Leptotrichia species in human infections II

ABSTRACT Leptotrichia species are non-motile facultative anaerobic/anaerobic bacteria that are found mostly in the oral cavity and some other parts of the human body, in animals, and even in ocean sediments. Valid species include L. buccalis, L. goodfellowii, L. hofstadii, L. honkongensis, L. shahii, L. trevisanii, and L. wadei. Some species require serum or blood for growth. All species ferment carbohydrates and produce lactic acid that may be involved with tooth decay. Acting as opportunistic pathogens, they are involved in a variety of diseases, and have been isolated from immunocompromised but also immunocompetent individuals. Mucositis, oral lesions, wounds, and abscesses may predispose to Leptotrichia septicemia. Because identification of Leptotrichia species by phenotypic features occasionally lead to misidentification, genetic techniques such as 16S rRNA gene sequencing is recommended. Early diagnosis and treatment of leptotrichia infections is important for positive outcomes. Over the last years, Leptotrichia species have been associated with several changes in taxonomy and new associations with clinical diseases. Such changes are reported in this updated review.


Introduction
Leptotrichia is one of four genera within the family Leptotrichiaceae.
Some species have been recovered from the human oral cavity, while others such as L. buccalis and L. goodfellowii have been recovered from dog bites [20] and guinea-pig wounds [6,21]. Based on 16S rDNAsequences comparisons Leptotrichia species were isolated from the hindgut of termites, fish, and even ocean sediments (Table 2) [3]. Most mammals may have their own versions of human oral species, which are typically host-species specific.

Genomics
The whole genomes of 12 Leptotrichia species have been completely sequenced [99,100]. A short description of these species and their genomic features are given in Table 1. In addition, a large variety of 16S rRNA gene Leptotrichia nucleotide sequences exists in various databases (e.g. in HOMD; www.homd.org), NCBI GenBank, RDP, DNA data Bank of Japan (DDBJ), and other private databases. For instance, a survey from the NCBI GenBank showed that >4,800 Leptotrichia nucleotide sequences were registered and deposited as of 7 August 2017. The sequences came from material collected from humans, animals, fish, and ocean sediment. A representative phylogenetic tree based on 4,800 Leptotrichia sequences showing the diversity of the species aligned by ClustalW is given in Figure 1. The phylogenetic tree was generated by neighbor joining based on 500 bootstrap replicates and reconstructed with MEGA7 software (www. megasoftware.net).   [79] (Continued )

Conserved signature inserts
Genome sequencing has provided insight into rich resources of molecular markers or signatures that are specific for different groups of bacteria. These novel molecular markers can be used to demarcate diverse bacterial taxa. An example is conserved signature inserts (CSIs) or deletions (i.e. indels) in protein sequences [100]. Members of the family Leptotrichiaceae are easily distinguished based on concatenated sequences for conserved proteins. Comparative analysis of Fusobacteria identified CSIs in proteins involved in a broad range of functions specific for the phylum. Some of these CSIs important proteins are uniquely present in the protein homologs of all sequenced members of Fusobacteria and thereby provide potential molecular markers for this phylum, which includes the family Leptotrichiacaeae. Further, it has been suggested that these specific CSIs provide evidence that could be used as novel tools for identifying and distinguishing members of the families Fusobacteriaceae and Leptotrichiaceae and other bacteria [100]. The gene sequences for many of the proteins containing these CSIs are highly conserved and based upon the conserved regions of the genes/ proteins, for which PCR primers can be designed.

Clinical importance of Leptotrichia species
Eribe and Olsen [2,3] reported previously that the clinical importance of Leptotrichia species remains unclear due to difficulties in isolation and identification of the organisms [2,3,70]. Recently, with modern molecular techniques and more awareness, more light has been shed on Leptotrichia species and their involvement in a variety of diseases. Leptotrichia species commonly colonize the mucous membrane of humans and animals, and are significant constituents of the microbiota of the human oral cavity, playing an important role in many diseases [2,3,100]. Table 2, a continuation of previous Table 1 [2], depicts 176 cases of Leptotrichia species presented in the current review. It shows where Leptotrichia species were isolated, the various sources they came from, which Leptotrichia species were detected, the polymicrobial species they are associated with, as well as their frequencies. As can be seen, Leptotrichia species are commonly present in the human and animal gastrointestinal tract, in the periurethral region, and in the genitalia of women [1][2][3]21,54,97].
In a previous review [3], it was concluded that Leptotrichia species were isolated and recovered from various sources, including patients who had gingivitis, necrotizing ulcerative gingivitis, adult/juvenile periodontitis, 'refractory periodontitis', Vincent's angina, noma, acute appendicitis, bacterial vaginosis, aortic aneurysms, cellulitis, phagedenic chancroid, saplpingitis, neutropenia, human immunodeficiency virus (HIV), leukemia, endocarditis, and human and animal infections [2,97]. It was suggested that Leptotrichia species are opportunistic pathogens. Current documentation and a review of the literature support this view.

