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

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

*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.

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.