Genomic characterization and assessment of the virulence and antibiotic resistance of the novel species Paenibacillus sp. strain VT-400, a potentially pathogenic bacterium in the oral cavity of patients with hematological malignancies

Background Paenibacillus sp. strain VT-400, a novel spore-forming bacterium, was isolated from patients with hematological malignancies. Methods Paenibacillus sp. strain VT-400 was isolated from the saliva of four children with acute lymphoblastic leukemia. The genome was annotated using RAST and the NCBI Prokaryotic Genome Annotation Pipeline to characterize features of antibiotic resistance and virulence factors. Susceptibility to antibiotics was determined by the Kirby–Bauer disc diffusion method. We used a mouse model of pneumonia to study virulence in vivo. Mice were challenged with 7.5 log10–9.5 log10 CFU, and survival was monitored over 7 days. Bacterial load was measured in the lungs and spleen of surviving mice 48 h post-infection to reveal bacterial invasion and dissemination. Results Whole-genome sequencing revealed a large number of virulence factors such as hemolysin D and CD4+ T cell-stimulating antigen. Furthermore, the strain harbors numerous antibiotic resistance genes, including small multidrug resistance proteins, which have never been previously found in the Paenibacillus genus. We then compared the presence of antibiotic resistance genes against results from antibiotic susceptibility testing. Paenibacillus sp. strain VT-400 was found to be resistant to macrolides such as erythromycin and azithromycin, as well as to chloramphenicol and trimethoprim–sulphamethoxazole. Finally, the isolate caused mortality in mice infected with ≥8.5 log10 CFU. Conclusions Based on our results and on the available literature, there is yet no strong evidence that shows Paenibacillus species as an opportunistic pathogen in immunocompromised patients. However, the presence of spore-forming bacteria with virulence and antibiotic resistance genes in such patients warrants special attention because infections caused by spore-forming bacteria are poorly treatable.


Background
Acute leukemia accounts for more than 10,000 deaths annually despite improved treatment regimens and novel cytostatic agents [1]. Pneumonia due to opportunistic Gram-positive Staphylococcus spp., Bacillus spp., and Enterococcus spp. is one of the leading causes of morbidity in these patients, as well as in patients with other forms of hematological malignancies, because of treatment-induced immunosuppression [2,3].
The oral cavity, which hosts more than 700 commensal bacterial species, is the main reservoir of microorganisms that cause aspiration pneumonia [4,5]. Thus, investigating the oral microbiome is essential to improve therapeutic strategies, especially for patients with hematological malignancies [6]. However, most commensal bacteria are not yet culturable, and molecular techniques based on cloning and sequencing the ribosomal 16S RNA have been used instead to identify species in the human microbiome [7]. Nevertheless, these techniques are prone to false negatives, such as when one bacterial species masks another, and thus underestimate bacterial diversity [8,9]. In a previous study, we described Paenibacillus sp. strain VT-400, a novel spore-forming bacterium isolated from the saliva of patients with acute lymphoblastic leukemia [10]. The strain has never been previously detected in humans.
Notably, spore-forming bacteria are poorly studied, and only a few such bacteria have been described and are associated with the human microbiota [11,12]. Spores tolerate high temperature, radiation, and noxious chemicals, harbor genes that confer antibiotic resistance, and allow bacteria to survive in unfavorable conditions [13,14]. Thus, spores contribute significantly to the persistence of infection and the spread of antimicrobial resistance [15]. Indeed, prophylactic treatments like oral rinses are poorly effective against spores, and are thus not sufficiently reduce the bacterial load in the oropharynx, or prevent aspiration pneumonia in at-risk patients, especially those with underlying pathologies such as hematological malignancies [16,17]. Therefore, identification and characterization of potentially infectious spore-forming microbial species are critical to improve the management or treatment of patients with acute leukemia.
Paenibacillus spp. was not known to cause human disease until recent reports implicated P. alvei, P. thiaminolyticus, and P. sputi in respiratory and urinary tract infection, as well as bacteremia in a patient on hemodialysis [18][19][20]. In this study, we describe Paenibacillus sp. strain VT-400, a novel bacterium isolated from the saliva of four children with hematological malignancies, and investigate its potential to cause pneumonia.

