مطالعه برخی خصوصیات استافیلوکوکسی‏ها‏ی جدا شده از شیر میش‏ها‏ی مبتلا به ورم پستان تحت بالینی در شهرکرد، ایران

نویسندگان

1 استادیار میکروبیولوژی، دانشگاه شهرکرد، ایران

2 دکتری حرفه ای دامپزشکی، دانشگاه شهرکرد، ایران

3 دانشیار مامایی و بیماری‏های تولید مثل، دانشگاه شهرکرد، ایران

4 دانشجوی کارشناسی ارشد باکتری شناسی، دانشگاه شهرکرد، ایران

چکیده

  مقدمه: استافیلوکوکسی‏ها‏ آنزیم‏ها‏ی فراوانی در محیط آزاد می‏کنند که برخی از آن‏ها نظیر کوآگولاز، بتا لاکتاماز، همولایزین‏ها‏ و بیوفیلم‏ها‏ جزء اندیس‏ها‏ی بیماری ‏ زایی این باکتری‏ها‏ محسوب می ‏ شوند. هدف مطالعه حاضر، جدا‏سازی و تشخیص استافیلوکوکوس اورئوس و استافیلوکوکوس‏ها‏ی کوآگولاز منفی از گوسفندان مبتلا به ورم پستان تحت بالینی و آزمایش تولید بیوفیلم، بتالاکتاماز و همولایزین‏ها‏ و نیز الگوی مقاومت آنتی بیوتیکی آن‏ها می‏باشد .   مواد و روش ‏‏ ها: در این مطلعه، 55 جدایه استافیلوکوکسی به‏‏دست آمد که 33 مورد آن (60 درصد) استافیلوکوکوس اورئوس و 22 مورد (40 درصد) استافیلوکوکسی کوآگولاز منفی تشخیص داده شد. فعالیت همولایتیک جدایه‏ها‏ با کشت آن‏ها بر سطح محیط ژلوز خون‏دار (تهیه شده با خون گاو) ارزیابی شد. تولید بیوفیلم با روش پلیت میکروتیتر ارزیابی و تولید بتا لاکتاماز با روش یودومتریک و حساسیت آنتی بیوتیکی با روش دیسک دیفیوژن تعیین شد .   نتایج: 26 مورد (8/78) از جدایه‏ها‏ی استافیلوکوکوس اورئوس و 9/59 درصد از استافیلوکوکسی‏ها‏ی کوآگولاز منفی قادر به تولید بیوفیلم بودند. از جدایه‏ها‏ی استافیلوکوکوس اورئوس دو مورد همولیز آلفا (06/6 درصد)، دو مورد همولیز بتا و 6 مورد (2/18 درصد) همولیز دلتا تولید کردند. از استافیلوکوکسی‏ها‏ی کوآگولاز منفی 6 جدایه (27/27) همولیز آلفا و 10 جدایه (45/45 درصد) همولیز دلتا تولید کردند. در جدایه‏ها‏ی استافیلوکوکوس اورئوس 26 مورد (8/78 درصد) و استافیلوکوکسی‏ها‏ی کوآگولاز منفی پنج مورد (72/22 درصد) تولید آنزیم بتا لاکتاماز کردند. استافیلو کوکسی‏ها‏ی جدا شده حساسیت پایینی نسبت به متی سیلین و استرپتومایسین نشان دادند .   بحث و نتیجه ‏ گیری: درصد بالایی از جدایه‏ها‏ همولایزین آلفا تولید کردند که نقش مهمی در تولید بیوفیلم توسط استافیلوکوکوس اورئوس ایفا می نماید. 21 جدایه از 33 جدایه (63/63 درصد) استافیلوکوکوس‏ها‏ قادر به تولید بیوفیلم بودند که می‏تواند اثرات بدی داشته باشد. چون به نظر می رسد تولید بیوفیلم نقش مهمی در ابقا این باکتری داشته باشد. 16 جدایه از 33 جدایه (5/48 درصد) استافیلوکوکوس اورئوس در آزمایش تولید بتا لاکتاماز مثبت شدند. صرف نظر مقاومت به متی سیلین تمامی این جدایه‏ها‏ حساسیت قابل توجهی به آنتی بیوتیک ‏ها‏ی بتا لاکتام مورد آزمایش نشان دادند. درصد بالای تولید همولیزین، بیوفیلم و بتا لاکتاماز بیانگر نقش مهم این عوامل حدت در بیماری‏زایی استافیلوکوکسی‏ها‏ی جدا شده از نمونه شیر‏ها‏ی ورم پستان میش است .

