mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Haemolytic and co-haemolytic (CAMP-like) activity in dermatophytes 3 _ €g en,1 Ramazan Gu € mral2 and Macit Ilkit Aylin Do 1 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey, 2Department of Microbiology, Gu€lhane Military Medical Academy, Ankara, Turkey and 3Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C ß ukurova, Adana, Turkey
Summary
Dermatophytes are some of the most common fungal pathogens in both humans and animals. These fungi release enzymes (e.g., keratinases) that play roles in their pathogenesis. Little is known about their haemolytic and co-haemolytic (CAMP-like) activities; however, in bacteria, these components play significant roles in pathogenesis. This study characterised these two factors in 45 dermatophyte strains (representing the genera Arthroderma, Epidermophyton, Microsporum and Trichophyton) using Columbia agar (CA) supplemented with 5% bovine, ovine and equine erythrocytes. Haemolysis was best observed on CA supplemented with ovine erythrocytes followed by equine and bovine erythrocytes, while CAMP-like reactions occurred using bovine and ovine but not equine erythrocytes. Haemolytic and CAMP-like activities were best observed using ovine and bovine erythrocytes in CA in 44 and 38 strains at 7 and 3 days respectively. Most dermatophytes recovered from both symptomatic and asymptomatic lesions had haemolytic and CAMP-like activities. We suggest that the haemolytic and CAMP-like activities are not correlated with ecological characteristics, isolation sites or clinical manifestations of dermatophytic fungi. We also believe that this study has the potential to contribute to the existing literature on dermatophytes and dermatophyte pathogenesis.
Key words: CAMP factor, dermatophytes, haemolysin, keratinophilic, virulence.
Introduction Dermatophytes are a group of filamentous fungi that can cause diseases on the glabrous skin, scalp and nails in humans and animals. Despite a global prevalence approaching 70% for dermatophytosis, particularly tinea pedis,1 a sophisticated understanding of how these organisms establish and maintain disease is lacking.2 Haemolysins are defined as exotoxins because of their capacity to lyse red blood cells.3 Correspondence: M. Ilkit, Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C ß ukurova, Adana 01330, Turkey. Tel.: +90 532 286 0099. Fax: +90 322 457 3072. E-mail: [email protected] Submitted for publication 21 June 2014 Revised 05 October 2014 Accepted for publication 15 October 2014
doi:10.1111/myc.12269
Although haemolysins play important roles in the pathogenesis of several bacteria,4 haemolytic activity is also observed in pathogenic fungi, such as Aspergillus5 and Candida species,6 and in dermatophytic fungi.7–10 Haemolysins are important virulence factors that mediate the severity of infectious diseases,11 and the loss of their activity often results in avirulence.12 Furthermore, they have cytotoxic effects on the membranes of erythrocytes13 and phagocytic cells14 and have pore-forming and lysis effects on other eukaryotic cells and cellular structures.3,15 Bacterial and fungal haemolysins differ in their biochemical and cytotoxic properties.3,4 The co-haemolytic effect was first described by Christie, Atkins and Munch-Peterson in 1944 and was named the ‘CAMP factor’, using the initials of the authors who first described the reaction.16 Streptococcus agalactiae (B group streptococci) produces a
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Haemolytic activities in dermatophytes
thermostable, extracellular, diffusible protein that acts synergistically with the Staphylococcus aureus b-lysin to produce a zone of enhanced lysis in ovine and bovine erythrocyte cultures but not in human, horse, rabbit or guinea pig erythrocytes.16,17 Bernheimer et al. [17] also described the mode of action of the S. agalactiae CAMP-haemolysin; namely, this ‘CAMP factor’ stably binds to membranes containing ceramides (e.g. the membranes of erythrocytes). This test is also useful in the identification of both S. agalactiae and many grampositive rods, including Listeria monocytogenes.18 Remarkably, in a clinical setting, interdigital tinea pedis or tinea unguium has the potential to cause severe bacterial complications, such as abscess, cellulitis, erysipelas and fasciitis.19 It is essential to understand dermatophytes in different ecological and clinical characteristics to fully comprehend bacteria–dermatophyte interactions. While previous studies have described the impacts that bacterial haemolysins4,11–15 and CAMP-like activities16,17 have on pathogenesis, there are limited data regarding haemolysins7–10 and CAMP-like activities20 on dermatophyte pathogenesis. It is also important to examine different species’ erythrocytes for lytic activity to identify the differences in activity among erythrocytes from different animal species. In this study, we investigated the haemolytic and CAMP-like reactions in well-characterised dermatophytic fungi recovered from both symptomatic and asymptomatic lesions using Columbia agar (CA) supplemented with 5% bovine (CBA), ovine (COA) and equine (CEA) erythrocytes.