Bacterial strain
Paenibacillus sp. strain VT-400 was isolated from the saliva of four children with acute lymphoblastic leukemia who were hospitalized at First Pavlov State Medical University, St. Petersburg, Russia. Unless stated otherwise, the isolate was grown on Columbia agar with 5 % sheep blood (BioMerieux, France) and were stored at −80 °C in Columbia broth (BioMerieux) supplemented with 50 % glycerol. The strain was screened for hemolytic activity by cultivation at 37 °C for 48 h on agar plates supplemented with 5 % sheep blood. Clearing and greenish zones around colonies were considered to indicate β-and α-hemolytic activity, respectively. Primary morphological characterization was performed by light microscopy (Axiostar, Zeiss, Germany), and Gram staining was performed using a kit (Merck, Darmstadt, Germany).
To generate inoculum for infecting mice, the strain was grown at 37 °C for 48 h on Columbia agar with 5 % sheep blood. Colonies picked from the plate were then grown for 18 h at 37 °C in 5 mL Columbia broth. Cells were harvested by centrifugation at 3000×g for 15 min (Eppendorf 5415 C centrifuge; Eppendorf Geratgebau GmbH, Hamburg, Germany), and suspended in an isotonic phosphate buffer (0.15 mM, pH 7.2). The turbidity of the suspension was adjusted using a McFarland standard.

Genome annotation and phylogenetic analysis
Whole-genome sequences from isolates of Paenibacillus sp. strain VT-400 were aligned using MUSCLE, and phylogenetic trees were constructed based on the Tamura-Nei distance model in PHYML version 3.0, with 1000 bootstrap replicates [21][22][23]. The most closely related Paenibacillus genomes were included in the analysis. The genome was annotated and mined for virulence factors and antibiotic resistance genes using Rapid Annotation using Subsystems Technology (RAST) and the NCBI Prokaryotic Genome Annotation Pipeline [24,25].

Pathogenicity in a mouse infection model
Adult C57BL/6 mice weighing approximately 20 g (Rappolovo, North-West region, Russia) were housed in individual cages in a facility free of known murine pathogens, and were provided feeding ad libitum. Animals were cared for in accordance with National Research Council recommendations, and experiments were executed in accordance with the Guide for the Care and Use of Laboratory Animals [27].
Animals were randomly designated into two groups of eight, which were used to measure overall survival and bacterial load. Mice were then anesthetized with 2 % isoflurane, and orally instilled with bacterial suspension as previously described [28]. Briefly, nares were blocked, and mice aspirated 50 µL Paenibacillus sp. strain VT400 into the lungs while being held vertically for 60 s. Mice received a total dose of 7.5 log 10 , 8.5 log 10 , or 9.5 log 10 CFU/mouse. Control mice were treated with sterile 50 µL phosphate-buffered saline. Overall survival was assessed over 7 days, while bacterial load was measured in the lungs and spleen of surviving mice 48 h post infection.

Microbiological assessment of infected lung and spleen
Bacterial load in the spleen and lungs was measured 48 h post infection. Briefly, surviving animals in groups designated for this assessment were euthanized by CO 2 and cervical dislocation. Lungs and spleen were collected and homogenized in 1 mL phosphate-buffered saline. As Paenibacillus sp. strain VT-400 was found to be resistant to chloramphenicol and trimethoprim, serial tenfold dilutions of tissue homogenates were plated on Columbia agar with 5 % sheep blood, 5 μg/mL chloramphenicol, and 10 μg/mL trimethoprim (Sigma Chemical Co., St Louis, MO, USA), and cultured at 37 °C. Colonies of spore-forming bacteria were counted after 48 h, and bacterial loads are reported as mean log 10 CFU/g tissue ± SD. Morphology was characterized by light microscopy (Axiostar, Zeiss), and cells were Gram stained using a kit (Merck).

