Intra-abdominal infection with Campylobacter curvus: case report and review of the literature

Background Campylobacter curvus is a Gram-negative bacteria associated with periodontal disease in humans. Cases of extra-oral manifestations of infection are rare with only six reported cases of extra-oral infection including this report that have been identified in the current literature. Molecular methods are generally used to identify C. curvus while optimal antibiotic choice and duration to treat extra-oral infections for this pathogen is unknown. Case presentation A 63-year-old male with a background history of alcoholic pancreatitis presented with fever and malaise who was found to have radiological intra-abdominal collections. Drainage of these collections identified C. curvus via matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry with high probability and identification further confirmed by whole-genome sequencing. Antibiotic susceptibility testing to erythromycin and ciprofloxacin of C. curvus was performed using E-test diffusion methods along with investigation for the presence of resistance genes. The patient was treated with intravenous piperacillin-tazobactam followed by ciprofloxacin for 4 weeks total with good clinical recovery. Conclusions Extra-oral manifestations with the pathogen C. curvus are rare with few cases described in the literature. There is minimal data on susceptibility patterns, optimal antibiotic treatment and duration. Treatment of extraintestinal C. curvus infections in humans should encompass both adequate source control and antibiotic therapy.


BACKGROUND
The Campylobacter genus comprises a large group of over 30 bacterial species with its more familiar members Campylobacter jejuni and Campylobacter coli implicated mainly in gastrointestinal disease [1]. Extra-intestinal infection with Campylobacter spp. including bacteremia, urinary tract infections and extraluminal collections are also known to occur with lesser-known members in the genus such as Campylobacter curvus emerging as pathogens causing infections in humans [2]. Originally identified as Wolinella curva in 1984 from human oral samples, C. curvus is a motile curved Gramnegative bacillus [3][4][5]. Following immunotypic and genetic analyses, W. curva was revised to C. curvus given homology to other Campylobacter species [6]. Notable for its association in periodontal disease and sporadic infectious diarrhoea, extra-oral infections with C. curvus are rare [7]. A review of the literature demonstrates only five case reports of extra-oral infection to date [8][9][10][11]. We describe here a case of C. curvus intra-abdominal infection including the methodology for identification and management as well as a comparison to the cases identified in the literature for management of extra-oral infections with this species.

CASE REPORT
A 63-year-old male presented with 4 weeks of malaise and fever in the setting of a prior pancreatic pseudocyst co-infection with Enterococcus faecalis, Klebsiella pneumoniae and mixed coagulase negative staphylococci, on a background of alcoholic pancreatitis. This infected pseudocyst had been OPEN ACCESS drained followed by 6 weeks of intravenous piperacillintazobactam and a further 2 weeks of oral ciprofloxacin with amoxicillin-clavulanate. Treatment was completed 5 months prior with complete clinical recovery, marked radiological reduction in the collections and resolution of inflammatory markers. His medical history was also notable for hypertension and alcoholic cardiomyopathy.
Examination at representation to hospital demonstrated right basal crackles in the lungs and a non-tender abdomen. Investigations demonstrated an elevated white cell count of 16.33×10 9 per litre with a predominant neutrophilia and a C-reactive protein of 156 mg l −1 (normal limits: 0-8 mg l −1 ). Other abnormalities included sodium, potassium and creatinine levels of 123 mmol l −1 , 5.3 mmol l −1 , and 201 μmol l −1 respectively. Liver function tests remained normal with a normal lipase level of 36 U l −1 . A computed tomography (CT) scan of his abdomen showed interval progression of pre-existing retroperitoneal and intraperitoneal collections with the largest collection measuring 129×58 mm (Fig. 1).
Ceftriaxone and metronidazole were commenced empirically, and CT-guided drains were inserted into the largest two collections. Sixty millilitres of purulent specimen was aspirated. Although there were no immediate complications, the patient later developed hypotension secondary to the underlying cardiomyopathy and transferred to the intensive care unit. Antimicrobial therapy was changed to piperacillintazobactam. Following haemodynamic stabilisation, he returned to the surgical ward 48 h later. A repeat CT scan showed near resolution of the two main collections.
Gram-stain of aspirated material demonstrated numerous polymorphic leukocytes and Gram-negative bacilli under high powered field microscopy ( Fig. 2). After 2 days of incubation, tiny white non-haemolytic colonies were cultured on horse blood agar under anaerobic conditions at 37 degrees Celsius (Fig. 3). The organism was oxidase-positive, catalasenegative, H 2 S non-producing, urease-negative, and motile. Using matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (Bruker; Preston, Victoria), the isolate was identified as C. curvus with a score of 2.143.
Antibiotic testing using E-test diffusion methods showed a ciprofloxacin and erythromycin minimum inhibitory concentration (MIC) of 0.19 ug ml −1 and 16 ug ml −1 respectively. The patient completed 10 days of intravenous piperacillintazobactam followed by oral ciprofloxacin for a further 3 weeks. At the time of antibiotic cessation, the patient had near radiological resolution of the drained collections and normalisation of his inflammatory markers. He remained clinically stable 2 months post-antibiotic cessation following hospital discharge.