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Study on some characteristics of Staphylococci isolated from sheep sub clinical mastitis milk in Shahrekord, Iran

نویسندگان [English]

  • azizollah Ebrahimi 1
  • Fatemeh Soleimani 2
  • Azar Moatamedi 2
  • Naser Shams 3
  • Sharareh Lotfalian 4
1 Assstant Professor of Microbiology, Institute of zoonotic diseases, Shahrekord University, Iran
2 DVM student, Shahrekord University, Iran
3 Associate Professor of Gynaecology, Shahrekord University, Iran
4 M.Sc. student of Bacteriology, Shahrekord University, Iran
چکیده [English]

  Introduction : Staphylococci release a large number of enzymes. Some of these, such as coagulase, beta lactamase, hemolysins and biofilms are considered indices of pathogenicity. The aim of the current study was based on the isolation and identification of Staphylococcus aureus and coagulase negative Staphylococci (CNS) strains from sheep sub clinical mastitis and examining their biofilm, beta lactamase, hemolysins production and antibiotic resistance pattern.   Materials and method s: 55 Staphylococci strains were isolated from seventy cases of sheep subclinical mastitis. Thirty three were determined as Staphylococcus aureus (60%) and 22 (40%) as CNS. The hemolytic activity was evaluated by plating Staphylococci strains on 5% bovine blood agar. The biofilm assay was performed by using micro titer plates . Beta Lactamase production was detected by test tube iodometric technique and susceptibility to antimicrobial agents was determined for isolated strains by the disk diffusion method.   Results : Twenty six (78.8%) S. aureus strains were biofilm producers. For CNS (59.9%) strains were positive in biofilm production. Two isolates (6.06%), of S. aureus were α, the same number β and 6 (18.2%) isolates were ∂ hemolysin producers. Six isolates of CNS (27.27%) were α and ten (45.45%) ∂ hemolysin producers . Sixteen S. aureus (48.5%) and five CNS (22.72%) isolates were positive in beta lactamase production. The isolated Staphylococci show a low sensitivity pattern to methicillin and streptomycin.   Discussion and conclusion : A high percentage of strains make α toxin that play a role in S. aureus biofilm formation. Twenty one out of 33 (63.63%) isolated Staphylococci were biofilm producers that can have deleterious effects because biofilm formation is thought to play an important role in the survival of virulent strains of Staphylococci. Sixteen out of 33 (48.5%) isolated S. aureus were positive in beta lactamase test, Excluding resistant to methicillin, all of these isolates show a marked sensitivities to other examined beta lactam drugs. High percentage of hemolysins, biofilm and beta lactamase production by isolated Staphylococci, suggest an important role of these virulence factors in the pathogenesis of isolated Staphylococci from mastitis sheep milk samples.  

کلیدواژه‌ها [English]

  • Mastitis
  • Sheep
  • Staphylococci
  • Biofilm
  • Beta lactamase
  • Hemolysin

Introduction

Sheep intra mammary infection (IMI) in its sub clinical form is the single most important factor affecting milk quality and quantity (1).

Several pathogens can cause mastitis but Staphylococcus spp. is the most frequently diagnosed causal microorganism of intra mammary infection (IMI) in sheep (2). Some species of this genus cause a variety of diseases by production of a series of enzymes and toxins, invasion of host cells and tissues.

Staphylococcal alpha-hemolysin or alpha-toxin is the most studied and characterized cytotoxin, and is considered a main pathogenicity factor because of its hemolytic, dermonecrotic and neurotoxic effects (3). Additionally, beta-hemolysin is a sphingomyelinase that is highly active against sheep and bovine erythrocytes (4), while delta -hemolysin as well as alpha-hemolysin induce pore formation, perturbing the cell membrane permeability (5).

The biofilms consisted of micro colonies encased in extracellular polysaccharide material.

