Investigation of antimicrobial susceptibility and resistance gene prevalence in Capnocytophaga spp. isolated from dogs and cats and characterization of novel class A β-lactamase CST-1

doi:10.21203/rs.3.rs-5285692/v1

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Investigation of antimicrobial susceptibility and resistance gene prevalence in Capnocytophaga spp. isolated from dogs and cats and characterization of novel class A β-lactamase CST-1

https://doi.org/10.21203/rs.3.rs-5285692/v1

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Purpose

Capnocytophaga spp., common inhabitants of the animal oral cavity, are zoonotic pathogens transmitted to humans through dog/cat bites and cat scratches. Appropriate antimicrobial therapy is essential for treat this zoonotic disease because of the rapid deterioration at disease onset; however, antimicrobial resistance of animal bite-associated Capnocytophaga spp. has not been fully investigated. We sought to understand the antimicrobial susceptibility and prevalence of resistance genes among Capnocytophaga sp. isolates obtained from dogs and cats.

Method

Minimum inhibitory concentrations (MICs) of nine antibiotics for 57 C. canimorsus, C. cynodegmi, C. canis, C. felis, C. stomatis, and C. catalasegens isolates were assayed by E-test. Resistance genes were detected by polymerase chain reaction, nucleotide sequencing, and whole-genome sequencing.

Results

The MICs of penicillin, ceftriaxone, cefepime, clindamycin, minocycline, nalidixic acid, and ciprofloxacin were high for some isolates. The MICs of imipenem and amoxicillin/clavulanic acid were low for all isolates. Known resistance genes bla_cfxA2, bla_OXA−347, emrF, and tetQ were detected using polymerase chain reaction. Mutation in the quinolone resistance-determining region of gyrA was also detected. Cst-1, a previously unreported gene, was identified using whole-genome analysis of two C. stomatis isolates. CST-1 was proposed as a class A, subclass A2, β-lactamase based on amino acid sequence and phylogenetic relationship. In recombination experiments, CST-1 inactivated penicillin and first- and second-generation cephems; however, sulbactam inhibited it.

Conclusion

Known and novel resistance genes are prevalent among Capnocytophaga spp. in animal oral cavities. The findings have significant clinical implications, especially in antimicrobial treatment.

Capnocytophaga spp.

zoonosis

antimicrobial resistance

β-lactamase

CST-1

The genus Capnocytophaga consists of gram-negative, capnophilic fusiform to rod-shaped, gliding bacteria that usually inhabit the human or animal oral cavities [1, 2]. Seven species of this genus (C. ochracea, C. gingivalis, C. sputigena, C. granulosa, C. haemolytica, C. leadbetteri, and C. periodontitidis) inhabit humans and are mainly associated with periodontal disease. In contrast, five species (C. canimorsus, C. cynodegmi, C. canis, C. felis, and C. catalasegens) inhabit dogs and cats [3–5]. In addition, the provisional novel species C. stomatis is thought to inhabit dogs and cats [6].

Among Capnocytophaga spp. distributed in the oral cavity of dogs and cats, C. canimorsus, C. cynodegmi, C. canis, C. felis, and C. stomatis have been recognized as zoonotic pathogens transmitted to humans via dog/cat bites and cat scratches [2, 7–9]. More than 500 cases have been reported worldwide, and there are some reports of 0.5–0.7 cases per million inhabitants per year or 4.1 cases per million inhabitants per year [3]. They can cause a wide range of infections, including wound and invasive infections, such as bacteremia, endocarditis, meningitis, and sepsis. In particular, C. canimorsus can cause fatal sepsis with a mortality rate of approximately 26% [2], and capsular serovar of C. canimorsus is thought to be associated with disease severity [10]. However, bacteremia associated with non-canimorsus Capnocytophaga (C. cynodegmi, C. canis, and C. stomatis) has also been reported [8].

Appropriate antimicrobial therapy is required to treat this zoonotic disease because of the rapid deterioration of systemic symptoms at disease onset. The rapid progression to high-grade bacteremia and sepsis after dog/cat bites is thought to contribute to the high mortality rate [1, 2]. Antimicrobial therapy is often initiated without waiting for the results of cultural susceptibility tests, thus evaluating the antimicrobial susceptibility of Capnocytophaga spp. inhabiting dogs and cats is vital. However, information regarding the susceptibility and prevalence of resistance genes among animal bite-associated Capnocytophaga spp. is insufficient.