Materials and methods Test organisms
Table 1 lists the clinical sources, geographical origins and reference numbers of the 45 dermatophyte strains used in this study: representatives of Arthroderma spp. (n = 7), Epidermophyton floccosum (n = 1), Microsporum audouinii (n = 2), M. canis (n = 2), M. ferrugineum (n = 1), M. gypseum (n = 1), Trichophyton mentagrophytes (n = 7) and T. rubrum (n = 20) complexes, T. schoenleinii (n = 2) and one of each strain T. concentricum and T. verrucosum. Most of these strains were obtained from the culture collection service at Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands. In addition, two M. canis and three T. interdigitale strains from the working collection of Macit Ilkit (MI) were included in the study (Table 1). These strains were previously identified by DNA sequencing. The fungal
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
names were assigned based on the current taxonomy and/or the CBS list of cultures (http://www.cbs. knaw.nl/databases). Before testing, all strains were subcultured on Sabouraud glucose agar (SGA, Merck, Darmstadt, Germany) at 27 °C for 10 days and were controlled for purity and identity.8,20 Haemolytic activity
Haemolytic activity was monitored by inoculating a 5 9 5 mm portion from the edge of a 10-day-old colony from each strain of the 45 dermatophytes that were maintained on SGA onto CBA, COA and CEA media (Besimik, Istanbul, Turkey) concurrently. The culture plates were incubated at 27 °C for 7 days. To increase haemolytic activity, the cultures were additionally incubated at 36 °C for 1–5 days. Haemolytic activity was measured macroscopically, and the test results were recorded each day.8 Qualitative CAMP test
The qualitative CAMP test was performed using the same CBA, COA and CEA media (Besimik), and the same study strains were inoculated concurrently. A needle was used for the punctiform inoculation of the dermatophyte cultures to be tested. After incubation at 27 °C for 3–7 days, the edge of a loop was used to streak a b-haemolytic strain (S. aureus ATCC 25923), an a-haemolytic strain (Streptococcus pneumoniae ATCC 6303) and a non-haemolytic strain (Enterococcus faecalis ATCC 29212) in straight lines across the plate at a distance of 10 mm from the border of the colony. The plates were then incubated at 27 °C for 1–7 days. The CAMP-like reactions were inspected daily for 7 days. A positive result was observed as a distinct arrowhead of haemolysis at the intersection of the tester strain and the test dermatophyte streaks.18,20
Results Different species’ erythrocytes were tested with CA media at different temperatures to show haemolytic activity. Please see Table 2 for the haemolytic activity data. Haemolysis was best observed on COA followed by CEA and CBA at 27 °C for 5 days and an additional incubation at 36 °C for 2–3 days. Incubation at 36 °C clearly induced haemolysis with all erythrocyte types. Most of the study strains (97.8%) showed haemolytic activity that produced a single zone of haemolysis (Fig. 1). However, only T. rubrum sensu stricto CBS 132249 produced a complete zone of haemolysis
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Table 1 Dermatophyte strains of Arthroderma, Epidermophyton, Microsporum and Trichophyton species used in this study. Species Arthroderma strains A. simii A. simii A. simii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii Epidermophyton floccosum Microsporum strains M. audouinii M. audouinii M. canis M. canis M. ferrugineum M. gypseum Trichophyton mentagrophytes T. erinacei T. erinacei T. erinacei T. interdigitale T. interdigitale T. interdigitale T. m.var.mentagrophytes T. rubrum complex T. fischeri T. fluviomuniense T. kuryangei T. megninii T. raubitschekii T. raubitschekii T. raubitschekii T. raubitschekii T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. soudanense T. violaceum T. violaceum T. violaceum T. yaoundeib Trichophyton strains T. concentricum T. schoenleinii T. schoenleinii T. verrucosum
CBS no.
MI no.
Clinical source
Geography
417.65 448.65 132352 117724 428.63 132252 138553 –
– – – – – 19654 19761 3027a
Chicken Poultry Tinea inguinalis Human Skin, foot sole Human, groin carrier Human, folliculitis –
India India Mersin, Turkey France Netherlands Adana, Turkey Adana, Turkey –
732.88 102894 – – 118548 130948
– – 19562 19563 – –
Hair and skin Hair of head and skin Tinea capitis Tinea capitis Hair Tinea pedis, human
Egypt Netherlands Izmir, Turkey Adana, Turkey China Iran
344.79 511.73 677.86 – – – 110.65
– – – 19670 19671 19672 –
Human, skin, arm Erinaceus europaeus (hedgehog) Human, nail Tinea pedis Tinea unguim Tinea unguium Human, pubic hair
Netherlands New Zealand Germany Adana, Turkey Adana, Turkey Adana, Turkey Netherlands
100081 592.68 518.63 389.58 202.88 287.86 125604 125605 392.58 138551 132251 132249 132250 138550 138552 436.63 253.88 119446 120322 677.82
– – – – – – – – – 19762 19651 19652 19653 19763 19764 – – – – –
Contaminant Human, skin – Human, nail Human, tinea pedis Human, skin Human, skin Human, skin Human, tinea pedis Human, folliculitis Human, groin carrier Human, groin carrier Human, groin carrier Human, folliculitis Human, folliculitis Skin, head Skin, head Tinea capitis Tinea capitis Scalp
– Rio Muni, Guinea – Utrecht, Netherlands Ontario, Toronto, Canada Toronto, Canada Adana, Turkey Adana, Turkey Rotterdam, Netherlands Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Africa (Country unknown) Netherlands Gabon Switzerland Netherlands (patient from Morocco)
448.61 564.94 138574 282.82
– – 19401 –
Legs and trunk Skin scales of head Human, favus Hair, skin
Netherlands Unknown Adana, Turkey Norway
a
This strain was obtained from the RSHCP (Refik Saydam Hygiene Center Presidency).
b
This T. yaoundei isolate was deposited as T. violaceum at CBS until January 2001.
MI, Macit Ilkit working collection.
followed by a small zone of complete haemolysis (bizonal lytic effect); the remaining study strains failed to produce this effect (Fig. 2). Remarkably, only one
42
strain, M. canis MI 19562, did not produce haemolysin, despite incubation at 36 °C for 7 days (Table 2). We also observed that three T. rubrum strains
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Haemolytic activities in dermatophytes
Table 2 Haemolytic and co-haemolytic reactions of dermatophytes on Columbia agar according to erythrocyte type and time interval
(days).
Species Arthroderma strains A. simii A. simii A. simii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii Epidermophyton floccosum Microsporum strains M. audouinii M. audouinii M. canis M. canis M. ferrugineum M. gypseum Trichophyton mentagrophytes T. erinacei T. erinacei T. erinacei T. interdigitale T. interdigitale T. interdigitale T. m.var. mentagrophytes T. rubrum complex T. fischeri T. fluviomuniense T. kuryangei T. megninii T. raubitschekii T. raubitschekii T. raubitschekii T. raubitschekii T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. soudanense T. violaceum T. violaceum T. violaceum T. yaoundei T. strains T. concentricum T. schoenleinii T. schoenleinii T. verrucosum
CBS no.
MI no.