Bacteria inside biofilms have increased resistance to antimicrobial agents. The production of biofilms was correlated with pathogenicity and virulence of bacteria (6).

Many common bacterial pathogens exist in animals as biofilms. Mastitis is a typical animal disease where bacterial biofilms are believed to be involved based on histopathologic and ultra structural appearance of the bacteria within tissue (7).

The public health significance of Staphylococci isolated from milk and dairy products is important because these products can be a source of toxins and antibiotic-resistant strains for humans (8).

To treat mastitis successfully, means knowing the prevalence and the antimicrobial susceptibility patterns of microorganisms isolated from the mammary gland.

The present study was undertaken in order to determine some phenotypic characteristics of Staphylococci isolates from sheep sub clinical mastitis in Shahrekord district in south west of Iran.

 

Materials and methods

 

Sample collection

 The study covered 16 months from March 2011 to June 2012. A preliminary analysis for presence of sub clinical mastitis was done in 600 randomly selected native lactating sheep from 16 free ranging flocks in Shahrekord district, south west of Iran, via California mastitis test (CMT) (9).

CMT positive milk samples were aseptically obtained in sterile 10 ml tubes. The ice box containing milk samples were sent to microbiology lab of Veterinary College of Shahrekord university for the isolation of Staphylococcus species. In laboratory aliquots of 0.01 ml (10) of milk were streaked on blood agar plates containing 5% defibrinated sheep blood, (BA), (Merck, Darmstadt, Germany). The incubation was done aerobically at 37 °C for 24-48 h. The presence of more than 3 colonies of a similar morphotype was accepted as positive bacteriological finding (10).

 

Identification of Staphylococcus species

One colony from similar morphotype colonies growing on above BA was subcultured on freshly prepared plates of mannitol salt agar (MSA), (Merck, Darmstadt, Germany) and incubated again aerobically at 37 °C for 24-48 h. Primary characterization of isolates was based on the gram stain, morphological and cultural characteristics. Colonies were tested with slide coagulase (using rabbit plasma) test (11).The catalase and oxidase tests were followed by examining the susceptibility profile of isolates towards bacitracin according to Quinn et al. (11).

The isolates were kept frozen at −70 °C in Tryptic soy broth containing 15% (v/v) glycerol, until the further examinations were carried out.

 

Assay for hemolytic activity

The hemolytic activity was evaluated by plating Staphylococci strains on 5% bovine blood for alpha and beta hemolysin production. The criteria for hemolysin identification were: complete lytic zone (transparent) with blurred edges for alpha-hemolysin and incomplete (non-transparent) lytic zone, which became complete with sharp edges after overnight incubation at 4 °C, for beta-hemolysin (12). Delta hemolysin was determined by using the synergistic hemolysis method described by Hébert and Hancock (13), Fig. 1.

 

 

Fig. 1- Delta hemolysin determined by using the synergistic hemolysis of beta-hemolytic S. aureus seeded vertically (center) and the samples to be tested perpendicularly. Positive strains showing an increase in their hemolysis area at the extremity close to the beta-hemolytic S. aureus. Photo is taken from our experiments.

 

Antimicrobial Susceptibility testing

For antimicrobial susceptibility testing, isolates were incubated in Tryptic soy broth at 37 °C for 24 h and the suspension was adjusted to a turbidity equivalent to a 0.5 McFarland standard. Susceptibility to antimicrobial agents was determined for isolated strains by the disk diffusion method on Mueller-Hinton (MH) agar, (Merck, Darmstadt, Germany), following the Clinical and Laboratory Standards Institute (CLSI), (14). The selected antibiotics for antibiogram were amoxicillin, kanamycine, penicillin, ciprofloxacine, tetracycline, gentamicin, methicillin, erythromycin, streptomycin and oxaciline.

Isolates were categorized as susceptible and resistant based upon interpretive criteria developed by the CLSI (14).

 

Biofilm and beta lactamase assays

Beta lactamase production was detected by test tube iodometric technique as described in reports (15).