Brief additional clinical information on
Leptotrichia species

L. buccalis
Recently, L. buccalis has been isolated from irreversible pulpitis, pulp necrosis, apical periodontitis [70], and dental plaques of both humans and guinea pigs with alveolar bone loss (Table 2) [21,56,71,90]. It has also been recovered from root canals of patients with or without other oral diseases, tissue fluids and subgingival plaque samples, and exudate with cellulitis after a dog bite (Table 2) [8,52,[72][73][74]77,90]. Furthermore, it has been recovered from the blood and amniotic fluid of a female patient and from the amniotic fluid of an afebrile pregnant woman with acute chorioamnionitis [4,78] (Table 2). It has also been detected in saliva, on the mucosal surface of patients with removable partial dentures, in periimplant crevicular fluids [34,76,79], and in biofilms ( Table 2) [75]. In addition, L. buccalis was isolated from the blood of an elderly woman who suffered from moderate normocytic anemia, acute myelogenous leukemia, and mucositis (Table 2) [15,87].
L. goodfellowii L. goodfellowii has been isolated from oral swabs of guinea pigs [21] and the gastric fluid of patients who suffered spontaneous stillborn child expulsion [85]. It has also been isolated from the blood of an amniotic fluid patient with a wound and respiratory difficulties [4], from a wound exudate of a healthy person with cellulitis after a dog bite [74], from saliva, plaque, and the mucosal surface of caries-active patients and diabetic smokers [56,90], and from the blood of patients with heart failure, diabetes, bladder cancer, pulmonary edema, and bronchopneumonia [11]. L. goodfellowii has been recovered from an immunocompetent endocarditis patient with bioprosthetic pulmonic valve and an aortic valve homograft suffering from fever and chronic night sweats (diaphoretic) ( Table 2) [12]. carcinoma HPV, and oral cavity squamous-cell carcinoma HPV [19] all contained Leptotrichia species. They were also isolated from the bile aspirate of fish with cholelithiasis (gallstone diseases) and Opisthorchis felineus (fish-borne liver fluke infections), in pancreatitis and hepatitis C [61], and in saliva from a Behçet's disease patient [64]. Wu et al. [57] reported recovery of Leptotrichia species, together with Veillonella parvula and Peptostreptococcus species in low amounts in cigarette smokers' mouthwash (Table 2) [57,90,91]. Also, human skin emanation samples and oropharyngeal samples of mite-food-sensitized children with rhinitis and asthma were found to contain Leptotrichia species [31,92].