Capnocytophaga spp. are typically susceptible to penicillin and other antibiotics commonly used as prophylaxis following animal bites [2]. However, the recent emergence of strains resistant to β-lactams and quinolones [11–13], including reports of carbapenem (the most important β-lactam for treating gram-negative bacterial infections)-resistant C. stomatis and its associated resistance gene bla_oxa−347, is becoming a concern [13]. Therefore, in this study, we investigated the antimicrobial susceptibility and prevalence of associated resistance genes in C. canimorsus, C. cynodegmi, C. canis, C. felis, and C. stomatis isolates collected from oral swabs of dogs and cats, identifying a previously unreported resistance gene.

Bacterial strains

A total of 57 isolates were used: 10 C. canimorsus, 31 C. canis, 2 C. felis, 5 C. cynodegmi, and 9 C. stomatis. These isolates were obtained from dental plaque swabs of dogs and cats in our previous study during 2011–2013 [14]. Details of the isolates are listed in Online Resource 1. Bacterial species were identified using ANI values obtained from NGS data analyzed using the DDBJ Fast Annotation and Submission Tool (DFAST pipeline [15]) or 16S rRNA sequence analysis.

Antimicrobial susceptibility

The minimum inhibitory concentrations (MICs) of piperacillin, ceftriaxone, cefepime, imipenem, amoxicillin/clavulanic acid, clindamycin, minocycline, nalidixic acid, ciprofloxacin, ampicillin, ampicillin/sulbactam, cephalothin, cefotaxime, cefoxitin, and aztreonam were determined using the E-test (Biomerieux, Marcy l'Etoile, France) according to the manufacturer’s instructions. Antimicrobials examined included drugs commonly used to treat dog and cat bites [2]. The interpretation criteria for Capnocytophaga spp. are not defined by the Clinical and Laboratory Standards Institute [16] or European Committee on Antimicrobial Susceptibility Testing [17]. For testing Capnocytophaga sp., E-test strips were placed on heart infusion with 5% sheep-blood agar plates and incubated at 35°C under 5% CO₂ for 48 h before reading the results. For testing E. coli, E-test strips were placed on Mueller–Hinton agar plates and incubated at 35°C under aerobic conditions for 20 h before reading the results. β-Lactamase-producing activity was detected via cefinase test using a cefinase disk (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.

Polymerase chain reaction detection and nucleotide sequence determination of antimicrobial resistance genes

Genomic DNA was extracted from gram-negative bacteria using the Illustra Bacteria GenomicPrep Mini Spin Kit (GE Healthcare, Chicago, Ilm USA) according to the manufacturer’s instructions.

The primers for polymerase chain reaction (PCR) detection and the sequences used are listed in Online Resource 2. β-Lactamase (bla_cfxA1, bla_cfxA2, bla_oxa-347, and bla_ybxI), macrolide resistance (ermF), and tetracycline resistance (tetQ and tetX) genes were detected using PCR with specific primers and confirmed by determining the nucleotide sequences of amplicons using the same primers. Chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of gyrA were detected through PCR amplification of gyrA and determination of the nucleotide sequences.

WGS analysis

Genomic DNA was extracted as described above. Library construction and sequencing on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) were provided as custom service by Eurofins Genomics (Tokyo, Japan). Genomic DNA samples were sheared into 350-bp fragments using a sonicator. The resulting DNA fragments were processed for adaptor ligation and amplification to generate libraries. The prepared libraries were subjected to paired-end 2 ×100 bp sequencing on the HiSeq 2500 platform. The reads were assembled using the CLC Genomic Workbench 11.0.1 (QIAGEN, Hilden, Germany). Coding sequence (CDS) annotation and taxonomic identification were performed using the DFAST pipeline [15].

Antimicrobial resistance genes were searched and identified using ResFinder 4.1 on the Center for Genomic Epidemiology website (http://genomicepidemiology.org/).

The nucleotide sequences and amino acid residues were aligned using ClustalX (ver. 2.1) with the parameters provided in ClustalW (ver. 2.6). A phylogenetic tree with 1,000 bootstrap replicates was constructed using the neighbor-joining (NJ) method. The resulting tree was drawn using the NJ plot software. A linear comparison of genetic loci was performed using EasyFig 2.2.3.