417.65 448.65 132352 117724 428.63 132252 138553 –
– – – – – 19654 19761 3027*
732.88 102894 – – 118548 130948
– – 19562 19563 – –
344.79 511.73 677.86 – – – 110.65
– – – 19670 19671 19672 –
100081 592.68 518.63 389.58 202.88 287.86 125604 125605 392.58 138551 132251 132249 132250 138550 138552 436.63 253.88 119446 120322 677.82
– – – – – – – – – 19762 19651 19652 19653 19763 19764 – – – – –
448.61 564.94 138574 282.82
– – 19401 –
Haemolytic activity at 27 °C/36 °C
CAMP-like activity at 27 °C
CBA
COA
CEA
CBA
/3 /3 /2 /3 6/2
/2 5/2 5/2 5/2 5/2 6/2 /3 6/2
/2 /2 /2 6/2 /3 /2 /3 6/2
+ + + + + + +
+* +*
/3 /2
5/2 5/2
+ + + +
+*
/2 /3 /5 5/2
7/2 /4 7/2
/3 /3 /3 7 /2 5/2 /4 5/2 5/2 /2 /2
/3
/3
COA
+* +*
/3 /3
/2 /5
5/2 5/2 5/2 7/2 /3 7/2 6/2
/2 /2 /2 7/2 /4 /2 7/2
+ + + + + + +
+* +*
/2 /2 5/2 /2 /2 /2 /2 /3 5/2 /3 5/2 5/2 /2 5/2 5/2 7/2 5/2 7/2 /2 5
/2 /2 /2 /2 /2 /2 /3 /3 /2 /3 7/2 5/2
+
+*
+ + +
+* +*
5/2 5 6 5/2
CEA
5/2 5/2 7/2 /2 /2 /2
+ +
+
+* +* +*
+ + + +
+
+*
+ + +
+ +
/2
/3
CBA, Colombia agar (CA) with 5% bovine erythrocytes; COA, CA with 5% ovine erythrocytes; CEA, CA with 5% equine erythrocytes; , weak reaction; +*, haemolysis incorporated; , negative; /, days with numbers before and after the slash indicating that the haemolytic reaction was first noted at 27 °C for 7 days and completed at 36 °C for 1–5 days.
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
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(I)
(a)
(b)
(c)
(a)
(b)
(c)
(II)
Figure 1 Arthroderma vanbreuseghemii CBS 117724 on Columbia agar incubated with 5% (a) equine, (b) bovine and (c) ovine erythrocytes produced a single zone of haemolysis (7 days/27 °C followed by additional incubation for 2 days/36 °C); (I) colony surface and (II) colony reverse.
(a)
(CBS 132249, CBS 132250 and CBS 132251) recovered from asymptomatic lesions and an Arthroderma vanbreuseghemii strain (CBS 132252) detected in a groin carrier had haemolytic activity. Next, we investigated CAMP-like haemolysis with dermatophytes to show bacteria–dermatophyte interactions. Remarkably, CAMP-like haemolysis was apparent from the third day and became incorporated and extreme on the fourth and fifth days; therefore,
44
(b)
Figure 2 Trichophyton rubrum CBS 132249 on CBA shows a bizonal effect haemolysis at 5 days/27 °C; (a) colony surface and (b) colony reverse.
CAMP-like haemolysis could not be clearly evaluated. In contrast to haemolytic activity results, CAMP-like haemolysis was best observed on CBA at 27 °C for 2–3 days (Table 2). A total of 38 of the 45 (84.4%) strains had CAMP factor activity (Fig. 3). The seven strains that did not produce a CAMP factor on CBA were M. audouinii (n = 2), members of the T. rubrum complex (n = 4) and T. concentricum (n = 1). In addition, one E. floccosum, one T. raubitschekii and three
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Haemolytic activities in dermatophytes
Figure 3 Trichophyton raubitschekii CBS 202.88 (a) and Microsporum canis MI 19562 (b) on Columbia agar with 5% bovine erythrocytes developed CAMP reactions in the zone of b-haemolytic Staphylococcus aureus; however, no reactions were observed with a-haemolytic Streptococcus pneumoniae and nonhaemolytic Enterococcus faecalis strains. All plates were photographed using transillumination.
T. rubrum sensu stricto strains displayed weak reactions on CBA. CAMP haemolysin was clearly observed on COA only in three strains, namely, T. erinacei CBS 511.73, T. schoenleinii CBS 564.94 and T. schoenleinii MI 19401. The CAMP factor was not observed on CEA (Table 2). This reaction only occurred when a dermatophyte strain was tested with a b-haemolytic strain (S. aureus) and resulted in complete haemolysis; no haemolysis occurred with a-haemolytic (S. pneumoniae) or nonhaemolytic (E. faecalis) strains. Remarkably, there was no correlation between haemolytic and co-haemolytic activities and the dermatophyte species or symptomatic and asymptomatic infections. Furthermore, no bacterial contamination was observed during the study period.