Leptotrichia and proinflammatory mediators
It is known that the systemic release of endotoxin and proinflammatory mediators from infected host tissue can contribute directly or indirectly to the sepsis syndrome associated with Leptotrichia [2,3,7]. Once activated, the immune system is hard to switch off, and sometimes it gets out of control, causing damage to other parts of the body. This 'self-inflicted' damage acts as trigger for various disease conditions [101]. Many types of Gram-negative bacteria secrete LPS that stimulates the immune system. A study by Langfeldt et al. [48] found that Leptotrichia was able to trigger the transcription level of proinflammatory interleukin (IL)-1β, IL-6, IL-8, and IL-10 in epithelial cells [48]. This suggests that Leptotrichia may play a key role during the transition from health to disease [54]. IL-1β modulates human cell differentiation, proliferation, and apoptosis, which regulate the release of other proinflammatory cytokines such as IL-6 and IL-8 [48]. In addition, IL-6 and IL-8 have the capacity to attract granulocytes and lymphocytes, thereby inducing the host cellular immune response. In contrast, IL-10 is designated as an anti-inflammatory mediator that prohibits excessive immune response by suppressing pro-inflammatory cytokine production and the antigen-presenting capacity of monocytes, macrophages, and dendritic cells [48]. Both pathogenic and commensal bacteria interfere with early host cell signalling for survival or promote bacterial infection by decreasing pro-inflammatory responses [48]. In an in vitro multispecies biofilm model with or without major periodontal pathogens, it was documented that such biofilms can upregulate IL-8 expression in gingival epithelial cells. The presence of the 'red-complex' species (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) resulted in even greater upregulation [48]. The data strongly argued that Leptotrichia may be crucially involved in the 'fine-tune' regulation of epithelial immune response to obtain homeostasis or propagate inflammatory response [48]. Jang et al. [102] reported that L. wadei, Fusobacterium nucleatum, and Campylobacter gracilis when co-cultured with human gingival fibroblasts highly upregulated the expression of antimicrobial chemokine peptides and the proinflammatory mediators IL-6 and IL-8, whereas the red-complex bacteria stimulated low levels or often suppressed expression of these factors [102].
New-onset patients with chronic rheumatoid arthritis harbored high levels of several pathogens, including Gemella morbillorum, Propionibacterium acnes, Streptococcus gordonii, and L. buccalis. This indicated that L. buccalis can be more specifically associated with multiple disease activity than so far realized [20,52]. Irrespective of periodontal disease status, the Leptotrichia OTU 87 (L. wadei) clone and Prevotella OTU 60 (P. intermedia) clone were the only clones observed in increased amount in patients with new-onset rheumatoid arthritis but were absent in healthy controls [20].
Thus, recent reports have proven the pathogenicity of Leptotrichia species. Inappropriate clinical situations can affect the protective function of the indigenous bacterial flora, which can lead to disruption by broad-spectrum antibiotic therapy [2][3][4]12,69,103], resulting in infection. Likewise, enhanced Leptotrichia proliferation and tissue invasion can culminate in bloodstream invasion and dissemination [2,3]. This occurs frequently when the patient's immune system is comprised with Leptotrichia species such as with cases involving L. buccalis, L. trevisanii, L. wadei, and L. goodfellowii. These species have been reported to act as opportunistic pathogens responsible for bloodstream infections in immunocompromised patients [2,4,5,15,17,33,74,85,87,103].
L. buccalis has been associated with chorioamnionitis and child loss during pregnancy [78]. The authors suggested that the development of chorioamnionitis was a result of hematogenous spread arising from the oral cavity [78]. Unique to bacteremia from other Leptotrichia species, L. goodfellowii showed an association with bacteremia secondary to endocarditis [11,12]. In contrast to previously reported cases of Leptotrichia bacteremia, the patient in this report was immunocompetent and had no history of endocarditis. For the first time, a case of L. goodfellowii bacteremia was recently reported in a Korean patient [11]. It is noteworthy that in a 62-month retrospective survey of 4,857 episodes of anaerobic bacteremia, Leptotrichia species were identified as the causative pathogens in 7.3% of cases [12,22].
In an examination of the relationship of the oral microbiota with subsequent risk of pancreatic cancer in a large nested case-control study, the authors reported that the carriage of oral pathogens, P. gingivalis and Aggregatibacter actinomycetemcomitans, was associated with a higher risk of pancreatic cancer [66]. They also found that a greater abundance of the phylum Fusobacteria was associated with decreased pancreatic cancer risk as well as its genus Leptotrichia [66]. Their finding was inconsistent with a recent cross-sectional study of eight patients, which found higher abundances of Leptotrichia and Porphyromonas in the saliva of pancreatic cancer patients compared to controls and those with other diseases, including non-cancerous pancreatic disease [18]. Torre et al. [18] concluded that the Leptotrichia and Porphyromonas ratio may serve as a potential pancreatic cancer biomarker. Based on their findings, pancreatic cancer may be detected at early stages by sampling individuals' saliva and looking at the ratios of Leptotrichia and Porphyromonas.