Cloning experiments

For cloning experiments, an In-Fusion HD Cloning Kit (TaKaRa, Kyoto, Japan) was used according to the manufacturer’s instructions. pET28a was used as a plasmid vector (Merck & Co., Rahway, NJ, USA). Briefly, a DNA fragment containing cst-1 (945 bp) from C. stomatis 11022B-2 was amplified using the primers pET28a_F1-FW and pET281_F1-RV (Online Resource 2). The DNA fragment containing the plasmid vector pET28a was amplified using primers pET28a_Vec_FW and pET28a_Vec_RV (Online Resource 2). After amplification, the cst-1 fragment was cloned into the pET28a plasmid fragment and BL21 (DE3)-competent E. coli (New England Biolabs, Ipswich, MA, USA). The nucleotide sequences of the inserted fragments were confirmed using the pET upstream primer (seq) and the T7 terminator primer (seq; Online Resource 2). The transformed bacteria were selected with 50 μg/mL kanamycin-containing Luria–Bertani agar plates. After the induction of protein expression using 0.5 mM isopropyl-β-D-thiogalactopyranoside-containing Luria–Bertani broth, MICs were assessed using the method described above. Cloning experiments were approved by the Recombinant DNA Experiments Safety Management Committee, Osaka Institute of Public Health, Japan.

Nucleotide sequence accession numbers

The annotated whole-genome DNA sequences of C. stomatis isolates 11022B-2 and 11058B-1 were submitted to GenBank/EMBL/DDBJ under accession numbers BOPK01000001-BOPK01000029 (11022B-2) and BOPJ01000001-BOPJ01000029 (11058B-1), respectively, associated with BioProject number PRJDB11150.

Antimicrobial susceptibility results

MIC (μg/mL) ranges of nine antibiotics (penicillin, ceftriaxone, cefepime, imipenem, amoxicillin/clavulanic acid, clindamycin, minocycline, nalidixic acid, and ciprofloxacin) for 57 Capnocytophaga sp. isolates from dogs and cats were evaluated. The MIC distributions and MIC₈₀ values are listed in Table 1.

The MIC₈₀ values ranged from 0.023 to 0.5, except for nalidixic acid (>256). For some or all isolates, the MICs of penicillin, ceftriaxone, cefepime, clindamycin, minocycline, nalidixic acid, and ciprofloxacin were more than 1. In contrast, MICs of imipenem and amoxicillin/clavulanic acid were less than 1 for all isolates.

Prevalence of antimicrobial resistance genes and QRDRs among Capnocytophaga spp.

Antimicrobial resistance genes detected using PCR and WGS and chromosomal mutations in QRDRs, along with MIC values, are summarized in Table 2.

PCR detection of known resistance genes: class A β-lactamase gene bla_cfxA2 was found in an isolate (C. cynodegmi), class D β-lactamase gene bla_OXA-347 in two isolates (C. canimorsus and C. canis), macrolide resistance gene emrF in two isolates (C. canimorsus and C. stomatis), and tetracycline resistance gene tetQ in three isolates (C. canimorsus, C. cynodegmi, and C. stomatis). The class A β-lactamase gene bla_cfxA1,class D β-lactamase gene bla_ybxI, and tetracycline resistance gene tetX were not found in any isolates. The presence of resistance genes was consistent with the elevated MIC values of the drugs to which the genes were associated.

For two isolates (C. stomatis 11022B-2 and 11058B-1), the MIC values of β-lactams were relatively high, although β-lactamase genes were not detected using PCR. Applying WGS contig sequences with ResFinder 4.1 and BLAST search, an unidentified resistance gene (945 bp) with approximately 70% identity to the CfxA family class A β-lactamase genes was found. The identity of the unidentified gene between the two C. stomatis isolates was 100%. Two C. stomatis isolates (C. stomatis 11022B-2 and 11058B-1), one bla_cfxA2 harboring isolate, and two bla_OXA-347harboring isolates were positive for cefinase. Thus, the unidentified resistance gene was assumed to be a β-lactamase gene and putatively named cst-1 (Capnocytophaga stomatis-1).

QRDRs for DNA gyrase gene A (gyrA) were detected in 17 isolates (4 from C. canimorsus, 2 from C. canis, 3 from C. cynodegmi, and 8 from C. stomatis). All QRDRs were substitutions of Ser83 to Pro83 and Asp86 to Glu86 (Pro83/Glu86). For 17 isolates with Pro83/Glu86, the MIC values of NA and CPFX were 64 to >256 and 0.38 to 4, respectively.

Comparative analysis of the amino acid sequences of CST-1

A phylogenetic tree was constructed by aligning the full-length amino acid sequence of CST-1 with the sequences of the related CfxA family and representative members of class A, subclass A2, β-lactamases [18,19] (Fig. 1). CST-1 was closely related to CfxA family members; however, it was classified as a branch different from the CfxA family cluster. CST-1 was also distinctly branched from other class A, subclass A2, β-lactamases.