Discussion Haemolysins and CAMP-like reactions are important for pathogenesis in other bacteria, but their importance with respect to dermatophytes has not been well characterised. This report presents the results of a study evaluating the impacts that different strains of dermatophytes have on haemolysis and CAMP-like activity using media containing different species erythrocytes. Here, 45 dermatophyte strains were grown on CA media containing either bovine, ovine, or equine erythrocytes, and haemolytic and CAMP-like activities were measured. Most strains exhibited haemolytic (97.8%) and CAMP-like (84.4%) activities. While haemolysins were most apparent on COA followed by CEA and CBA after 7 days, CAMP-like activity was unique to bovine erythrocytes and was best observed at 3 days. Surprisingly, isolates from both symptomatic and asymptomatic infections and from a variety of different sources induced haemolysis. We suggest that this study also has the potential to
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
(a)
(b)
improve the methodology for measuring dermatophyte haemolysis and CAMP-like activity. Although dermatophytes can trigger both haemolytic7–10 and CAMP-like reactions,20 the underlying mechanisms remain unknown. In this study, M. audouinii (CBS 732.88 and CBS 102894), T. fluviomuniense (CBS 592.86), T. raubitschekii (CBS 287.86), T. rubrum (CBS 132251) and T. concentricum (CBS 448.61) exhibited haemolytic activity but no CAMPlike activity. This antagonist effect can be explained by the lack of bacteria–dermatophyte interactions, but the biochemical mechanisms need to be highlighted. To our knowledge, Ozegovic and Grin [10] were the first to study haemolytic activities in dermatophyte species on blood agar supplemented with bovine, ovine and equine erythrocytes, but no detailed media descriptions were included. Notably, haemolysis was observed on all three types of media; the haemolytic zones were more developed with equine erythrocytes when the pH was adjusted from 7.0–6.5. The authors also noted that haemolysis developed more quickly (5 days vs. 15–21 days) at a higher incubation temperature (37 °C vs. 26 °C).10 In another study using heart infusion agar supplemented with 5% bovine, ovine and equine erythrocytes, the haemolytic activities of dermatophytes were investigated for geophilic species, including 10 strains each of M. cookei, M. gypseum, T. ajelloi and T. terrestre. Of these species, the latter three exhibited haemolytic activity.5 Later, Solgun et al. [9] examined 79 anthropophilic T. rubrum isolates on COA and found that while 71 (89.9%) isolates exhibited haemolysis, the reaction was absent in eight (10.1%) isolates.9 Similar to our results, anthropophilic,8–10 zoophilic8,10 and geophilic5,7,10 species showed haemolytic activity. Schaufuss and Steller [8] examined four dermatophyte strains, T. rubrum, T. mentagrophytes, T. equinum and T. verrucosum. The authors observed a bizonal
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lytic effect on T. rubrum and T. equinum strains after cultivation on CBA, COA and CEA media; however, in this study, we observed a bizonal lysis effect on only one (CBS 132249) of seven T. rubrum sensu stricto strains. This effect was observed as zones of complete and incomplete haemolysis, indicating the selection of two distinct cytolytic factors. In addition, the colonies of T. mentagrophytes and T. verrucosum in Schaufuss and Steller’s study [8] were surrounded by a single zone of complete haemolysis, as observed herein. The authors explicitly noted that haemolysins may play important roles in the balance between the cellular immunity of the host and the capacity of the fungus to diminish the immune response. Schaufuss et al. [20] investigated 76 dermatophyte strains representing the three anamorphic genera and showed that all strains undergo CAMP-like haemolytic reactions. Consistent with this study, the authors also detected reactions with ovine and bovine (but not equine) erythrocytes. The authors suggested that this result was directly related to the sphingomyelin content in the membrane phospholipids, which accounts for 51% of the total phospholipid content in ovine and bovine erythrocytes and for only 13.5% in equine erythrocytes. They observed that with b-haemolytic strains (S. aureus, S. intermedius or L. ivanovii), dermatophytes induced a distinct zone of complete haemolysis; however, no CAMP-like reaction was noted with non-b-haemolytic strains (S. hyicus or S. epidermidis), as in our study. Similarly, we observed that most of the dermatophyte strains expressed CAMP-like haemolysins. We also suggest that keratinophilic fungus with a negative CAMP test could still be dermatophytic and would require further testing. The main shortcomings of this study include (i) the low number of asymptomatic isolates investigated and (ii) the care required during the preparation of the study media, as haemolysis would have been undetectable if the prepared agar was too thin. In another study, Schaufuss et al. [21] isolated and partially purified a soluble haemolysin from the culture supernatant of T. mentagrophytes CBS 388.58. An earlier report also stated that haemolytic activity is correlated with the severity and chronicity of dermatophytosis, but the erythrocyte sources in that study was not specified.22 While it is known that haemolysins modulate the pathogenesis of dermatophytes and are correlated with more severe and recurrent infections,22 the effect of CAMP-like haemolytic activity produced from bacteria and dermatophyte interactions is less clear. Understanding these interactions may provide us with a better understanding of the concomitant clinical
46
image of bacterial and dermatophytic diseases, such as erysipelas and tinea pedis. Thus, future studies should include a collection of clinically isolated dermatophytes from chronic or recurrent presentations and their concomitant diseases. In conclusion, we suggest that dermatophytes have the unique capacity to exert haemolytic and CAMPlike activities regardless of their ecological characteristics, isolation site or clinical manifestations. These two activities may play important roles in the pathogenicity and adaptation of dermatophytes to human hosts. Because ovine and bovine erythrocytes exhibited clear zones of haemolysis and CAMP-like haemolytic reactions, respectively, both activities can be easily monitored on CA. We believe that further studies will improve our current knowledge of the biochemical characteristics of these toxins and the role that haemolysins play in host–microbe interactions.
Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the manuscript.
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Ilkit M, Durdu M. Tinea pedis: the etiology and global epidemiology of a common fungal infection. Crit Rev Microbiol doi: 10.3109/ 1040841x.2013.856853. White TC, Oliver BG, Gr€ aser Y, Henn MR. Generating and testing molecular hypothesis in the dermatophytes. Eukaryot Cell 2008; 7: 1238–45. Nayak AP, Green BJ, Beezhold DH. Fungal hemolysins. Med Mycol 2013; 51: 1–16. Bernheimer AW. Cytolytic toxins of bacterial origin. Science 1968; 159: 847–51. Wartenberg D, Lapp K, Jacobsen ID et al. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein. Int J Med Microbiol 2011; 301: 602–11. Guzel AB, Kucukgoz-Gulec U, Aydın M et al. Candida vaginitis during contraceptive use: the influence of methods, antifungal susceptibility and virulence patterns. J Obstet Gynaecol 2013; 33: 850–6. Gip L, Palsson G. On the hemolytic activity of geophilic dermatophytes. Mycopathol Mycol Appl 1970; 2: 221–3. Schaufuss P, Steller U. Haemolytic activities of Trichophyton species. Med Mycol 2003; 41: 511–6. Solgun G, Fındık D, T€ urk-Da gı H, Arslan U. Trichophyton rubrum klinik izolatlarının hemolitik aktivitesi ve antifungal ilacßlara in vitro duyarlılı gının saptanması [Determination of hemolytic activity and in vitro antifungal susceptibility of Trichophyton rubrum clinical strains]. Mikrobiyol Bul 2011; 45: 159–67. Ozegovic L, Grin EI. H€ amolytische Eigenschaft der Dermatophyten [Haemolytic property of dermatophytes]. Mykosen 1967; 10: 325–30. V azquez-Boland JA, Kuhn M, Berche P et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 2001; 14: 584–640. Hof H. Virulence of different of Listeria monocytogenes serovar 1/2a. Med Microbiol Rev 1984; 173: 207–18.
