Introduction
Capnocytophaga canimorsus and
Capnocytophaga cynodegmi normally reside in the oral flora of healthy cats and dogs, but can be transmitted to humans via bites or scratches.
C. canimorsus can cause wound infections, but also invasive infections, including sepsis, meningitis, or endocarditis [
4], whereas
C. cynodegmi has mostly been associated with wound infections [
9,
13,
23]. Recently, an additional
Capnocytophaga species named
Capnocytophaga canis was found in the oral flora of healthy dogs [
22]. In addition, we also recently described a fourth animal-associated
Capnocytophaga species that was given the name
Capnocytophaga stomatis [
26]. The pathogenic potential of
C. canis is unknown, whereas
C. stomatis was found in a wound specimen from a dog bite [
26].
The treatment of wound infections after cat- and dog-bites involves cleaning and debridation and also antibiotics as prevention or treatment, unless the wound is superficial. For dog-bites, the first-line treatment is amoxicillin+clavulanic acid, and for cat-bites the first-line therapy is penicillin [
12]. If the patient is allergic to penicillin, the recommendation is trimethoprim–sulfametoxazol or doxycycline [
21]. Clindamycin or daily injections with ceftriaxone—in cases of low compliance—have been proposed [
15]. These antibiotics are considered to cover the most common bacteria related to cat and dog bites, including
Pasteurella,
Staphylococcus,
Streptococcus as well as anaerobic
Fusobacteria spp. and
Bacteroides fragilis. In addition, the suggested empirical treatment is thought to cover for
C. canimorsus and
C. cynodemi [
20].
However, available data on antibiotic susceptibility testing (AST) is scarce for
C. canimorsus and
C. cynodegmi. The related human
Capnocytophaga spp., which are found in the oral flora of healthy individuals, are usually susceptible to penicillin. However, penicillin resistance also occurs in these bacteria, and some reports have described beta-lactamase production among clinical isolates of human
Capnocytophaga spp., including
C. sputigena and
C. ochracea [
10,
17]. However, whether animal-associated
Capnocytophaga species produce beta-lactamases is not known.
Thus, given the lack of available AST data, we set out to describe the phenotypic susceptibility patterns for the strains in our collection to a range of clinically relevant antibiotics. Our strain collection included C. canimorsus and C. cynodegmi as well as the novel Capnocytophaga species C. canis and C. stomatis. In order to obtain a mechanistic understanding of the phenotypic results, we coupled the AST data to bioinformatic analysis of whole genome sequences.
Discussion
Here we set out to describe the antibiotic susceptibility patterns of a collection of animal bite associated
Capnocytophaga isolates, including the recently discovered
C. canis and
C. stomatis. In addition, we linked the phenotypic results with full genome sequences obtained in a previous project [
26]. A major finding in this work is the identification of a beta-lactamase in four strains of animal bite associated
Capnocytophaga species. This enzyme, designated blaOXA347, is associated with phenotypic resistance to penicillin, cephalosporins, and also to imipenem. Given this broad substrate specificity, it could indeed be classified as a carbapenemase [
8]. Notably, blaOXA347 was only found in wound-associated strains and not in blood culture isolates, suggesting a possible negative effect on virulence and fitness. The enzyme also appears to be connected to the bacterial chromosome and not found on a plasmid, which significantly lowers the transmission potential to other
Capnocytophaga strains. However, the fact that the gene is 100% identical at the nucleotide level in the four strains that represent two different species indicates that it has been horizontally transferred.
It is possible that commensal bacteria in the oral flora of cats and dogs serve as a reservoir for antibiotic resistance genes. In humans, for example,
Capnocytophaga species (
C. ochracea, C. sputigena) are often resistant to penicillin and cephalosporins by virtue of several different beta-lactamases [
6,
10]. In line with this analogy, it is possible that blaOXA347 could be transmitted via transposons to the more virulent and invasive
C. canimorsus, although no such multidrug resistant
C. canimorsus have been described. Notably, this mechanism of transmission was recently shown for the chromosomally encoded blaNDM-1 carbapenemase gene in
Acinetobacter baumanii [
14] and OXA48 carbapenemase in highly virulent
E. coli [
2].