Figure 2 shows an alignment of the amino acid sequence of CST-1 with those of representative members of class A, subclass A2, β-lactamases, proposed previously [18]. CST-1 had three major motifs (70SxxK, 130SDN, and 234KTG) associated with catalytic mechanisms and/or substrate binding common in class A β-lactamases. CST-1 also harbored a Ω-loop motif specific to subclass A2 β-lactamases containing the E166 residue involved in catalytic mechanism and/or in substrate binding.

Comparison of genetic structures around cst-1

Genetic regions around cst-1 were compared between C. stomatis 11022B-2 and 11058B-1 isolates (Fig. 3.). Upstream of cst-1, the mobilization protein gene, hypothetical protein-coding sequence, and site-specific integrase gene were commonly present. The homology of the common regions between the two isolates was 100%, and no specific IS or transposition motif was found in the IS finder database (https://www-is.biotoul.fr/index.php) search. However, the genetic compositions upstream and downstream of these common regions were completely different.

cst-1 could be chromosomally encoded because of the presence of rRNA and tRNA genes in the same contig as cst-1 in C. stomatis 11022B-2 and 11058B-1. cst-1 appeared to be located at different chromosomal positions in the two isolates. The GC content of a series of site-specific integrase gene-hypothetical protein coding sequence-mobilization protein-cst-1 was 47%, which was higher than that of C. stomatis 11022B-2 and 11058B-1 whole genomes (approximately 36%). Thus, cst-1 may have been acquired from other bacteria via an integron cassette.

In the BLAST search, a β-lactamase gene (putatively named as cst-2), which differed from cst-1 in only one amino acid, was found in Bacteroides stercoris strain TF03-6 (accession number NZ_QSSV01) detected from human feces in Shenzhen, China, in 2012. Although the nucleotide sequences and genetic composition upstream of cst-2 have not been fully deposited in the NCBI database, the presence of an IS5 family transposase between cst-2 and the mobilization protein gene was characteristic.

Properties of CST-1 β-lactamase

To clarify the resistance phenotype of cst-1, full-length cst-1 (945 bp) was transfected into the plasmid vector pET28a and inserted into non-β-lactamase-producing E. coli BL21 (DE3). The MIC values of nine β-lactam antibiotics alone or in combination with a β-lactamase were determined using an E-test (Table 3). The cst-1-positive C. stomatis 11022B-2 and 11058B-1 with 11021B-1, in which none of the resistance genes were detected, and the cst-1transconjugant E. coli BL21 (DE3) [CST-1/pET28a], pET28a plasmid-inserted E. coli BL21 (DE3), and E. coli BL21 (DE3) were tested.

The MIC values of penicillin, ampicillin, and cephalothin for C. stomatis 11022B-2 and 11058B-1 increased remarkably compared with those for C. stomatis 11021B-1. Furthermore, the MIC values of cefepime and cefoxitin for C. stomatis 11058B-1 presented a certain extent of increase. For both C. stomatis 11022B-2 and 11058B-1, the addition of sulbactam decreased the MIC value of ampicillin.

The bla_CST-1transconjugant E. coli BL21 (DE3) [CST-1/pET28a] had significantly high MIC values of penicillin, ampicillin, cephalothin, and cefoxitin compared to pET28a plasmid-inserted E. coli BL21 (DE3) and E. coli BL21 (DE3). The MIC value of ampicillin decreased with the addition of sulbactam.

In this study, we analyzed the antimicrobial susceptibility of 57 dog and cat-derived Capnocytophaga sp. isolates, the largest cohort examined in similar research to date. The MIC values of penicillin, ceftriaxone, cefepime, clindamycin, minocycline, nalidixic acid, and ciprofloxacin were high in some strains (Table 1). We identified known resistance genes previously reported in Capnocytophaga spp. using PCR, including the β-lactamase genes bla_cfxA2 and bla_oxa−347, macrolide resistance gene emrF, and tetracycline resistance gene tetQ. Mutations in the QRDR of gyrA were also detected, with substitutions from amino acid positions 80 to 86 acting as primary targets for fluoroquinolones in Capnocytophaga sp. [12]. The consistency between resistance gene presence and antimicrobial susceptibility suggests these genes are functional in the strains, except for tetQ (Table 2).