DISCUSSION
Campylobacter spp. are a group of bacteria with its most familiar members C. jejuni and C. coli known to cause primarily gastrointestinal infection in humans [13]. Lesserknown species in the genus such as C. curvus are emerging as potential pathogens causing human disease. C. curvus is a non-fermenting, oxidase positive, microaerophilic Gramnegative bacillus that produces pinpoint colonies on blood agar when cultured under microaerophilic conditions [7]. Further biochemical characteristics of C. curvus are shown in Table 1. Isolation of C. curvus in human samples outside the oral cavity is rare and its relationship to human pathology is poorly understood [3]. Found as commensal bacteria in the human mouth, notably periodontal pockets of diseased gums, a causal role for these organisms in periodontal disease has not been clearly established [14].
Humans are known hosts to C. curvus with rates of carriage presumed low. One study of human faecal specimens investigating presumed gastroenteritis identified C. curvus in only one of 320 samples [15]. In a similar microbiome study, C. curvus DNA from intestinal biopsies was found in only 1.4 and 4.6 % of patients with ulcerative colitis and healthy controls respectively [16]. C. curvus as a cause of infectious diarrhoea is rare with C. curvus isolated in only 0.0004-0.05 % of children with infectious gastroenteritis [3,17]. This was similar to another study of infectious diarrhoea whereby C. curvus was isolated in only 15 patients over a 4 year period [7].
Extra-oral infection with C. curvus is rare. A search of the literature using the PubMed and EMBASE library databases for articles on C. curvus that have been published to date was conducted. Search terms included 'Campylobacter curvus' OR 'Wolinella curva' . Twenty-three and sixty-three results based on these terms were found in the PubMed and Embase databases respectively with only five case reports of extra-oral infection, comprising two cases of liver abscesses, two cases of pulmonary infection and a case of chorioamnionitis ( Table 2) [8][9][10][11].
In four cases, C. curvus was isolated on bacterial media when cultured under anaerobic conditions. All five cases prior to this report required molecular detection methods for identification of C. curvus with 16S rRNA sequencing the main method used. Three cases with infected collections were polymicrobial and in the case of C. curvus-associated chorioamnionitis, Haemophilus parainfluenza was also detected via molecular methods. In contrast to predominantly monomicrobial infections with C. foetus, C. curvus appears to exhibit co-habitation with other bacteria leading to polymicrobial infection similar to C. showae [13,18]. Whilst C. curvus was the only pathogen isolated in this patient's collection, this may be attributable to pre-treatment with antibiotics or selective pressures from previous therapy.
Whilst prior studies relied on molecular techniques to identify C. curvus, the isolate from this study was identified using MALDI-TOF MS with a high probability score. MALDI-TOF MS correctly identifies up to 91 and 83 % of all non-jejuni/ coli Campylobacter strains and C. curvus isolates respectively [19]. The isolate demonstrated identification of C. curvus with a score of 2.143 suggesting a high probability match. Whole-genome sequencing was used to confirm speciation and based on the genomic context from pan-genomes against other Campylobacter spp., the isolate appeared most related to C. curvus (Fig. 4).