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Elek SD, Levy E. Distribution of hemolysins in pathogenic and non-pathogenic staphylococci. J Pathol Bacteriol 1950; 62: 541–54. Kingdom GC, Sword CP. Effects of Listeria monocytogenes hemolysin on phagocytic cells and lysosomes. Infect Immun 1970; 1: 356–62. Bhakdi S, Tranum-Jensen J. Mechanism of complement cytolysis and the concept of channel-forming proteins. Philos Trans R Soc Lond B Biol Sci 1984; 306: 311–24. Christie R, Atkins NE, Munch-Peterson E. A note on a lytic phenomenon shown by group B streptococci. Aust J Exp Biol Med Sci 1944; 22: 197–200. Bernheimer AW, Kinder R, Avigad LS. Nature and mechanism of action of the CAMP protein of group B streptococci. Infect Immun 1979; 23: 838–44. York MK, Traylor MM, Hardy J, Henry M. CAMP factor tests (standart and rapid) and the reverse CAMP test. In: Garcia LS (ed), Clinical
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Microbiology Procedures Handbook, 3rd edn. Washington, DC: ASM Press, 2010: section 3.17.8. Vanhooteghem O, Szepetiuk G, Paurobally D, Heureux F. Chronic interdigital dermatophytic infection: a common lesson associated with potentially severe consequences. Diabetes Res Clin Pract 2011; 91: 23–25. Schaufuss P, Brasch J, Steller U. Dermatophytes can trigger cooperative (CAMP-like) haemolytic reactions. Br J Dermatol 2005; 153: 584–90. Schaufuss P, M€ uller F, Valentin-Weigand P. Isolation and characterization of a haemolysin from Trichopyton mentagrophytes. Vet Microbiol 2007; 122: 342–9. L opez-Martinez R, Manzano-Gayosso R, Mier T, Mendez-Tovar R, Hern andez-Hern andez F. Exoenzymes of dermatophytes isolated from acute and chronic tinea. Rev Latinoam Microbiol 1994; 36: 17–20.
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Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Haemolytic and co-haemolytic (CAMP-like) activity in dermatophytes 3 _ €g en,1 Ramazan Gu € mral2 and Macit Ilkit Aylin Do 1 Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey, 2Department of Microbiology, Gu€lhane Military Medical Academy, Ankara, Turkey and 3Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C ß ukurova, Adana, Turkey
Summary
Dermatophytes are some of the most common fungal pathogens in both humans and animals. These fungi release enzymes (e.g., keratinases) that play roles in their pathogenesis. Little is known about their haemolytic and co-haemolytic (CAMP-like) activities; however, in bacteria, these components play significant roles in pathogenesis. This study characterised these two factors in 45 dermatophyte strains (representing the genera Arthroderma, Epidermophyton, Microsporum and Trichophyton) using Columbia agar (CA) supplemented with 5% bovine, ovine and equine erythrocytes. Haemolysis was best observed on CA supplemented with ovine erythrocytes followed by equine and bovine erythrocytes, while CAMP-like reactions occurred using bovine and ovine but not equine erythrocytes. Haemolytic and CAMP-like activities were best observed using ovine and bovine erythrocytes in CA in 44 and 38 strains at 7 and 3 days respectively. Most dermatophytes recovered from both symptomatic and asymptomatic lesions had haemolytic and CAMP-like activities. We suggest that the haemolytic and CAMP-like activities are not correlated with ecological characteristics, isolation sites or clinical manifestations of dermatophytic fungi. We also believe that this study has the potential to contribute to the existing literature on dermatophytes and dermatophyte pathogenesis.
Key words: CAMP factor, dermatophytes, haemolysin, keratinophilic, virulence.
Introduction Dermatophytes are a group of filamentous fungi that can cause diseases on the glabrous skin, scalp and nails in humans and animals. Despite a global prevalence approaching 70% for dermatophytosis, particularly tinea pedis,1 a sophisticated understanding of how these organisms establish and maintain disease is lacking.2 Haemolysins are defined as exotoxins because of their capacity to lyse red blood cells.3 Correspondence: M. Ilkit, Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C ß ukurova, Adana 01330, Turkey. Tel.: +90 532 286 0099. Fax: +90 322 457 3072. E-mail: [email protected] Submitted for publication 21 June 2014 Revised 05 October 2014 Accepted for publication 15 October 2014
doi:10.1111/myc.12269
Although haemolysins play important roles in the pathogenesis of several bacteria,4 haemolytic activity is also observed in pathogenic fungi, such as Aspergillus5 and Candida species,6 and in dermatophytic fungi.7–10 Haemolysins are important virulence factors that mediate the severity of infectious diseases,11 and the loss of their activity often results in avirulence.12 Furthermore, they have cytotoxic effects on the membranes of erythrocytes13 and phagocytic cells14 and have pore-forming and lysis effects on other eukaryotic cells and cellular structures.3,15 Bacterial and fungal haemolysins differ in their biochemical and cytotoxic properties.3,4 The co-haemolytic effect was first described by Christie, Atkins and Munch-Peterson in 1944 and was named the ‘CAMP factor’, using the initials of the authors who first described the reaction.16 Streptococcus agalactiae (B group streptococci) produces a
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thermostable, extracellular, diffusible protein that acts synergistically with the Staphylococcus aureus b-lysin to produce a zone of enhanced lysis in ovine and bovine erythrocyte cultures but not in human, horse, rabbit or guinea pig erythrocytes.16,17 Bernheimer et al. [17] also described the mode of action of the S. agalactiae CAMP-haemolysin; namely, this ‘CAMP factor’ stably binds to membranes containing ceramides (e.g. the membranes of erythrocytes). This test is also useful in the identification of both S. agalactiae and many grampositive rods, including Listeria monocytogenes.18 Remarkably, in a clinical setting, interdigital tinea pedis or tinea unguium has the potential to cause severe bacterial complications, such as abscess, cellulitis, erysipelas and fasciitis.19 It is essential to understand dermatophytes in different ecological and clinical characteristics to fully comprehend bacteria–dermatophyte interactions. While previous studies have described the impacts that bacterial haemolysins4,11–15 and CAMP-like activities16,17 have on pathogenesis, there are limited data regarding haemolysins7–10 and CAMP-like activities20 on dermatophyte pathogenesis. It is also important to examine different species’ erythrocytes for lytic activity to identify the differences in activity among erythrocytes from different animal species. In this study, we investigated the haemolytic and CAMP-like reactions in well-characterised dermatophytic fungi recovered from both symptomatic and asymptomatic lesions using Columbia agar (CA) supplemented with 5% bovine (CBA), ovine (COA) and equine (CEA) erythrocytes.
