Streptobacillus sp . isolated from a cat with pneumonia

Case presentation: Following the necropsy of a domestic cat, bacteria displaying substantial characteristics of Streptobacillus sp. were cultured from pneumonic lung tissue. Streptobacilluslike morphological features observed included strictly microaerophilic pleomorphic Gramnegative rods with bulbar swellings that grew exclusively in the presence of serum. Significant shared biochemical properties included negative reactions for cytochrome oxidase, catalase, urease, nitrate reduction and indole production, as well as broad antimicrobial susceptibility. These characteristics are all indicative of Streptobacillus moniliformis. However, 16S rRNA gene sequencing revealed only 98 % sequence homology to type strain DSM 12112. A mass spectrometry analysis confirmed the affiliation of the domestic cat isolate described in this study with bacteria of the genus Streptobacillus, but matrix-assisted laser desorption/ionization time-offlight mass spectrometry indicated that it differed from nine reference strains of Streptobacillus moniliformis isolated from various sources and host species.


Introduction
The genus Streptobacillus (Leptotrichiaceae, Fusobacteriales) comprises the monotypic Streptobacillus moniliformis (Elliott, 2007;Gaastra et al., 2009), one of two aetiological organisms causing the bacterial zoonosis rat-bite fever (RBF).RBF is predominantly transmitted through rodent bites and scratches or through direct or indirect contact with rodent faeces and urine (Bleich & Nicklas, 2008;Hayashimoto et al., 2008;Torres et al., 2003).The zoonosis generally referred to as RBF involves two similar yet distinct syndromes caused by two different pathogens: Spirillum minus and Streptobacillus moniliformis.Spirillum minus infection [due to lack of type or reference strains, it is not listed in the Approved List of Bacterial Names at http://www.bacterio.net/;(Elliott, 2007;Gaastra et al., 2009)], also known as sodoku, is more prevalent in Asia than in Western Europe and is not covered in this study.However, Streptobacillus moniliformis is a primary pathogen in humans and may cause RBF or food-borne Haverhill fever.The acute disease symptoms in humans include fever, malaise, muscle pain, articular inflammation, septicaemia, and maculopapular, petechial or pustular rash after exposure to the bacteria through a bite or scratch by rats or through the ingestion of water or food contaminated by rat excrement (Gaastra et al., 2009).Other rodent species like gerbils, squirrels, spinifex hopping mice and guinea pigs are also susceptible to Streptobacillus moniliformis infection, and mice may straindependently develop clinical disease.Rats are usually asymptomatic (Wullenweber et al., 1990), but abscess formation caused by Streptobacillus moniliformis infection has been described (Rohde et al., 2008).Approximately 50-100 % of wild rats carry Streptobacillus moniliformis in their oro-or nasopharynx and shed the pathogen with saliva and urine (Ditchfield et al., 1961;Elliott, 2007;Washburn, 1995).Nevertheless, isolation of Streptobacillus moniliformis from rat urine and faeces is not always successful (Freundt, 1956;Wullenweber et al., 1990).Streptobacillus infection has also been reported on a few occasions from livestock (calves, a pig and domestic turkey), as well as from zoo animals (non-human primates and a koala) (Boyer et al., 1958;Gaastra et al., 2009;Glu ¨nder et al., 1982;Gourlay et al., 1982;Mohamed et al., 1969;Russell & Straube, 1979;Smallwood, 1929;Valverde et al., 2002;Yamamoto & Clark, 1966).Most reports on isolates derived from livestock lack genetic data and are focused mainly on pathogen morphology or zoonotic symptoms alone.Pathological findings in infected ruminants include pneumonia in calves and isolations from seminal vesicles of bulls, whilst domestic turkeys also display polyarthritis, synovitis, tendon sheath swellings and joint lesions.Notably, a bite by an asymptomatic pig led to RBF-like disease in a woman (the epidemiological data is summarized by Gaastra et al., 2009).Carnivorous animals regularly feeding on rats and mice, such as dogs, weasels and ferrets, are known reservoirs of Streptobacillus moniliformis and are therefore possible vectors for human infection (Gaastra et al., 2009;Gordon & Jones, 1999;Peel, 1993;Wouters et al., 2008).The role of cats as carriers and transmitters of Streptobacillus moniliformis to humans remains speculative, despite their frequent exposure to rats.The only known case study lacks culture-based or serological confirmation (Gascard et al., 1967).Detection of Streptobacillus moniliformis often seems to be attributed to colonization rather than infection, thus stressing the role of resident or latent oral flora in wild or pet rodents or in dogs and cats.Recently, a closely related Streptobacillus sequence, COT-370, was detected in a canine oral microbiome project (Dewhirst et al., 2012).Currently, only two reports of clinical infection by Streptobacillus moniliformis in dogs exist (Das, 1986;Ditchfield et al., 1961).One dog was reported as displaying acute gastroenteritis, arthritis, skin rash, pneumonia and endocarditis, symptoms similar to those of Haverhill fever (Ditchfield et al., 1961).The other case described Streptobacillus moniliformis from an abscess aspirate (Das, 1986).
We report here for the first time, to our knowledge, an infection in a cat displaying clinical symptoms of fatal pneumonia which could be attributed to a streptobacillary infection.