The WGS data with a ResFinder search of two strains that showed high MIC values for β-lactams, including penicillin, but no known β-lactamase revealed a novel β-lactamase candidate, cst-1 (Table 2). Recently, a new β-lactamase was identified based only on the sequence data; however, a lack of functional data is considered to minimize the usefulness of this information. Therefore, transferring a novel β-lactamase gene to a non-β-lactamase-producing strain and confirming its resistance phenotype is recommended [20]. We conducted recombination experiments using cst-1 transconjugant E. coli (CST-1/pET28a), showing that CST-1 can inactivate penicillin and first- and second-generation cephems and is inhibited by sulbactam (Table 3). Therefore, CST-1 could be a class-A penicillinase. Based on amino acid sequence homology, presence of major functional motifs, and phylogenetic relationship, CST-1 was classified as a class A, subclass A2, β-lactamase [18, 19] (Figs. 1, 2).

Some class A, subclass A2, β-lactamase genes are chromosomally encoded and naturally produced, whereas others are transferable-element-encoded. For instance, genes encoding a CfxA-type enzyme in Capnocytophaga spp. have been identified in a mobilizable transposon [18, 21]. cst-1 could be encoded in a transferable-element and inserted at separate chromosomal locations as a gene cassette containing integrase and mobilization protein genes (Fig. 3.). cst-1 may have originally been derived from another species via conjugative transmission because the BLAST search showed that the amino acid sequence of the mobilization protein located upstream of cst-1 had more than 95% homology with the “mobV family relaxase” of Bacteroides, Parabacteroides, and other bacteria. Mob family relaxases are associated with the conjugative transfer of DNA in gram-negative bacteria [22]. Moreover, site-specific integrases associated with cst-1 from Bacteroides and Parabacteroides were identified using the BLAST search in this study.

The cst-1-positive strains showed variable MIC values for β-lactams (Table 3). This phenomenon may be associated with the cst-1 expression level, regulatory gene expression, or drug efflux pump expression. Tamanai-Shacoori et al. [23] reported that human oral clinical isolates of Capnocytophaga spp. show variable susceptibility to β-lactams with variable bla_cfxA expression levels.

The first study on OXA-347 in animal bite-associated C. cynodegmi and C. stomatis reported a high MIC of imipenem for bla_OXA−347-harboring strains [13]. Our findings suggest that OXA-347 is unlikely to be a carbapenemase, given the low MIC values of imipenem for bla_OXA−347-harboring C. canimorsus and C. canis strains, compared to high MICs of penicillin (Table 2). The reason for this discrepancy remains unclear, highlighting the need for further investigation owing to the remarkable clinical implications of determining whether OXA-347 is a carbapenemase.

In conclusion, we found that for some dog/cat-derived C. canimorsus, C. canis, C. felis, C. cynodegmi, and C. stomatis isolates, the MIC values of some antimicrobials are high. Imipenem-or amoxicillin/clavulanic acid-resistant strains were not found; however, strains resistant to penicillin, which are important drugs for animal bites and wounds, were detected. The findings have significant clinical implications, especially in antimicrobial treatment. We detected various resistance genes previously found in animal- or human-derived Capnocytophaga spp. and a novel class A, subclass A2, β-lactamase gene, bla_CST−1, from two C. stomatis isolates from cats.

A limitation of this study is that the isolates were obtained from a limited area. Analyzing isolates from not only dogs and cats but also human patients would be beneficial. Future research should include functional analysis of individual resistance genes to further clarify the antimicrobial resistance, epidemiology, and clinical implications of Capnocytophaga spp. zoonotic infections in both humans and animals.

Funding

This work was supported by the Health and Labor Science Research Grant (HSLRG) numbers H30-Shinkogyousei-Shitei-001 and 21HA2001; Japan Society for the Promotion of Science (JSPS) KAKENHI grant number JP19K06433; and Japan Agency for Medical Research and Development (AMED) grant number JP23fk0108615.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, and data collection and analysis were performed by Kaoru Umeda and Michio Suzuki. The first draft of the manuscript was written by Kaoru Umeda and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are not publicly available due to individual privacy but are available from the corresponding author on reasonable request.

Ethics approval

Cloning experiments were approved by the Recombinant DNA Experiments Safety Management Committee, Osaka Institute of Public Health, Japan. Ethics application was not required according to the guidelines of the Ethical Committee, Osaka Institute of Public Health, Japan.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

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Tables 1 to 3 are available in the Supplementary Files section.

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Investigation of antimicrobial susceptibility and resistance gene prevalence in Capnocytophaga spp. isolated from dogs and cats and characterization of novel class A β-lactamase CST-1

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