The biofilm assay was performed by using micro titer plates as described by Tendolkar et al (16). Interpretation of biofilm production was according to the criteria's described by Stepanovic et al. (17). Based on these criteria's ODc (optical density cut-off value) is defined as: average OD of negative control + 3 × SD (standard deviation) of negative control, and the biofilms producers are categorized as: negative ≤ ODc, weak ODc < ~ ≤ 2 × ODc, moderate 2 × ODc < ~ ≤ 4 × ODc and strong biofilm producer > 4 × ODc. While "~ " stands for average of sample ODs.

 

Results

 

From 600 examined lactating sheep seventy (11.66%) were CMT positives which followed by bacteriological examinations. Cultures from fifty five CMT positive samples (78.6%) lead to isolation and identification of Staphylococci. Out of these 55 isolates, 33 (60℅) were S. aureus and 22 (40℅) were coagulase negative Staphylococci, (CNS). Details for hemolysins, biofilm and beta lactamase production by isolated Staphylococci is appeared in table 1.

Table 1: Hemolysin, biofilm and β-lactamase production by isolated Staphylococci from sub clinical mastitis sheep

Hemolysin

α no. (%)

β no. (%)

∂ no. (%)

αβno. (%)

S. aureus

2(6.06)

2(6.06)

6(18.18)

0(0)

CNS*

6(27.2)

0 (0)

10(45.4)

0 (0)

Biofilm

Strong no. (%)

Medium no (%)

Weak no (%)

Total no. (%)

S. aureus

0(0)          

6(18.1)

20(60.6)

26(78.8)

CNS

0(0)          

1(4.5)

12(54.5)

13(59.09)

β-lactamase

Strong no. (%)

Medium no. (%)

Weak no. (%)

Total no. (%)

S. aureus

8(24.2)

6(18.1)

2(6.06)

16(48.4)

CNS

2(9.09)

2 (9.09)

1(4.5)

5(22.7)

 

*Coagulase negative Staphylococci

 

In total 8 Staphylococci isolates were α hemolysin positives out of them six (75%) were biofilm producers simultaneously.

Totally thirty nine out of 55 (70.9%) isolated Staphylococci were biofilm producers, out of them 16 isolates (41%) were positive in beta lactamase test.

Out of 33 isolates of S. aureus 16 (48.4) MRSA and 5 (15.1%) MSSA were recorded. The numbers for 22 isolates of CNS were 0 and 10 (45.4%) respectively.

Table 2 shows the result of overall antimicrobial susceptibility patterns irrespective of ß-lactamase production.

 

Table 2: Antibiotic susceptibility responses of isolated Staphylococci from sub clinical mastitis sheep irrespective of ß-lactamase production.

 

Staphylococci

S. aureus CNS

Antibiotics

 

S

no (%)

NS

no (%)

S

no (%)

NS

no (%)

Amoxicillin

32 (96.96)

0 (0.0)

22 (100)

0 (0.0)

Kanamycine

26 (78.8)

0 (0.0)

17 (77.2)

0 (0.0)

Penicillin

27 (81.8)

0 (0.0)

18 (81.8)

0 (0.0)

Ciprofloxacine

33 (100)

0 (0.0)

22 (100)

0 (0.0)

Tetracycline

25 (75.5)

4 (12.1)

20 (90.9)

0 (0.0)

Gentamicin

28 (84.8)

0 (0.0)

20 (90.9)

0 (0.0)

Methicillin

5 (15.1)

16 (48.4)

10 (45.4)

0 (0.0)

Erythromycin

11 (33.3)

20 (60.6)

18 (81.8)

2 (9.1)

Streptomycin

16 (48.4)

2 (6)

7 (31.8)

2 (9.1)

Oxacilin

26 (78.7)

7 (21.2)

18 (81.8)

2 (9.1)

Ofloxacine

29 (87.8)

0 (0.0)

21 (95.4)

0 (0.0)

S= sensitive   NS = non sensitive

The S. aureus isolates show a low sensitivity pattern to methicillin, erythromycin and streptomycin, while for CNS isolates only methicillin and streptomycin show this pattern.

 

Discussion and conclusion

 

Sub clinical mastitis in small ruminant average from 5 to 30% (2), this rate covers our result of 11.66%, but is higher than our previous report of 4.75% (18).

