Phenotypic and Genotypic Traits of Pasteurella multocida subsp. septica Isolates From the Wounds of Two Patients Due to Dog or Cat Biting, 2023

We describe the phenotypic and genotypic traits of Pasteurella multocida subsp. septica isolates from the dog/cat bite wounds of two patients in 2023. A 79-year-old man with diabetes mellitus and cerebral infarction who was bitten by a dog on his left hand developed deep inflammation under the tendon between his left fourth and fifth fingers. The patient’s condition was resolved with antimicrobial treatment and surgical intervention. Another patient, a healthy 49-year-old woman who was bitten by a cat on her left hand, developed superficial inflammation of the left thumb and index finger. The patient’s condition improved with antimicrobial treatment without surgical intervention. The isolates from the two patients had similar biochemical properties, and the antimicrobial susceptibility data for both isolates indicated erythromycin resistance. Genotypic analyses revealed clade 2 on the dendrogram of repetitive sequence-based fingerprinting, capsule serogroup cap genotype A, and hsf-1-nanH-pmHAS (virulence-associated genes). Our observations show that the two isolates have similar phenotypic and genotypic traits, regardless of differences in patient background, biting pets, wound inflammation, or the necessity of surgical intervention.


Introduction
Pasteurella multocida was first isolated by Pasteur in 1881 from an epidemiological case of fowl cholera. This species is a Gram-negative nonmotile coccobacillus that is found in many animals' oral cavities and gastrointestinal tracts (including those of cats and dogs). P. multocida is divided into three subspecies of multocida, septica, and gallicida, based on the internal sequences of the superoxide dismutase gene (sodA) with 16S rRNA gene sequences [1], because the 16S rRNA gene sequences are similar among P. multocida. In a bacteriological analysis of infected wounds caused by dog or cat bites, the Emergency Medicine Animal Bite Infection Study Group [2] found that P. canis (previously known as P. multocida biotype 6, revealing a positive ornithine decarboxylation test, a negative urease test, and variable acid production from trehalose and d-xylose) was the most common species isolated from dog bite wounds, whereas P. multocida subsp. multocida and P. multocida subsp. septica were the most common pathogens isolated from cat bite wounds. Herein, we report the phenotypic and genotypic traits of P. multocida subsp. septica isolates from the wounds of two patients bitten by a dog and a cat, respectively, in 2023.

First case
A 79-year-old man with diabetes mellitus and cerebral vascular infarction visited our emergency room in 2023. Two days before the visit, he had been bitten by a pet dog on his left hand. The fourth and fifth fingers of the left hand were swollen, and purulent discharge drained from the wounds. On admission, the patient had a temperature of 36.4 °C, a blood pressure of 132/76 mmHg, a respiratory rate of 14 breaths/min, an oxygen saturation of 97% (room air), and a heart rate of 118 beats/min. Physical examination was notable for swelling, redness, and tenderness in the fourth and fifth fingers ( Figure 1).  The patient was diagnosed with purulent tendovaginitis. Two sets of blood cultures using the BacT/ALERT3D system (bioMérieux Japan Ltd., Tokyo, Japan) and aerobic/anaerobic pus cultures were obtained because the patient was immunocompromised. Gram stain of the exudate showed Gram-positive cocci and Gram-negative rods. Intravenous ampicillin/sulbactam (12 g/day) was initiated, and the patient underwent emergency surgery for an early-stage exploratory incision and drainage. The operative findings included non-purulent effusion and inflammation that seemed to undermine the tendon. A non-purulent effusion culture was obtained during surgery; however, no microorganisms were observed on Gram staining. As there was no obvious degeneration of the tendon, a tendon synovectomy was performed to remove the inflamed synovial membrane. The pus culture collected on admission grew P. multocida, Cutibacterium acnes, Fusobacterium nucleatum, and aerobic Gram-positive cocci, whereas no microorganisms grew on the two sets of blood cultures. Anaerobic C. acnes and F. nucleatum were identified using the Rapid ID32A test kit (bioMérieux Japan Ltd.). The effusion culture collected during surgery was positive for P. multocida. The patient's condition improved, and he was discharged on day 11. Antimicrobial treatment was changed to a three-week course of oral amoxicillin/clavulanate (amoxicillin 1500 mg/day with clavulanate 375 mg/day). The patient remained well without recurrence or sequelae.

Second case
A healthy 49-year-old woman's left hand was bit by a pet cat seven hours before her presentation to our hospital in 2023. She presented with a swollen index finger and had a temperature of 36.1 °C, a blood pressure of 122/74 mmHg, a respiratory rate of 12 breaths per minute, an oxygen saturation of 98% (under room air), and a heart rate of 72 beats/min. Physical examination revealed swelling, redness, and tenderness of the left thumb and index finger ( Figure 2).

FIGURE 2: Appearance viewed from back of the hand of the second case
Laboratory tests did not reveal any remarkable findings. A small volume of pus was draining from the index finger. A pus culture was obtained; however, no microorganisms were observed on the Gram staining. Cellulitis was diagnosed because the patient did not exhibit any Kanavel cardinal signs of flexor sheath infection. Oral administration of amoxicillin/clavulanate (amoxicillin 1500 mg/day with clavulanate 375 mg/day) was initiated as outpatient treatment. Careful observation of the patient confirmed that her condition had not worsened. The pus culture grew P. multocida, and the treatment was successful in resolving the redness. The patient remained well without recurrence or sequelae during the follow-up period of one month.

Discussion
The phenotypic and genotypic traits are important when evaluating epidemiological evidence and associated virulence when selecting appropriate antimicrobial(s). Table 2 shows the phenotypic and genotypic traits of the P. multocida subsp. septica isolates (strains PA97 and PA98) from the wounds of our two patients who had been diagnosed with tendovaginitis (in the first case) and cellulitis (in the second case).
Isolate no.  ID TEST HN-20 Rapid system (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) was used to assess the biochemical reactions except for sorbitol fermentation.
API RAPID ID 32 E (bioMérieux Japan Ltd., Tokyo, Japan) was applied to evaluate the sorbitol fermentation.
1 Key biochemical properties of P. multocida subsp. septica to distinguish from P. multocida subsp. multocida are shown. 2 Antimicrobial susceptibility testing was done using disk diffusion and broth microdilution methods based on the Clinical and Laboratory Standards Institute document M45-A2 (2016).
The biochemical properties of strains PA97 and PA98 were similar, except for sorbitol fermentation ( Table 2). Table 3 depicts the AST data using the disk diffusion and broth microdilution methods against both isolates, along with erythromycin resistance, revealing similar AST patterns against PA97 and PA98.  Minimum inhibitory concentrations (μg/mL) of antimicrobials were determined using the broth microdilution method (Dry Plate Eiken DP44; Eiken Chemical Co., Ltd., Tokyo, Japan).
There were similar antimicrobial susceptibility data against PA97 and PA98 isolates.
Additionally, genotypic analyses yielded the same cluster (clade 2) on the UPGMA dendrogram of ERIC-PCR fingerprinting ( Figure 3) and indicated that cap genotype A contributed to the capsule serogroup and that the VAG profile contained hsf-1-nanH-pmHAS. PA60 and PA57 were found to be outliers on the UPGMA dendrogram.
info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.