Materials and methods Test organisms
Table 1 lists the clinical sources, geographical origins and reference numbers of the 45 dermatophyte strains used in this study: representatives of Arthroderma spp. (n = 7), Epidermophyton floccosum (n = 1), Microsporum audouinii (n = 2), M. canis (n = 2), M. ferrugineum (n = 1), M. gypseum (n = 1), Trichophyton mentagrophytes (n = 7) and T. rubrum (n = 20) complexes, T. schoenleinii (n = 2) and one of each strain T. concentricum and T. verrucosum. Most of these strains were obtained from the culture collection service at Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands. In addition, two M. canis and three T. interdigitale strains from the working collection of Macit Ilkit (MI) were included in the study (Table 1). These strains were previously identified by DNA sequencing. The fungal
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
names were assigned based on the current taxonomy and/or the CBS list of cultures (http://www.cbs. knaw.nl/databases). Before testing, all strains were subcultured on Sabouraud glucose agar (SGA, Merck, Darmstadt, Germany) at 27 °C for 10 days and were controlled for purity and identity.8,20 Haemolytic activity
Haemolytic activity was monitored by inoculating a 5 9 5 mm portion from the edge of a 10-day-old colony from each strain of the 45 dermatophytes that were maintained on SGA onto CBA, COA and CEA media (Besimik, Istanbul, Turkey) concurrently. The culture plates were incubated at 27 °C for 7 days. To increase haemolytic activity, the cultures were additionally incubated at 36 °C for 1–5 days. Haemolytic activity was measured macroscopically, and the test results were recorded each day.8 Qualitative CAMP test
The qualitative CAMP test was performed using the same CBA, COA and CEA media (Besimik), and the same study strains were inoculated concurrently. A needle was used for the punctiform inoculation of the dermatophyte cultures to be tested. After incubation at 27 °C for 3–7 days, the edge of a loop was used to streak a b-haemolytic strain (S. aureus ATCC 25923), an a-haemolytic strain (Streptococcus pneumoniae ATCC 6303) and a non-haemolytic strain (Enterococcus faecalis ATCC 29212) in straight lines across the plate at a distance of 10 mm from the border of the colony. The plates were then incubated at 27 °C for 1–7 days. The CAMP-like reactions were inspected daily for 7 days. A positive result was observed as a distinct arrowhead of haemolysis at the intersection of the tester strain and the test dermatophyte streaks.18,20
Results Different species’ erythrocytes were tested with CA media at different temperatures to show haemolytic activity. Please see Table 2 for the haemolytic activity data. Haemolysis was best observed on COA followed by CEA and CBA at 27 °C for 5 days and an additional incubation at 36 °C for 2–3 days. Incubation at 36 °C clearly induced haemolysis with all erythrocyte types. Most of the study strains (97.8%) showed haemolytic activity that produced a single zone of haemolysis (Fig. 1). However, only T. rubrum sensu stricto CBS 132249 produced a complete zone of haemolysis
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Table 1 Dermatophyte strains of Arthroderma, Epidermophyton, Microsporum and Trichophyton species used in this study. Species Arthroderma strains A. simii A. simii A. simii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii Epidermophyton floccosum Microsporum strains M. audouinii M. audouinii M. canis M. canis M. ferrugineum M. gypseum Trichophyton mentagrophytes T. erinacei T. erinacei T. erinacei T. interdigitale T. interdigitale T. interdigitale T. m.var.mentagrophytes T. rubrum complex T. fischeri T. fluviomuniense T. kuryangei T. megninii T. raubitschekii T. raubitschekii T. raubitschekii T. raubitschekii T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. soudanense T. violaceum T. violaceum T. violaceum T. yaoundeib Trichophyton strains T. concentricum T. schoenleinii T. schoenleinii T. verrucosum
CBS no.
MI no.
Clinical source
Geography
417.65 448.65 132352 117724 428.63 132252 138553 –
– – – – – 19654 19761 3027a
Chicken Poultry Tinea inguinalis Human Skin, foot sole Human, groin carrier Human, folliculitis –
India India Mersin, Turkey France Netherlands Adana, Turkey Adana, Turkey –
732.88 102894 – – 118548 130948
– – 19562 19563 – –
Hair and skin Hair of head and skin Tinea capitis Tinea capitis Hair Tinea pedis, human
Egypt Netherlands Izmir, Turkey Adana, Turkey China Iran
344.79 511.73 677.86 – – – 110.65
– – – 19670 19671 19672 –
Human, skin, arm Erinaceus europaeus (hedgehog) Human, nail Tinea pedis Tinea unguim Tinea unguium Human, pubic hair
Netherlands New Zealand Germany Adana, Turkey Adana, Turkey Adana, Turkey Netherlands
100081 592.68 518.63 389.58 202.88 287.86 125604 125605 392.58 138551 132251 132249 132250 138550 138552 436.63 253.88 119446 120322 677.82
– – – – – – – – – 19762 19651 19652 19653 19763 19764 – – – – –
Contaminant Human, skin – Human, nail Human, tinea pedis Human, skin Human, skin Human, skin Human, tinea pedis Human, folliculitis Human, groin carrier Human, groin carrier Human, groin carrier Human, folliculitis Human, folliculitis Skin, head Skin, head Tinea capitis Tinea capitis Scalp
– Rio Muni, Guinea – Utrecht, Netherlands Ontario, Toronto, Canada Toronto, Canada Adana, Turkey Adana, Turkey Rotterdam, Netherlands Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Adana, Turkey Africa (Country unknown) Netherlands Gabon Switzerland Netherlands (patient from Morocco)
448.61 564.94 138574 282.82
– – 19401 –
Legs and trunk Skin scales of head Human, favus Hair, skin
Netherlands Unknown Adana, Turkey Norway
a
This strain was obtained from the RSHCP (Refik Saydam Hygiene Center Presidency).
b
This T. yaoundei isolate was deposited as T. violaceum at CBS until January 2001.