Ethical approval and consent
Ethical approval was granted by the First State I. P. Pavlov Medical University Ethics Committee (501/M2013). In accordance with ethical approval, consent to use human biological material was assumed following completion of consent forms.

Statistics
Survival was compared by Kaplan-Meier analysis logrank test. Differences in bacterial load were evaluated by one-way analysis of variance in SigmaStat version 2.03 (SPSS, Inc., San Rafael, CA). A P value <0.05 was considered significant.

Phylogenetic analysis
Paenibacillus sp. strain VT 400, which has never been detected in humans before, was isolated for the first time from the saliva of pediatric patients with acute lymphoblastic leukemia. In a previous study, whole-genome sequencing was performed on Illumina HiSeq 2500, with 125-fold average coverage [10]. Assembly generated 116 contigs spanning 6,986,122 bp, with G+C content 45.8 %.
On the basis of these analyses, the strain was identified as a novel species for which Paenibacillus sp. strain VT 400 was assigned, and its genome was deposited in GenBank under accession number LELF01000000. Phylogenetic analysis based on 16S rRNA demonstrated that Paenibacillus sp. strain VT 400 is clearly distinguished from other species, as well as from other strains of P. amylolyticus (Fig. 1).

Microbiological characteristics of Paenibacillus sp. strain VT 400
Paenibacillus sp. strain VT 400 is Gram-positive, aerobic, spore-forming, rod-shaped, and motile via peritrichous flagella [10]. Colonies growing on sheep blood agar are smooth, white pearl in color, and from 0.5 to 1 mm in diameter after 24 h at 37 °C in an aerobic atmosphere. β-hemolysis was observed around colonies growing on blood agar plates. The type strain is deposited in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (Braunschweig, Germany) under accession number DSM 100755.

Genes encoding virulence factors and in vivo pathogenicity
Analysis of the genome revealed a large number of genes encoding virulence factors that may contribute to pathogenicity (Table 1) [29]. Most are degradative enzymes and adhesins that may facilitate infection, including proteases, phospholipases, ureases, chitinases, and endopeptidases [30]. Significantly, we found chemotaxis proteins that were previously shown to contribute to bacterial virulence [31]. A couple of toxins or putative toxins were also detected, as well as superantigen CD4+ T-cellstimulating antigen, which causes severe symptoms and septic shock [32].
We used a mouse model of pneumonia to study virulence in vivo. Mice were challenged with 7.5 log 10 -9.5 log 10 CFU, and survival was monitored over 7 days (Fig. 2). All animals exhibited typical signs of acute infection within 24 h, including hypothermia, piloerection, breathing difficulty, narrowed palpebral fissures, trembling, and reduced locomotor activity. There was a direct correlation between severity of symptoms and dose. Accordingly, mortality depended on dose as well, with mortality observed within 48 h in mice exposed to 8.5 log 10 and 9.5 log 10 CFU Paenibacillus sp. strain VT 400.
Bacterial load was also measured in the lungs and spleen of surviving mice 48 h post infection (Table 2). To confirm the presence of Paenibacillus sp. strain VT 400, tissues were homogenized and plated on selective media. Spore-forming bacteria were identified by microscopy. There was approximately 2.47 log 10 more CFU/g of infected lung tissue in the high-dose group than in the low-dose group (P < 0.05). In addition, the data indicated that Paenibacillus sp. strain VT 400 spread from the lungs to the spleen, in which bacterial load was also dose-dependent. Taken together, the data suggest that mortality is due to, at least in part, progressive bacterial invasion and dissemination.
Moreover, analysis of the Paenibacillus sp. strain VT 400 genome revealed an array of proteins involved in or essential for sporulation (Table 3). Phylogenetic analysis indicated that these genes are conserved and are closely related to other members of the Bacillaceae family [33].