Interestingly, a BLAST search revealed that blaOXA347 has a 100% homology to a beta-lactamase in
Myroides odoratimimus (data not shown). This is an environmental gram-negative bacteria found in soil and water, which in rare cases may cause infections in immunocompromised individuals [
7].
M. odoratimimus is commonly resistant to beta-lactams, including cephalosporins and carbapenems [
16]. Several chromosomally encoded beta-lactamases have been described in this bacterial species [
1], and it is possible that it has transmitted blaOXA347 to bacteria in the dog oral flora, although this remains to be proven.
In addition, a neighbour in the phylogenetic tree is
Schewanella algae, a non-fermentative, gram negative opportunistic bacteria, which may cause skin and soft-tissue infections in immunocompromised individuals.
Schewanella algae has also been shown to be resistant to cefalosporins and to imipenem [
24]. In addition, whole genome sequencing has demonstrated the presence of several beta-lactamases in the
S. algae genome [
5]. Thus, it is possible that the environmental bacteria
M. odoratimimus and
S. algae may have transmitted blaOXA-347 to
C. cynodegmi and
C. stomatis in the oral flora of cats and dogs.
This study has several strengths. First, it is the most updated and largest analysis of antibiotic susceptibility in animal bite associated
Capnocytophaga species in more than 25 years [
3,
25]. Second, we have included the recently discovered novel species
C. canis and
C. stomatis, for which there is no data on antibiotic susceptibility available. Notably, blaOXA347 was found in two isolates of
C. stomatis, which make this species a focus for future studies in the area of bacterial resistance. Finally, we have combined detailed phenotypic testing with whole genome analysis, which has not previously been performed for
Capnocytophaga species.
There are also several weak points. Although the study includes 24 isolates (whole genomes) and 16 isolates (phenotypic analysis), it is a small study compared to studies on many other bacterial species. This has to be taken into account when interpreting the data, and the full picture of the epidemiology of blaOXA347 in animal bite associated Capnocytophaga species remains to be shown. Another limitation includes the bioinformatic analysis, which was performed utilizing the web-based program ResFinder. Interestingly, ResFinder rapidly identified blaOXA347 and tet(X) in several isolates. However, the phenotypic antibiotic resistance patterns indicated that there are several additional resistance genes encoded in the genomes of these bacteria. Taken together, this discrepancy indicates that more detailed bioinformatic work needs to be done to describe the full resistome on the genome level. Until efficient and easy-to-use bioinformatics tools are readily available for clinicians, phenotypic testing will still have a place in the clinical laboratory.
It is noteworthy that all
C. canimorsus strains tested had a very low MIC value for penicillin, the first-line drug for infections after cat or dog-bites. This finding is in line with previous studies [
3,
25]. Based on our data (Table
1), penicillin or cephalosporins or in the case of type I allergy to PC, clindamycin or tetracycline appear to be effective against invasive infections with
C. canimorsus. For wound infections, the situation is more complex, and there is broad resistance to beta-lactams in four isolates. However, no resistance to clindamycin was found, and tetracycline appears to be a good alternative. Thus, we suggest that clinicians become aware of the possibility of beta-lactam resistance in wound-associated
Capnocytophaga species, and that full AST is performed in case of animal bite associated wounds. Likewise, the presence of a beta-lactam resistant
C. canimorsus is unlikely, but—given the current data—a future possibility, which needs to be considered in clinical bacteriological laboratories. This is particularly important, since
Capnocytophaga are fastidious bacteria that grow slowly and can be difficult to identify, especially in wound specimens where a polymicrobial flora dominates.
To conclude, we demonstrated the antibiotic susceptibility profile of clinical Capnocytophaga isolates, and have identified a chromosomally encoded betalactamase (blaOXA347) with broad substrate specificity in animal associated Capnocytophaga species. This is a novel finding, and points to a role of the normal flora of pet animals as a reservoir of bacterial resistance genes, although the clinical relevance remains to be established. We suggest that further research is warranted in order to obtain more knowledge on the epidemiology, bacterial resistance, and clinical outcomes of Capnocytophaga infections.