Genomic analysis reveals the presence of a class D beta-lactamase with broad substrate specificity in animal bite associated Capnocytophaga species

Eur J Clin Microbiol Infect Dis (2017) 36:657–662 DOI 10.1007/s10096-016-2842-2 ORIGINAL ARTICLE Genomic analysis reveals the presence of a class D beta-lactamase with broad substrate specificity in animal bite associated Capnocytophaga species S. Zangenah 1 & A. F. Andersson 2 & V. Özenci 1 & P. Bergman 1 Received: 29 September 2016 / Accepted: 8 November 2016 / Published online: 1 December 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Capnocytophga canimorsus and Capnocytophga cynodegmi can be transmitted from cats and dogs to humans, and can cause a wide range of infections including wound infections, sepsis, or endocarditis. We and others recently discovered two new Capnocytophaga species, C. canis and C. stomatis, mainly associated with wound infections. The first-line treatment of animal bite related infections is penicillin, and in case of allergy, doxycycline and trimethoprim/sulfamethoxazole. However, there is a lack of antibiotic susceptibility patterns for animal bite associated Capnocytophaga species. Thus, we set out to study the antibiotic profiles against animal bite associated Capnocytophaga species isolated from wound and blood cultures after cat and dog bites and coupled the findings to whole genome sequencing data. A total of 24 strains were included in the study. Phenotypic analysis of antibiotic resistance was performed with E-tests. The web-based tool ‘Resfinder’ was used to identify resistance genes in the whole genome dataset. Two strains of C. cynodegmi and two strains of the recently discovered C. stomatis were resistant to penicillin (MIC > 24 mg/L) and cephalosporins (MIC > 24 mg/L), and three out of these strains also exhibited resistance to imipenem (MIC = 32 mg/ L). Genomic analysis revealed that these strains carried a class D beta-lactamase gene, which has not previously been found in Capnocytophaga spp. A class D beta lactamase with broad * P. Bergman 1 Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, Huddinge, Sweden 2 Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, KTH Royal Institute of Technology, Stockholm, Sweden substrate specificity was found in animal bite associated Capnocytophaga species, which could have important implications when treating wound infections after cat and dog bites. It also suggests that pet animal bacteria can harbour resistance genes with relevance for human infections. 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 dogbites, 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. 658 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. Eur J Clin Microbiol Infect Dis (2017) 36:657–662 clavulanate (amox/clav). Briefly, bacteria were thawed from stocks and sub-cultured on HBA plates, incubated at 35 °C in 5% CO2 for 48 hours. Thereafter, a loop of bacteria was suspended in sterile PBS to an optical density of McFarland 0.5 × 108 CFU/ml [18], and spread on blood agar plates immediately prior to the application of the E-test strips [11]. Plates were incubated at 35 °C in 5% CO2 for 48 hours before reading the results. MIC values were interpreted according to the EUCAST antimicrobial guidelines for non-species-related breakpoints [19]. Identification of antibiotic resistance genes The software tool ResFinder from the Center for Genomic Epidemiology was used for identification of antimicrobial resistance genes in the genomic sequences of 24 strains of Capnocytophaga spp. FASTA files of complete genomes for all strains were used as search strings in the program. The threshold for ID was set to 98% and the minimum length was set to 60% [29]. Phylogenetic analysis Material and methods Bacterial strains In total, 24 isolates were included in the study, including n = 9 C. canimorsus, n = 9 C. cynodegmi, n = 3 C. stomatis and n = 1 C. canis, and n = 2 reference strains (C. canimorsus, ATCC 35978 and C. cynodegmi, ATCC 49045). C. canis was isolated from a human wound specimen, and is thus here defined as a clinical isolate. These isolates have been described in detail in previous studies from our group [26–28]. The genomes for all 24 strains were used in the bioinformatic analyses, whereas 16 strains were subjected to antibiotic susceptibility testing based on consistent and reproducible growth patterns. Reagents Horse blood agar plates (HBA) and phosphate buffered saline (PBS) were manufact (...truncated)