is due to successful identification and treatment protocol, frequent multidrug therapy, specificity for control, contact precautions, active surveillance and adjunctive control measures adoption (Aqib et al., 2018). Vancomycin resistance shown in the isolates is in line with previous studies. Vancomycin resistance is an emerging issue in clinical isolates of S. aureus and their number is increasing day by day. This might be due to the acquired resistance as happened in case of methicillin (Marques et al., 2013). Vancomycin resistance in S. aureus is due to acquired transposon Tn1546, from vancomycin-resistant Enterococcus faecalis, causing changes in the structure of cell wall and cellular metabolism of isolates (Gardete and Tomasz, 2014). Glycopeptide antibiotics such as Vancomycin are last resort for the severe clinical infections of MDR S. aureus in whole world. But the continuous use of Vancomycin for handling of MDR S. aureus infections has caused a decrease in Vancomycin sensitivity in many countries. Following the identification of Vancomycin intermediate-resistant S. aureus (VISA) strains for the first time in Japan in 1997, glycopeptide-resistant staphylococci strains have been major concern for the researchers as well as clinicians. A new Vancomycin resistance defined as hetero resistant VISA (hVISA) was also identified in the same year as the VISA strains (Rağbetli et al., 2016). Vancomycin resistance in S. aureus when investigated at genomic level shows that the development of vanA gene is encoding this resistive behavior (Marques et al., 2013). The excellent response to gentamicin observed during the study is supported by observations in previously conducted trial. The decease uses of gentamicin in late 1990's and apparent shift in strains of clinical isolates of S. aureus are major factors for increased gentamicin susceptibility (Gardete and Tomasz, 2014). Ampicillin resistance in clinical isolates has been reported in many studies as more than 90% isolates of animal origin are resistant to ampicillin and most susceptibility is observed in the case of tetracycline (Aqib et al., 2017). Saba et al. (2017) reported that all S. aureus isolated from public places and hospitals are 100% resistant to ampicillin, oxacillin, tetracycline and trimethoprim + sulfamethoxazole. Marques et al. (2007) reported that S. aureus isolates are highly resistant to ampicillin and harboring blaZ gene encoding for such resistive behavior. Yılmaz and Aslantaş (2017) also reported the genes involved in antibiotic resistance. Aqib et al. (2017) reported 80% of S. aureus isolates are resistant to ampicillin. Kumar et al. (2010) reported that 96.6% of S. aureus isolates are resistant to ampicillin. High resistance to Fusidic acid in clinical isolates of S. aureus can be explained on the basis of results of existing literature. Edslev et al. (2018) reported Fusidic acid in the category of antibiotics to which S. aureus isolates are highly resistant. Tremendous resistive response of S. aureus to Fusidic acid is due to mutations in fus gene islands resulting in amino acid substitutions of protein encoded. Due to this, 3 to 6 % increase in resistant clinical isolates per year is observed (Cosandey et al., 2016). Increasing Fusidic acid resistance in S aureus might be important for three reasons. First, it might mean that systemic Fusidic acid can no longer be used in situations where it is clinically indicated. Second, failure of topical treatment may be occurring, especially in primary care where treatment is often empiric, and third, resistance to Fusidic acid might be linked to other antibiotic resistances, therefore favoring spread of multiple antibiotic resistant S aureus such as MRSA (Dobie and Gray, 2004). Increasing trend of cefotaxime resistance in S. aureus isolates of animal origin has been reported that mutations in existing S. aureus isolates genome can result into extensive clinical resistance (Tomasz et al., 1989). Ishii et al. (1995) isolated and studied Toho-1 gene which encodes for cefotaxime hydrolytic enzymes and reported that replacements in such genes specifies substrate molecules. Ishii et al. (1995) also reported that more than 80% of S. aureus isolates are resistant to cefotaxime. The main reason for this could be irrational exposure of pathogen to antibiotics in clinics.
We found deceasing susceptibility trends to potentiated penicillin which is due to genetic mutation in penicillin binding proteins encoding genes. This results in altering the binding capacity of drug to the receptor proteins, leading to higher MIC value of drug for required action (Munita et al., 2015). Rağbetli et al. (2016) reported 100% penicillin resistance in clinical isolates of S. aureus. Carfora et al. (2015) reported that S. aureus is developing abilities to hydrolyze penicillin, oxacillin and cephalosporins which is now being proven by molecular studies and genes isolation of the enzymes playing important role in drug resistance. Bille et al. (1991) studied that the modified penicillin binding proteins affinity by clavulanic acid combination is the root cause of potentiated Amoxicillin spectrum maintenance which is losing its efficacy. The reasons for such response are irrational use, over and under-dosing, and continuous exposure of Amoxycillin clavulanate to microbes in the field. Oxytetracycline is one of the first line treatment choice of field workers. Rubin et al. (2011) also reported the same results showing that more than 85% S. aureus isolates are sensitive to tetracyclines. Oppliger et al. (2012) also reported that S. aureus isolates from farm workers and animal products handlers have 100% susceptibility to oxytetracycline.

CONCLUSION
Present study found higher prevalence of biofilm producing S. aureus in buffalo and cattle milk. Significant association of risk factors are also increasing which alarms emergence of resistant strains. The spectrum of antibiotic efficacy got narrowed. Some of antibiotics like Cefoxitin were found effective despite of the factor of biofilm which is prominent finding. On the other hands, resistant strains of non-biofilm S. aureus were noted against wider range of antibiotics. The pattern of antibiotics response is altering which requires immediate attention.

Statement of conflict of interest
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e
Altered Pattern of Antibiogram and Biofilm Character in S. aureus