MI, Macit Ilkit working collection.
followed by a small zone of complete haemolysis (bizonal lytic effect); the remaining study strains failed to produce this effect (Fig. 2). Remarkably, only one
42
strain, M. canis MI 19562, did not produce haemolysin, despite incubation at 36 °C for 7 days (Table 2). We also observed that three T. rubrum strains
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Table 2 Haemolytic and co-haemolytic reactions of dermatophytes on Columbia agar according to erythrocyte type and time interval
(days).
Species Arthroderma strains A. simii A. simii A. simii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii A. vanbreuseghemii Epidermophyton floccosum Microsporum strains M. audouinii M. audouinii M. canis M. canis M. ferrugineum M. gypseum Trichophyton mentagrophytes T. erinacei T. erinacei T. erinacei T. interdigitale T. interdigitale T. interdigitale T. m.var. mentagrophytes T. rubrum complex T. fischeri T. fluviomuniense T. kuryangei T. megninii T. raubitschekii T. raubitschekii T. raubitschekii T. raubitschekii T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. rubrum T. soudanense T. violaceum T. violaceum T. violaceum T. yaoundei T. strains T. concentricum T. schoenleinii T. schoenleinii T. verrucosum
CBS no.
MI no.
417.65 448.65 132352 117724 428.63 132252 138553 –
– – – – – 19654 19761 3027*
732.88 102894 – – 118548 130948
– – 19562 19563 – –
344.79 511.73 677.86 – – – 110.65
– – – 19670 19671 19672 –
100081 592.68 518.63 389.58 202.88 287.86 125604 125605 392.58 138551 132251 132249 132250 138550 138552 436.63 253.88 119446 120322 677.82
– – – – – – – – – 19762 19651 19652 19653 19763 19764 – – – – –
448.61 564.94 138574 282.82
– – 19401 –
Haemolytic activity at 27 °C/36 °C
CAMP-like activity at 27 °C
CBA
COA
CEA
CBA
/3 /3 /2 /3 6/2
/2 5/2 5/2 5/2 5/2 6/2 /3 6/2
/2 /2 /2 6/2 /3 /2 /3 6/2
+ + + + + + +
+* +*
/3 /2
5/2 5/2
+ + + +
+*
/2 /3 /5 5/2
7/2 /4 7/2
/3 /3 /3 7 /2 5/2 /4 5/2 5/2 /2 /2
/3
/3
COA
+* +*
/3 /3
/2 /5
5/2 5/2 5/2 7/2 /3 7/2 6/2
/2 /2 /2 7/2 /4 /2 7/2
+ + + + + + +
+* +*
/2 /2 5/2 /2 /2 /2 /2 /3 5/2 /3 5/2 5/2 /2 5/2 5/2 7/2 5/2 7/2 /2 5
/2 /2 /2 /2 /2 /2 /3 /3 /2 /3 7/2 5/2
+
+*
+ + +
+* +*
5/2 5 6 5/2
CEA
5/2 5/2 7/2 /2 /2 /2
+ +
+
+* +* +*
+ + + +
+
+*
+ + +
+ +
/2
/3
CBA, Colombia agar (CA) with 5% bovine erythrocytes; COA, CA with 5% ovine erythrocytes; CEA, CA with 5% equine erythrocytes; , weak reaction; +*, haemolysis incorporated; , negative; /, days with numbers before and after the slash indicating that the haemolytic reaction was first noted at 27 °C for 7 days and completed at 36 °C for 1–5 days.
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
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(I)
(a)
(b)
(c)
(a)
(b)
(c)
(II)
Figure 1 Arthroderma vanbreuseghemii CBS 117724 on Columbia agar incubated with 5% (a) equine, (b) bovine and (c) ovine erythrocytes produced a single zone of haemolysis (7 days/27 °C followed by additional incubation for 2 days/36 °C); (I) colony surface and (II) colony reverse.
(a)
(CBS 132249, CBS 132250 and CBS 132251) recovered from asymptomatic lesions and an Arthroderma vanbreuseghemii strain (CBS 132252) detected in a groin carrier had haemolytic activity. Next, we investigated CAMP-like haemolysis with dermatophytes to show bacteria–dermatophyte interactions. Remarkably, CAMP-like haemolysis was apparent from the third day and became incorporated and extreme on the fourth and fifth days; therefore,
44
(b)
Figure 2 Trichophyton rubrum CBS 132249 on CBA shows a bizonal effect haemolysis at 5 days/27 °C; (a) colony surface and (b) colony reverse.
CAMP-like haemolysis could not be clearly evaluated. In contrast to haemolytic activity results, CAMP-like haemolysis was best observed on CBA at 27 °C for 2–3 days (Table 2). A total of 38 of the 45 (84.4%) strains had CAMP factor activity (Fig. 3). The seven strains that did not produce a CAMP factor on CBA were M. audouinii (n = 2), members of the T. rubrum complex (n = 4) and T. concentricum (n = 1). In addition, one E. floccosum, one T. raubitschekii and three
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Figure 3 Trichophyton raubitschekii CBS 202.88 (a) and Microsporum canis MI 19562 (b) on Columbia agar with 5% bovine erythrocytes developed CAMP reactions in the zone of b-haemolytic Staphylococcus aureus; however, no reactions were observed with a-haemolytic Streptococcus pneumoniae and nonhaemolytic Enterococcus faecalis strains. All plates were photographed using transillumination.
T. rubrum sensu stricto strains displayed weak reactions on CBA. CAMP haemolysin was clearly observed on COA only in three strains, namely, T. erinacei CBS 511.73, T. schoenleinii CBS 564.94 and T. schoenleinii MI 19401. The CAMP factor was not observed on CEA (Table 2). This reaction only occurred when a dermatophyte strain was tested with a b-haemolytic strain (S. aureus) and resulted in complete haemolysis; no haemolysis occurred with a-haemolytic (S. pneumoniae) or nonhaemolytic (E. faecalis) strains. Remarkably, there was no correlation between haemolytic and co-haemolytic activities and the dermatophyte species or symptomatic and asymptomatic infections. Furthermore, no bacterial contamination was observed during the study period.
