Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers

The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains’ ability to produce β-haemolysin was evaluated with phenotypic methods (hot–cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.


Introduction
Staphylococcus pseudintermedius belongs to the coagulasepositive staphylococci and together with Staphylococcus intermedius and Staphylococcus delphini constitutes SIG group (''Staphylococcus intermedius group''). S. pseudintermedius colonizes skin and mucosal membranes of animals, notably dogs and cats, and constitutes their opportunistic pathogen [5]. This species is prevalent in a veterinary hospital environment [15,25,32], which might be connected with the fact that people having frequent contact with animals (especially pets' owners or veterinary personnel) usually become carriers of this species of bacteria [1,13,18,19]. Human infections due to S. pseudintermedius occur usually in immunocompromised patients; however, their frequency has been still increasing [29,31]. Infections in humans, such as catheter-borne bacteremia [3], sinusitis [27], infective endocarditis [20], non-hospital pneumonia [17] and wound infection after bone marrow transplantation [23] have already been noted. Further increase in the number of infections is highly possible, due to the fact that S. pseudintermedius is well equipped with various virulence factors i.e. coagulase, protease, enterotoxins, SIET exfoliative toxin, Luk-I leukotoxin and haemolysins (mainly b, but some strains also d and a) [2,8,11].
The haemolysin type that has been most precisely described in the literature is staphylococcal b-haemolysin produced by Staphylococcus aureus and it constitutes a benchmark to haemolysin studies in other species [7]. In S. pseudintermedius, similarly to S. intermedius, b-haemolysin is considered to be produced constitutively [24]. b-haemolysin (sphingomyelinase) has a unique mechanism of action, such that it hydrolyses one of cell membrane lipids (sphingomyelin) to ceramides and phosphorylcholine, leading to cell lysis due to cell membrane destabilization [16]. Additionally, it stimulates the process of biofilm formation in vivo [14], exhibits cytolytic activity against human monocytes and macrophages [30], and it inhibits chemotaxis [28].
The activity of b-haemolysin is usually tested using sheep erythrocytes due to significant amount of sphingomyelin in their cytoplasmic membranes. The haemolytic effect is reinforced by lowering the incubation temperature, which prompts the characteristic hot-cold effect [26]. Co-haemolysis in reverse CAMP test and other CAMP-like tests is another method to test for the presence of b-haemolysin [21]. Molecular analyses detecting hlb gene, coding for b-haemolysin are becoming also more and more frequently used [10,11].

Methods
Bacterial Strains 51 clinical strains of Staphylococcus pseudintermedius (13 obtained from humans and 38 from animals, mainly from dogs) were analysed, as well as 6 clinical strains of Staphylococcus epidermidis isolated from humans used as a negative control (this species does not produce b-haemolysin). All the tested strains were obtained from hospital and veterinary laboratories in Lodz, Poland. Strains were identified with MALDI-TOF system (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Analysis) [4] and with genotypic method previously described by Sasaki et al. [22]. Staphylococcus aureus ATCC Ò 25923 and S. intermedius ATCC Ò 29663 reference strains were obtained from ATCC (LGC Standards) collection.

Hot-Cold Effect
Analysed strains were incubated on a 5 % sheep blood agar at 37°C for 24 h. Afterwards, the haemolysis effect was tested for. Subsequently, they were incubated at 4°C for the next 16 h and analysed again. The enlargement of haemolysis zone around bacterial colonies after incubation at 4°C (''double'' haemolysis) was considered as a positive result.

Reverse CAMP Test
In the middle of the 5 % sheep blood agar, reference strain of Streptococcus agalactiae (producing CAMP factor) was inoculated. Analysed strains were inoculated perpendicularly to the reference strain. Afterwards, the culture was incubated in 37°C for 24 h. An enlarged haemolysis zone near the reference S. agalactiae strain (''arrowhead'') was considered as a positive result.

DNA Isolation
Genomic DNA isolation was performed from overnight bacterial culture according to Genomic Mini AX BAC-TERIA SPIN (A&A Biotechnology) protocol.

PCR Reactions
In order to determine the hlb gene presence in genomic DNA, PCR reactions were conducted with 2 different pairs of primers: one recommended in the literature [11] and another one, which was newly designed in the CLC Main  Table 1.

Statistical Analysis
Statistical analysis was performed using STATISTICA 10 software (Statsoft).

Results
The results of phenotypic and genotypic analyses for S. pseudintermedius strains are shown in Table 2.
b-haemolysin was phenotypically detected (hot-cold effect and reverse CAMP test) in 61 % of analysed S. pseudintermedius and none of S. epidermidis negative control strains. One of S. pseudintermedius strains did not produce b-haemolysin. In 35 % of S. pseudintermedius strains, b-haemolysin production was detected only by hot-  cold effect, whereas the reverse CAMP test was negative (Fig. 1). The presence of hlb gene, in a PCR reaction with primers described in the literature [11], was confirmed only in 2 (4 %) S. pseudintermedius strains, whereas in the reaction with newly designed primers-in 50 (98 %) analysed S. pseudintermedius strains. The absence of hlb gene was detected only in SPI 323 animal strain which was also negative in phenotypic b-haemolysin test.
The hlb gene was detected in none of S. epidermidis control strains, nor in the S. aureus ATCC Ò 25923 and S. intermedius ATCC Ò 29663 reference strains. Results were negative in PCR reactions when both the described in the literature and the newly designed primers were used (Fig. 2).
The statistical analysis based on the v 2 test showed that the relationship between hot-cold effect and hlb gene presence in the PCR reaction with the newly designed primers was statistically significant (P = 0.00000).
PCR reactions conducted in this study with primers described by Gharsa et al. [11] showed positive results only in 2 (4 %) out of 51 analysed S. pseudintermedius strains. This contradicts previous results showing common phenotypic demonstration of b-haemolysin presence, as well as previously described genotypic studies proving hlb gene presence in strains able to produce b-haemolysin [9,11,12].
On the basis of the S. pseudintermedius ED99 complete genome deposited in Genbank, we designed a new pair of primers for hlb gene, which enable the analysis of S. pseudintermedius strains. PCR searching results completely confirmed phenotypic hot-cold test outcome, which proved to be more reliable than the reverse CAMP test.
The results described in this paper contest previous studies on the possibility of searching for S. pseudintermedius virulence genes using hlb primers described for S. aureus [11], because they seem to be inadequate for S. pseudintermedius strains. This result also contests the credibility of previously published analyses of bacterial strains from SIG group. Primers proposed in this study for searching for b-haemolysin hlb gene in S. pseudintermedius seem to be much more accurate in the detection of this virulence factor in bacterial strains of this species. Preliminary studies on the newly designed primers showed also negative hlb searching results for S. aureus and S. intermedius reference ATCC strains. This suggests that after further analysis, the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification. That would be extraordinarily desirable because of numerous difficulties in the differentiation among the species of the SIG group.

Compliance with Ethical standards
Conflicts of interest None.
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