The prevalence of bacterial isolates from clinically normal small ruminant's milk is affected by factors such as different hygiene and management practices followed on each farm, age and parity of the animal and type of milking (19,20).

In total twenty one out of 33 (63.63%) isolated Staphylococci were biofilm producers. This phenomenon can have deleterious effects because biofilm formation is thought to play an important role in the survival of virulent strains of Staphylococci. Moreover, biofilm formation has been shown to be positively correlated with resistance to antimicrobial agents (21). We don’t recommend methicillin, erythromycin and streptomycin for treating ewe's mastitis cases because of low sensitivity pattern of recovered agents to these antimicrobials.

Eight out of 33 (24.248%), of our S. aureus isolates were α, hemolysin producers, out of them, 6 (75%) were biofilm producers simultaneously. Nicky and Toole (22) showed a role for α hemolysin in S. aureus biofilm formation and that this toxin appears to be required for cell-to-cell interactions. A high percentage of strains make this toxin, and it is toxic to a wide range of mammalian cells, but environmental factors appear to play a role in alpha-hemolysin expression (3).

Only 6% of S. aureus isolates were positive in β hemolysin production that is much lower from bovine mastitis isolates reported elsewhere (4). Regarding ovine isolates published data is scarce.

Six out of 33 (18.18%) of our S. aureus isolates were ∂ hemolysin producers, This is much lower from reports that recorded 80%- 97% ∂ hemolysin production by S. aureus isolates (23). This toxin is capable of causing membrane damage in a variety of mammalian cells, as well as sub cellular structures such as membrane-bound organelles, spheroplasts, and protoplasts (24).

George et al, reported absent of delta-hemolysin expression in S. aureus isolates suggests that relative gene function is suppressed in these isolates (25).

Ten out of 22 (45.45%) isolates of CNS were ∂ hemolysin producers. Reports estimated a 40%- 80% of CNS have the ability to produce this toxin (26), with a detergent action on the membranes of various cell types. It is indicated that different genes might be responsible for the production of this toxin in different CNS species (27).

Sixteen out of 33 (48.5%) isolated S. aureus were positive in beta lactamase test, excluding resistant to methicillin, all of these isolates show a marked sensitivities to other examined beta lactam drugs. The sensitivities may be due to high level of penicillin receptors (PBPs) or high accessibility of these receptors due to a lack of permeability barriers created by autolytic enzymes in the cell wall, which can result in killing bacteria and contribute to high sensitivity rates seen in ß-lactamase producers.

In conclusion, the high percentage of hemolysins, biofilm and beta lactamase production by isolated Staphylococci obtained in this work; suggest an important role of these virulence factors in the pathogenesis of isolated Staphylococci from ewe's mastitis milk samples. The S. aureus isolates show a high sensitivity pattern to most examined antibiotics. Methicillin followed by erythromycin and streptomycin were found to be low active drugs against isolates from mastitis milk samples.

References

 (1)  Leitner G, Chaffer M, Shamay A, Shapiro FMerin UEzra E et al. Changes in milk composition as affected by sub clinical mastitis in sheep. J. Dairy Sci. 2004; 87 (1): 46–52.

(2)  Contreras A, Sierra D, Sanchez A, Corrales JC, Marco JC, Paape MJ. et al. Mastitis in small ruminants. Small Rumin. Res. 2007; 68 (1): 145–53.

(3)  Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus, Clin. Microbiol. Rev. 2000; 13 (1): 16–34.

(4)  Larsen HD, Aarestrup FM, Jensen NE. Geographical variation in the presence of genes encoding super antigenic exotoxins and β-hemolysin among Staphylococcus aureus isolated from bovine in Europe and USA, Vet. Microbiol. 2002; 85 (1): 61–7.

(5)  Butt H, Dunstan RH, Mcgregor NRRoberts TKZerbes MKlineberg IJ. An association of membrane-damaging toxins from coagulase-negative Staphylococci and chronic orofacial muscle pain, J. Med. Microbiol. 1998; 47 (7): 577–84.

(6)  Saginur R, Stdenis M, Ferris W, Shawn D. AFrancis CCraig L. et al. Multiple combination bactericidal testing of staphylococcal biofilms from implant associated infections. Antimicrob. Agents Chemother.2006; 50 (1):55-61.