Discussion Haemolysins and CAMP-like reactions are important for pathogenesis in other bacteria, but their importance with respect to dermatophytes has not been well characterised. This report presents the results of a study evaluating the impacts that different strains of dermatophytes have on haemolysis and CAMP-like activity using media containing different species erythrocytes. Here, 45 dermatophyte strains were grown on CA media containing either bovine, ovine, or equine erythrocytes, and haemolytic and CAMP-like activities were measured. Most strains exhibited haemolytic (97.8%) and CAMP-like (84.4%) activities. While haemolysins were most apparent on COA followed by CEA and CBA after 7 days, CAMP-like activity was unique to bovine erythrocytes and was best observed at 3 days. Surprisingly, isolates from both symptomatic and asymptomatic infections and from a variety of different sources induced haemolysis. We suggest that this study also has the potential to
© 2014 Blackwell Verlag GmbH Mycoses, 2015, 58, 40–47
(a)
(b)
improve the methodology for measuring dermatophyte haemolysis and CAMP-like activity. Although dermatophytes can trigger both haemolytic7–10 and CAMP-like reactions,20 the underlying mechanisms remain unknown. In this study, M. audouinii (CBS 732.88 and CBS 102894), T. fluviomuniense (CBS 592.86), T. raubitschekii (CBS 287.86), T. rubrum (CBS 132251) and T. concentricum (CBS 448.61) exhibited haemolytic activity but no CAMPlike activity. This antagonist effect can be explained by the lack of bacteria–dermatophyte interactions, but the biochemical mechanisms need to be highlighted. To our knowledge, Ozegovic and Grin [10] were the first to study haemolytic activities in dermatophyte species on blood agar supplemented with bovine, ovine and equine erythrocytes, but no detailed media descriptions were included. Notably, haemolysis was observed on all three types of media; the haemolytic zones were more developed with equine erythrocytes when the pH was adjusted from 7.0–6.5. The authors also noted that haemolysis developed more quickly (5 days vs. 15–21 days) at a higher incubation temperature (37 °C vs. 26 °C).10 In another study using heart infusion agar supplemented with 5% bovine, ovine and equine erythrocytes, the haemolytic activities of dermatophytes were investigated for geophilic species, including 10 strains each of M. cookei, M. gypseum, T. ajelloi and T. terrestre. Of these species, the latter three exhibited haemolytic activity.5 Later, Solgun et al. [9] examined 79 anthropophilic T. rubrum isolates on COA and found that while 71 (89.9%) isolates exhibited haemolysis, the reaction was absent in eight (10.1%) isolates.9 Similar to our results, anthropophilic,8–10 zoophilic8,10 and geophilic5,7,10 species showed haemolytic activity. Schaufuss and Steller [8] examined four dermatophyte strains, T. rubrum, T. mentagrophytes, T. equinum and T. verrucosum. The authors observed a bizonal
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lytic effect on T. rubrum and T. equinum strains after cultivation on CBA, COA and CEA media; however, in this study, we observed a bizonal lysis effect on only one (CBS 132249) of seven T. rubrum sensu stricto strains. This effect was observed as zones of complete and incomplete haemolysis, indicating the selection of two distinct cytolytic factors. In addition, the colonies of T. mentagrophytes and T. verrucosum in Schaufuss and Steller’s study [8] were surrounded by a single zone of complete haemolysis, as observed herein. The authors explicitly noted that haemolysins may play important roles in the balance between the cellular immunity of the host and the capacity of the fungus to diminish the immune response. Schaufuss et al. [20] investigated 76 dermatophyte strains representing the three anamorphic genera and showed that all strains undergo CAMP-like haemolytic reactions. Consistent with this study, the authors also detected reactions with ovine and bovine (but not equine) erythrocytes. The authors suggested that this result was directly related to the sphingomyelin content in the membrane phospholipids, which accounts for 51% of the total phospholipid content in ovine and bovine erythrocytes and for only 13.5% in equine erythrocytes. They observed that with b-haemolytic strains (S. aureus, S. intermedius or L. ivanovii), dermatophytes induced a distinct zone of complete haemolysis; however, no CAMP-like reaction was noted with non-b-haemolytic strains (S. hyicus or S. epidermidis), as in our study. Similarly, we observed that most of the dermatophyte strains expressed CAMP-like haemolysins. We also suggest that keratinophilic fungus with a negative CAMP test could still be dermatophytic and would require further testing. The main shortcomings of this study include (i) the low number of asymptomatic isolates investigated and (ii) the care required during the preparation of the study media, as haemolysis would have been undetectable if the prepared agar was too thin. In another study, Schaufuss et al. [21] isolated and partially purified a soluble haemolysin from the culture supernatant of T. mentagrophytes CBS 388.58. An earlier report also stated that haemolytic activity is correlated with the severity and chronicity of dermatophytosis, but the erythrocyte sources in that study was not specified.22 While it is known that haemolysins modulate the pathogenesis of dermatophytes and are correlated with more severe and recurrent infections,22 the effect of CAMP-like haemolytic activity produced from bacteria and dermatophyte interactions is less clear. Understanding these interactions may provide us with a better understanding of the concomitant clinical
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image of bacterial and dermatophytic diseases, such as erysipelas and tinea pedis. Thus, future studies should include a collection of clinically isolated dermatophytes from chronic or recurrent presentations and their concomitant diseases. In conclusion, we suggest that dermatophytes have the unique capacity to exert haemolytic and CAMPlike activities regardless of their ecological characteristics, isolation site or clinical manifestations. These two activities may play important roles in the pathogenicity and adaptation of dermatophytes to human hosts. Because ovine and bovine erythrocytes exhibited clear zones of haemolysis and CAMP-like haemolytic reactions, respectively, both activities can be easily monitored on CA. We believe that further studies will improve our current knowledge of the biochemical characteristics of these toxins and the role that haemolysins play in host–microbe interactions.
Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the manuscript.
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