Leptotrichia in dental caries
Among the many microbial species residing in oral biofilms (plaque) at the tooth surface [104], mutans streptococci have long been recognized as primary contributors in the etiology of dental caries [104]. The pathogenicity of organisms such as Streptococcus mutans and S. sobrinus is attributable in part to (i) the capacity of these species to produce extracellular glucan(s) from dietary sucrose that facilitate microbial adherence to the tooth surface, and (ii) the fermentation of sucrose to lactic acidthe causative agent in the demineralization of tooth enamel [104]. There is supporting evidence that the genus Leptotrichia is highly saccharolytic [1][2][3]11,[104][105][106], implying that it ferments a wide variety of mono-and disaccharides to lactic acid similar to S. mutans. This property may implicate the participation of Leptotrichia species in tooth decay [1][2][3]11].

Association between Leptotrichia and halitosis
Leptotrichia has also been associated with halitosis (oral malodor) [42,65,81,82]. Most of the species within the core microbiome of the tongue-coating biofilm are Gram-negative anaerobic bacteria that are adaptable to the tongue-coating environment ( Table 2) [81]. Malodor is foul-smelling breath from the oral cavity in humans [42]. Most malodor originates from the host's tongue plaque and is without any clear signs of disease, which is defined as physiologic oral malodor [42]. Malodorants are produced by the tongue plaque resident on the large surface area of the tongue. Some bacteria inside tongue plaque can produce amino acids and peptide by-products as well as food debris to putrefy, thus producing malodorants [42]. The unpleasant oral odor results from volatile sulfur compounds (VSCs), including hydrogen sulphide (H 2 S), methyl mercaptan (CH 3 SH), other thiols, and dimethyl sulphide ((CH 3 ) 2 S) involved and associated with halitosis [42]. Of the three major VSCs involved in oral malodor, (CH 3 ) 2 S is the main contributor to halitosis [81], whereas CH 3 SH is more pathogenic than H 2 S and is associated with periodontal disease [81]. It has been inferred that the reason for halitosis might be cooperative polymicrobial activity, which includes Leptotrichia species interactions rather than the effect of a single pathogen [81]. There is also evidence supporting that Leptotrichia species are present in increased abundances in people with oral malodor, despite a lack of H 2 S production [81,82]. Yang et al. reported that L. wadei was positively correlated with H 2 S concentrations [42] and concluded that Leptotrichia spp. and Prevotella spp. were found to be strongly associated with oral malodour [42], although direct proof of production was not provided. This bacterium was detected in relatively high abundance in all the halitosis tongue-coating samples and was inferred to be involved in halitosis [81,82], likewise L. hofstadii in some subjects [81,82]. Bacteria such as Peptostreptococcus stomatis and Prevotella shahii isolated from tongue coatings of diseased persons together with L. wadei were also suggested to be candidate halitosis pathogens [81] ( Table 2).

Leptotrichia in co-existence with other microbes
The human oral cavity has an indigenous microbiota known to include a robust community of microorganisms. Leptotrichia species are present in the salivary milieu and coexist with virus/bacteriophages in this environment, together with other microbes, for example Veillonella [76]. Their interrelationships remain elusive. Leptotrichia, Clostridium, and Citrobacter were found as the most abundant bacteria in the herbivorous fish gut [58]. Previous studies have reported that Clostridium, Citrobacter, Leptotrichia, Bacillus, and Enterobacter are important cellulose-degrading bacteria in herbivorous fish [58]. It was suggested that these bacterial species might play significant roles in their host's digestive system. Herbivorous fish harbored abundant cellulose-degrading bacteria, including Clostridium, Citrobacter, and Leptotrichia (Table 2) [58]. L. hofstadii was considered and reported as a potential biomarker for dental caries in association with Campylobacter showae and Parvimonas micra [69,84]. Leptotrichia species were found together with Fusobacterium and Campylobacter species in patients with colorectal carcinoma. This polymicrobial signature was associated with overrepresentation of numerous host genes, including the gene for encoding the proinflammatory chemokine IL-8 [40].
Leptotrichia species were reported in close association with fungi, including species of Saccharomyces, Aspergillus, Zygosaccharomyces, Pichia, Saccharomycopsis, Talaromyces, Eurotium, Fomitopsis, Trichosporon, Candida albicans, C. parapsilosis, and C. tropicalis, and other species from liquor [39], gastric fluid [16], the saliva of HIV patients [46], sputum [50], blood, and saliva [60] ( Table 2). The importance of these associations remains unknown. Leptotrichia species, together with Delftia species and Actinobacteria species, were significantly correlated with individuals attacked by malaria mosquitoes [31]. Leptotrichia species, L. wadei, and Streptococcus species were isolated together with C. albicans from dental plaque samples of patients with or without rampant caries [67,89]. The authors postulated that these pathogenic species and dysbiosis of the oral microbial community might have contributed to the pathogenesis of rampant caries in their patient. Leptotrichia spp. and Lautropia spp. were found to increase significantly in oral lichen planus (OLP) patients [88]. The argument for this was that as OLP is an immune-related disease, the elevated colonization of these bacteria might be related to the local immune dysfunction of OLP, which again suggested that OLP is associated with dysbiosis of the oral microbiome [88]. Kawanami et al. [24] suggested that in a severe pneumonia patient, isolated L. wadei and other Leptotrichia species, together with mixed oral bacteria (Enterococcus faecalis, E. casseliflavus, Veillonella parvula, V. atypica, V. dispar, Prevotella nanceiensis, Streptococcus spp. clones, Delftia sp. clone, Lactobacillus sp. clone, Syntrophococcus sp. clone, Clostridium sp. clone, and Actinomyces sp. clone), played important roles ( Table 2) [24].