Susceptibility testing and antibiotic MIC reports for C. curvus strains are limited due to the rarity of the infection. Reported MICs in the literature are variable but include: bacitracin >128 µg ml −1 , ceftriaxone >32 µg ml −1 , cefuroxime >256 µg ml −1 , chloramphenicol 2-4 µg ml −1 , ciprofloxacin 0.047 µg ml −1 , clindamycin 0.5-1 µg ml −1 , erythromycin 2 µg ml −1 , gentamicin 2 µg ml −1 , metronidazole 0.38-2 µg ml −1 , and penicillin 32 µg ml −1 [4,8]. Susceptibility to β-lactams in Campylobacter spp are variable with minimal data in particular to piperacillin-tazobactam [20]. One study examining C. coli/C. jejuni found low resistance to amoxicillin but high resistance to piperacillin thought secondary to poor penetration due to the bulky side chain in the latter [21]. E-test diffusion testing of the isolate demonstrated an MIC of 0.19 µg ml −1 for ciprofloxacin and 16 µg ml −1 for erythromycin. Although interpretation for these MIC values is not available for non-jejuni/coli Campylobacter spp due to limited data on C. curvus, we interpreted the isolate to be ciprofloxacin susceptible and erythromycin resistant by extrapolating EUCAST breakpoints for C. jejuni/C. coli species [22]. No gene could be detected in the draft genome that would confer antimicrobial resistance. Nevertheless, the draft genome harboured several sets of virulence genes encoding components of the . Notably, resistance via upregulation of motility and down regulation of metabolism following treatment with erythromycin has been found in C. jejuni [23].
Limited data exists for C. curvus susceptibility to erythromycin but this isolate appeared consistent to other non-jejuni/ coli species that show erythromycin resistance. This is potentially through mutations in the 23S RNA or via efflux pumps such as cmeABC [20,24]. The gene cluster cmeABC, which confers intrinsic low level erythromycin resistance in C. coli/ jejuni, was not detected in the isolate in this study but detection may have been hampered by the genetic distance between C. curvus and C. jejuni/coli or limited characterisation studies for this species of campylobacter.
The patient in this case report continued antibiotic therapy with ciprofloxacin following broad-spectrum antibiotics. Although C. jejuni/coli species readily acquire resistance to fluoroquinolones limiting its use, whether this applies to C. curvus is unclear [24]. The optimal duration of treatment following source control is uncertain, ranging between 2 days to 6 weeks. Given the propensity for C. curvus to form biofilms and its persistence in inhospitable environments, a prolonged treatment course may be prudent to ensure clearance of infection. The patient was treated with 3 weeks of ciprofloxacin following drainage [25].

CONCLUSION
Extraoral manifestations of C. curvus are rare with infections predominantly due to polymicrobial collections. Identification of C. curvus is classically reliant on molecular techniques due to its fastidious nature but the MALDI-TOF MS may be adequate in identifying most C. curvus isolates. Management  Fig. 4. Phylogenetic tree of isolate recovered from the reported case with other known Campylobacter species. The maximum likelihood tree was constructed using alignment of gene-to-gene comparison.
of extraoral infections require good surgical debridement or removal of the infection with a prolonged treatment course of antibiotics.

Funding information
The authors received no specific grant from any funding agency.
Author contributions T.P.C.K. is the main author of this article. T.P.C.K. and G.D.L. were responsible for clinical treatment of the patient. T.M. is the clinical scientist who isolated the organism and assisted with the biochemical and susceptibility testing. L.E.X.L. is the senior medical scientist who conducted whole genome sequence and assisted in writing the genomic analysis.