Case report
In January 2013, a cat that had been found dead on a dairy farm was submitted for necropsy.Post-mortem examination revealed an acute bronchopneumonia with pronounced acute oedema and congestion of the lungs and a marbled and beige-coloured myocardium with multifocal haemorrhages on the endo-and epicardium.Liver, spleen and kidneys showed acute congestion.No abnormalities such as wounds, scratches, ulcers or rashes were noted on the skin, which are common sequelae of RBF in humans.Histopathological examination of the lungs revealed an acute suppurative to fibrinous, focally necrotizing pneumonia with multifocal desquamation of type II pneumocytes and alveolar macrophages (Fig. 1).In the heart, an acutely suppurative and fibrinous endocarditis and myocarditis with acute congestion, oedema and single necrotizing foci were noticed (Fig. 2).

Diagnosis
Upon necropsy, viral and molecular examinations were carried out for the presence of feline calicivirus (cell culture), feline herpesvirus 1, Mycoplama spp.and  Chlamydia spp.using PCR, all with negative results.A lowgrade mixed concomitant flora that included Streptococcus canis, Pseudomonas sp. and a-haemolytic streptococci was isolated from liver, spleen, lung, heart, kidney and intestines.No growth of mycoplasmas could be observed in samples from lung tissue, but cultures for haemophilic bacteria showed high growth of strictly microaerophilic organisms on Columbia blood agar with 5 % sheep blood (CSBA; Oxoid) after 3 days of incubation at 37 u C in the presence of 10 % CO 2 .Colonies were tiny, drop like, shiny and slightly convex, measuring 0.120.4mm in diameter.Colonies appeared slightly a-haemolytic on CSBA (after 3 days of incubation) but developed a faint b-haemolysis after 5 days.Some colonies showed the typical 'fried-egg' appearance seen in L-forms.Microscopic morphological features were indicative of Gram-negative, pleomorphic, fusiform to filamentous, non-spore forming, non-encapsulated, non-acid-fast rods that were arranged in chains and clumps, also sometimes displaying irregular, lateral bulbar swellings.Conventional biochemical testing of isolate 131000547 from this study (all substrates Becton Dickinson) revealed broad accordance with the known pattern (Edwards & Finch, 1986;Wullenweber, 1995) (Table 1).Key reactions were negative for cytochrome oxidase, catalase, urease, nitrate reduction and indole production.In liquid medium (e.g.trypticase soy broth), with the addition of 20 % cattle serum (Oxoid), streptobacillary growth could be detected after 2-4 days as typical 'puff-ball' or 'bread crumb-like' appearance (Elliott, 2007;Gaastra et al., 2009).
Antimicrobial susceptibility testing was performed by disc diffusion on CSBA and respective discs (Oxoid) following DIN 58940 (German standard) criteria after 72 h of incubation at 37 u C in a microaerophilic atmosphere.Isolate 131000547 turned out to be inhibited in vitro by penicillin, ampicillin, oxacillin, amoxicillin-clavulanate, cephalotin, cephalexin, cefazoline, erythromycin, lincomycin, streptomycin, gentamicin, neomycin, tetracycline, enrofloxacin, florfenicol and chloramphenicol.Intermediate resistance to ciprofloxacin and resistance to polymyxin B and sulfamethoxazole-trimethoprim was also revealed.
For PCR analysis, thermolysates of freshly subcultured isolate 131000547 as well as strain NCTC 11941 (positive Table 1.Biochemical and phenotypical characteristics of Streptobacillus moniliformis as determined from the literature (Edwards and Finch, 1986;Elliott, 2007;Gaastra et al., 2009;Kimura et al., 2008;Wullenweber, 1995) control) were centrifuged (12 000 g, 10 min) and 1 ml of the supernatants was used for subsequent PCR with S5 (59-CATACTCGGAATAAGATGG-39) and AS2 (59-GCTTAGCTCCTCTTTGTAC-39) primers (Kimura et al., 2008).Species-specific PCR resulted in a distinct 269 bp band for isolate 131000547 (Fig. 3).Amplification of the 16S rRNA gene yielded a sequence length of 1425 bp using primers rRNA A (59-AGAGTT-TGATCATGGCTCAG-39) and rRNA H [59-AAGGAG-GTGATCCAGCCGCA-39 (Edwards et al., 1989)].Briefly, DNA was extracted from a bacterial pure culture (Maxwell 16 FFPE Plus LEV Purification kit; Promega).The PCR product was purified with a QIAquick PCR Purification kit (Qiagen) and sequenced by GATC Biotech (Konstanz, Germany).Sequence analysis carried out by BLAST   The comparison of MALDI-TOF MS spectra obtained by the direct smear and the extraction method for sample preparation revealed differences between the cat's isolate compared with nine reference strains of Streptobacillus moniliformis from various sources and host species (Fig. 4).Nevertheless, mass spectrometry could confirm the affiliation of isolate 131000547 to the genus Streptobacillus.