Identification of Leptotrichia species
Identification of Leptotrichia species can be problematic in terms of culturing because some strains are strictly anaerobic or facultative anaerobic, while others prefer growth under the influence of CO 2 . Leptotrichia species usually stain Gram-negative, but fresh cells may be Gram-positive. Old cells may even stain both ways, leading to misclassification.
Due to the insufficiency of databases, identification of Leptotrichia species by conventional biochemical assays may be difficult and challenging, since most species are not recorded in databases. Most databases contain only one or two species known as L. buccalis or Leptotrichia species. Schrimsher et al. [9] reported cases of misidentification of L. trevisanii sepsis where all the isolates were unidentified by biochemical tests. One of the isolates was misidentified as Sphingomonas paucimobilis [9] and another as Clostridium acetobutylicum [13]. A report from Lim et al. [11] showed misidentification of L. trevisanii as Capnocytophaga spp. and L. buccalis by the Vitek 2 system [11], or as unidentified using this system. In addition, the MALDI-TOF MS system may struggle in the identification of Leptotrichia species [11]. The VITEK MS database has no known Leptotrichia species, making their identification impossible and underestimated. Lim et al. [11], however, reported that the Bruker Biotyper System (Bruker Daltonics, Billerica, MA), which contains some Leptotrichia species in their database, gave successful identification [11]. It is of general interest that more database development and strain accumulation are made to enable the precise identification of Leptotrichia species [11]. To avoid misclassification of Leptotrichia species, application of 16S rRNA gene identification is recommended because of its reliability and feasability. HOMD with its large amount of genetic data from oral bacteria is probably the most reliable database to use.

Clustered regularly interspaced short palindromic repeats in Leptotrichia
There is evidence that almost all Archaea and about half of Bacteria possess clustered regularly interspaced short palindromic repeats (CRISPRs). These are segments containing short repetitions of base sequences. The unique sequences between the repeats match the DNA of the virus preying on the bacterium. CRISPRs are part of the bacterial immune system. CRISPR-associated proteins (Cas) are adaptive immune systems for Archaea and Bacteria defending microbes against foreign genetic elements (e.g. virus) via DNA or RNA-DNA interference [107,108]. Most Cas proteins are grouped into two functional modules: (i) the adaptation module, which delivers genetic materials into CRISPR arrays generating CRISPR RNAs (crRNAs); and (ii) the effector module, which is guided by crRNA that targets and cleaves invading nucleic acids [107]. Up-to-date characterized CRISPR-Cas systems consist of Cas1 and Cas2, which are exclusively involved in spacer acquisition [107]. C2c2 is the sole effector protein that uses a crRNA guide to achieve interference, targeting RNA [107]. Targeting C2c2 to mRNA prevents gene expression [107], suggesting that CRISPR-Cas systems and C2c2 can be used for development of a new molecular RNA-targeting tools [107], including tools for Leptotrichiaceae. C2c2 from L. shahii was documented to provide interference against RNA phage [108].

Disclosure statement
No potential conflict of interest was reported by the authors.