Rapid killing of Capnocytophaga canimorsus and Capnocytophaga cynodegmi by human whole blood and serum is mediated via the complement system

Zangenah and Bergman SpringerPlus (2015)4:517 DOI 10.1186/s40064-015-1308-9 Open Access RESEARCH Rapid killing of Capnocytophaga canimorsus and Capnocytophaga cynodegmi by human whole blood and serum is mediated via the complement system Salah Zangenah1,2 and Peter Bergman1,2* Abstract Purpose: Capnocytophaga canimorsus (Cani) and Capnocytophaga cynodegmi (Cyno) are found in the oral cavities of dogs and cats. They can be transmitted to humans via licks or bites and cause wound infections as well as severe systemic infections. Cani is considered to be more pathogenic than Cyno, but the pathophysiological mechanisms are not elucidated. Cani has been suggested to be resistant to serum bactericidal effects. Thus, we hypothesized that the more invasive Cani would exhibit a higher degree of serum-resistance than the less pathogenic Cyno. Methods: Whole blood and serum bactericidal assays were performed against Cani- (n = 8) and Cyno-strains (n = 15) isolated from blood and wound-specimens, respectively. Analysis of complement-function was performed by heat-inactivation, EGTA-treatment and by using C1q-depleted serum. Serum and whole blood were collected from healthy individuals and from patients (n = 3) with a history of sepsis caused by Cani. Results: Both Cani and Cyno were equally susceptible to human whole blood and serum. Cani was preferentially killed by the classical pathway of the complement-system whereas Cyno was killed by a partly different mechanism. Serum from 2/3 Cani-infected patients were deficient in MBL-activity but still exhibited the same killing effect as control sera. Conclusion: Both Cani and Cyno were readily killed by human whole blood and serum in a complement-dependent way. Thus, it is not likely that serum bactericidal capacity is the key determinant for the clinical outcome in Cani or Cyno-infections. Keywords: Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Serum killing assay, Whole blood killing assay, Classical pathway, Alternative pathway, Complement system, Mannose binding lectin deficiency Background The gram negative bacteria Capnocytophaga canimorsus (Cani) and Capnocytophaga cynodegmi (Cyno) constitute a significant part of the oral flora of dogs and cats (Suzuki et al. 2010). Cani was first described in 1976 and originally named dysgonic fermenter 2 (DF-2) (Bobo and Newton 1976). In the same report a ‘DF-2 like’ bacteria, *Correspondence: 1 Div of Clinical Microbiology, F68, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Huddinge, Stockholm, Sweden Full list of author information is available at the end of the article mainly associated with wound infections, was described. Later, DF2 was named Capnocytophaga canimorsus (‘canimorsus’ is latin for ‘dogbite’) and “DF-2 like” Capnocytophaga cynodegmi (‘cynodegmi’ is greek for ‘dogbite’) (Brenner et al. 1989). Cani can cause severe infections, including sepsis, meningitis or endocarditis, after contact with dogs or cats (Oehler et al. 2009). The clinical picture is characterized by a rapid onset a few days after animal contact, often with a fulminant course (Oehler et al. 2009) and sometimes with neurological sequelae (Gasch et al. 2009). In contrast to Cani, Cyno is not as well described, but the general notion is that Cyno mainly © 2015 Zangenah and Bergman. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zangenah and Bergman SpringerPlus (2015)4:517 is found in wound infections and rarely cause invasive infections. Most Cani-strains are susceptible to empirical treatment with ampicillin together with clavulanic acid (Oehler et al. 2009; Butler 2015). The diagnostic microbiology is notoriously difficult, mainly due to the fastidious nature and slow growth on standard agar media. Cani and Cyno are facultative anaerobic bacteria that grow best on blood and hematin agar plates. In addition, the presence of 5–10 % CO2 and 48 h of incubation is needed for efficient growth and for typical colony morphology. After 18–24 h of incubation on blood agar plates, colonies are small (<0.5 mm) and may be irregular in shape. After 48 h, the colonies become visible (1–3 mm) and can vary in size and shape, within the same isolate or species. In gram staining, the bacteria are thin and fusiform with pointed ends (Brenner et al. 1989). Traditional methods require several days for diagnosis, but the introduction of novel methods, such as MALDI-TOF, in clinical bacteriology has significantly shortened the time to diagnosis (Zangenah et al. 2012). Patient with congenital or acquired asplenia or with a dysfunctional spleen, alcoholics and elderly people constitute risk groups for severe Cani infections (Shahani and Khardori 2014; Ugai et al. 2014). However, there are only a few reports on Cani pathogenesis. For example, Cani has been shown to block the release of proinflammatory cytokines from monocytes (Shin et al. 2007). Recently, it was shown that Cani has a deglycosylation system that degrades exposed sugars on human IgG and on epithelial cell surfaces (Renzi et al. 2011). Finally, Cani has been described to be ‘serum resistant’ to 10 % New Zealand rabbit serum (Butler et al. 1985) and to normal human serum (Shin et al. 2009), but Cani has also been shown to be sensitive to normal human serum (Hicklin et al. 1987). Thus, the interactions between Cani and the immune system are not completely understood but it is possible that one or several of the described immune evasive features could contribute to the high virulence previously reported for infections with Cani. In contrast to Cani, there is very limited knowledge on the role of human immunity in Cyno-infections. Cyno is more often reported in the case of wound infections and there is only two reports on invasive Cyno-infection (Khawari et al. 2005; Sarma and Mohanty 2001), whereas there are many case reports on invasive Cani-infections. Thus, if serum survival is a key virulence trait of Cani, it should be more resistant in serum than the presumably less virulent Cyno. To answer this question, we used a collection of clinical isolates of Cani and Cyno collected at the Karolinska University Laboratory between 2007 and 2010 and used traditional whole blood and serum bactericidal assays. The role of the complement system Page 2 of 9 was dissected by the use of heat inactivation, calcium chelation and C1q-depleted sera. In addition, sera from patients deficient in mannose binding lectin (MBL-) activity were used. Finally, sera from patients with a previous history of sepsis with Cani were used in an attempt to elucidate pathophysiological mechanisms. Method (...truncated)