(7)  Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science 1999; 284 (5418): 1318-22.

(8)  Thatcher FS, Simon W. The resistance of Staphylococci isolated from cheese to various antibiotics, Can. J. Public Health. 1955; 46 (10): 407-09.

(9)  Keisler DHM, Andrews L, Moffatt R J. Subclinical mastitis in ewes and its effect on lamb performance. J Anim Sch. 1992; 70 (6):1677-1681.

(10)             Deinhofer M, Pernthaner A. Staphylococcus spp. as mastitis-related pathogens in goat milk, Vet Microbiol. 1995; 43 (2-3): 161-66.

(11)             Quinn PJ, Carter ME, Markey B, Carter GR. Clinical Veterinary Microbiology. Section 2. Bacteriology, 8. Staphylococcus species. London, UK: Wolf/Mosby, 1994; 118-26.

(12)             Balows A, Hausler WJ, Kenthl JR, Isenberg HHD, Shadomy HJ. Manual of clinical microbiology 5th ed. AMS 1991; 228-29.

(13)             Herbert GA, Hancock G. Synergistic hemolysis exhibited by species of Staphylococci. J. Clin. Microbiol 1985; 22 (3): 409-15. 

(14)             Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing 2011; M100-S21 Vol. 31.

(15)             David M. L, Derek F. J. B. Detection of β-lactamase-mediated resistance. J. antimicrob. chemother. 2001; 48 (suppl 1): 59-64.

(16)             Tendolkar PM, Arto S, Baghdayan M.S, Gilmore N.S. Enterococcal surface protein enhances biofilm formation by Enterococcus faecalis. Infect. Immun. 2004; 72 (10): 6032-39.

(17)             Stepanovic SVuković DHola VDi Bonaventura GDjukic SCirkovic I. et al. Quantification of biofilm in micro titer plates: overview of testing conditions and practical recommendations for assessment of biofilm production by Staphylococci. APMIS 2007; 115 (8): 891-99.

(18)             Ebrahimi A, Lotfalian S, Karimi S. Drug resistance in isolated bacteria from milk of sheep and goats with sub clinical mastitis in Shahrekord district. IJVR. 2007; 8 (18): 76-9.

(19)             Boscos C, Stefanakis A, Alexopoulos C,  Samartzi F. Prevalence of sub-clinical mastitis and influence of breed, parity, stage of lactation and mammary gland bacteriological status on Coulter Counts and California Mastitis Test in the milk of Saanen and autochronous Greek goats. Small Rum. Res. 1996; 21 (2): 139 –47.

(20)             Gebrewahid, TT, Abera BH, Menghistu HT. Prevalence and etiology of subclinical mastitis in small ruminants of Tigray regional state, north Ethiopia. Vet.World. 2012; 5 (2):103-09.

(21)             Moretro THermansen LHolck ALSidhu MSRudi KLangsrud S. Biofilm formation and the presence of the intercellular adhesion locus ica among Staphylococci from food and food processing environments. Appl Environ Microbiol. 2003; 69 (9): 5648–55.

(22)             Nicky CC,’Toole GAO. Alpha toxin is required for biofilm formation by Staphylococcus aureus. J. Bacteriol. 2003; 185 (10): 3214-17.

(23)             Dinges M, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clin.Microbiol.Rev. 2000; 13 (1): 16–34.

(24)             Dongyou L. Molecular detection of human bacterial pathogens. 1th ed. CRC Press 2011; 309- 10.

(25)             Sakoulas GEliopoulos GMMoellering RC JrWennersten CVenkataraman LNovick RP, et al. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob Agents Chemother. 2002; 46 (5): 1492–502.

(26)             Herbert GA, Hemolysins and other characteristics that help differentiate and biotype Staphylococcus lugdunensis and Staphylococcus schleiferi. J.Clin.Microbiol. 1990; 28 (11): 2425–31.

(27)             Marconi C, Cunha MLRS, Araujo JrJP, Rugolo LMSS. Standardization of the PCR technique for the detection of delta toxin in Staphylococcus spp. J. Venom. Anim. Toxins incl. Trop. Dis. 2005; 11 (2): 117-28.