Discussion
Only two case reports involving dogs focus on susceptibility and clinical relevance of Streptobacillus moniliformis in companion animals.One of these dogs displayed Haverhill fever-like symptoms including acute gastroenteritis, arthritis, skin rash, pneumonia and endocarditis after feeding on presumably contaminated items from rubbish (Ditchfield et al., 1961).The other case report involved a scapula abscess aspirate (Das, 1986) for which growth characteristics and antibiotic susceptibility testing were not fully in accordance with those of Streptobacillus moniliformis.We present here the first evidence for a streptobacillary infection in a domestic cat that succumbed to fatal pneumonia and endocarditis.In light of the extremely high prevalence of Streptobacillus moniliformis in rats (close to 100 %; Gaastra et al., 2009) and the regular exposure of domestic cats to wild rodents, it is striking that no other incidences of streptobacillary infections in domestic felines exist.In humans, the risk of acquiring a streptobacillary infection after a rat bite or scratch is indicated as 10 % (Hagelskjaer et al., 1998), whereas the subsequent mortality rate of an untreated bite can be as high as 13 % (Wullenweber, 1995).It is possible that, much like in humans, infections are underdiagnosed in pets due to the oversight of rodent bites, occurrence of non-specific clinical symptoms, fastidious growth, non-notifiable disease and broad chemotherapeutic susceptibility.
A hypothesis presented recently is that dogs may be naturally colonized by other, as-yet-undescribed species of Streptobacillus (Dewhirst et al., 2012).The cat described in this study originated from a farm with an abundance of rats and, similarly, could have simply aspirated an uncommon strain of Streptobacillus moniliformis through the oral cavity.Comparable case reports exclusively link streptobacillosis to respiratory infection in both a guinea pig and a koala (Kirchner et al., 1992;Russell & Straube, 1979).Although no direct morphological features consistent with pulmonary streptobacillosis were detected during histopathological examination, no infectious agents for pneumonia other than Streptobacillus moniliformis could be cultivated or detected by culture, necropsy or histopathology.This suggests a major role for this pathogen as the causative agent.Unfortunately, as only lung tissue was cultivated microaerophilically, we cannot completely rule out a septicaemic cause of streptobacillary infection.Although no routine tests for feline leucosis virus and feline immunodeficiency virus were carried out in this case, no characteristic signs of viral infection such as emaciation or tumoral growth were detected.Biochemical properties of the isolate examined in this study were consistent with assigned field and reference strains of Streptobacillus moniliformis (Table 1), but phenotypic variations within this species are known to occur (Edwards and Finch, 1986;Elliott, 2007;Gaastra et al., 2009;Kimura et al., 2008;Wullenweber, 1995).Some previous studies have also noticed the occurrence of haemolysis in Streptobacillus moniliformis (Ditchfield et al., 1961;Wullenweber et al., 1992).With respect to previous antimicrobial studies, our isolate did not significantly differ from Streptobacillus moniliformis.It showed resistance only to polymyxin B and sulfamethoxazole-trimethoprim, and intermediate resistance to ciprofloxacin, suggesting a good starting point for chemotherapeutic treatment.Besides a broad susceptibility of Streptobacillus moniliformis, specific strains of this pathogen have been reported to show resistance to nalidixic acid, polymyxin B, cephalosporins, aminoglycosides, sulfamethoxazol-trimethoprim, cotrimoxazole, tobramycin, norfloxacin, levofloxacin and ciprofloxacin (Cunningham et al., 1998;Frans et al., 2001;Freunek et al., 1997;Rygg & Bruun, 1992;Wullenweber, 1995) or intermediate sensitivity to fluoroquinolones (Rygg & Bruun, 1992;Wullenweber, 1995).In addition to classic diagnostic tools such as biochemistry, morphology and molecular analysis, this is the first instance where MALDI-TOF MS was employed to identify Streptobacillus moniliformis.Whereas all field and reference strains could clearly be assigned with high accuracy, isolate 131000547 clustered closely but could not correctly be identified at the species level (Fig. 4).This can be explained by the presence of only a single strain represented by 24 spectra in the manufacturer's database, therefore not providing detailed coverage of the full spectral variance of this pathogen (M.Timke, personal communication).However, the possibility exists that the pathogen represents an undescribed species of Streptobacillus, as we found additional circumstantial evidence regarding phenotype (e.g.fermentation of cellobiose and arginine) and genotype (16S rRNA gene).Additional studies to clarify this situation are currently underway.
RBF represents a significant public health threat that might be underdiagnosed in species other than the known host animals, in particular in domestic pets.Despite frequent prey-mediated close contact to Streptobacillus moniliformis, cats appear not to be particularly prone to infection.We have shown for the first time here that domestic cats are nevertheless susceptible to streptobacillary infection and clinical disease.Future research should elucidate the role of domestic cats as a reservoir for Streptobacillus moniliformis and their immune response towards this pathogen.