Analysis of drug resistance genes and antimicrobial susceptibility testing
Genome analysis also revealed that Paenibacillus sp. strain VT 400 harbors different antibiotic resistance genes (Table 4). A total of 96 genes were major facilitator superfamily (MFS) plasma membrane transporters, 18 were multidrug ATP-binding cassette (ABC) transporters [34,35]. Four genes were identified as multidrug ABC transporter permeases, eight as multidrug and toxic compound extrusion (MatE) transporters, and two as small multidrug resistance (SMR) proteins [36,37]. A multidrug drug metabolite transporter (DMT) was also detected [38]. Moreover, the Paenibacillus sp. strain VT 400 genome also contains genes that confer resistance to specific antibiotics. Finally, genes encoding resistance to tellurium, tunicamycin, and bleomycin were also present. These compounds are used to treat hematological malignancies [39,40].
The antibiotic susceptibility of Paenibacillus sp. strain VT 400 was then tested against an array of antimicrobials commonly used to treat nosocomial pneumonia [41]. As can be seen from Table 5, the strain was resistant to macrolides such as erythromycin and azithromycin, as well as to chloramphenicol and trimethoprim-sulfamethoxazole. However, it was sensitive to β-lactams, aminoglycosides, glycopeptides, tetracyclines, lincosamides, and fluoroquinolones.

Discussion
Bacteria that colonize the oral cavity are important pathogenic agents of pneumonia and other opportunistic infections, especially in immunocompromised hosts. We have now identified one such bacterium, Paenibacillus sp. strain VT 400, a novel species that was isolated from children with acute leukemia [28].
Whole-genome analysis indicated that this spore-forming bacterium harbors known virulence factors such as hemolysin, degradative enzymes, adhesins, and flagella. Moreover, CD4+ T-cell-stimulating antigen, a superantigen that causes toxic shock, is also present, along with other virulence determinants such as peptidases, ureases, lipases, and chitinases. Chemotaxis proteins were also found, suggesting that the isolate, which is motile, is capable of chemotaxis [42].
The detection of a strain such as Paenibacillus sp. strain VT 400 in patients with hematological malignancies is a critical result, especially in light of in vivo studies. In these experiments, mice intranasally challenged with at least 8.5 log 10 CFU of the isolate died from pneumonia, and were found to have infected lungs as well as spleen, indicating dissemination of the infection. Taken together, the data suggest that the strain not only presents genetic features of pathogenic bacteria, but may indeed trigger a life-threatening infection.
In addition, the genome of Paenibacillus sp. strain VT 400 features numerous multidrug efflux transporters known to confer intrinsic and acquired resistance to many antibiotics used in clinical practice [43]. These proteins catalyze uptake, efflux, diffusion, solute exchange, and other mechanisms of bacterial defense against xenobiotics [44,45]. In addition, these transporters are not drug-specific and are associated with multidrug resistance [46]. Moreover, the isolate contains two SMR efflux pumps, which are hallmarks of nosocomial infections and imply that Paenibacillus sp. strain VT 400 is most likely a circulating hospital strain, or a strain circulating among hematology patients [47]. SMR efflux pumps confer nosocomial antibiotic resistance and poor sensitivity to biocidal quaternary ammonium compounds [48,49]. Notably, SMR proteins have never been previously found in Paenibacillus.
Nevertheless, many resistance genes of Paenibacillus sp. strain VT 400 are not expressed, in accordance with the idea that many mutations do not lead to resistant phenotype [57]. Sporulation, such as in Paenibacillus sp. strain VT 400, preserves and disperses genetic material such as antibiotic resistance genes to overcome harsh environmental conditions [58,59]. These spores may be particularly hazardous to immunocompromised patients.

Conclusions
This study expands the number of poorly characterized Paenibacillus spp. that may cause pulmonary disease in humans [18]. We provide virulence and antibiotic resistance data based on draft genomes and antimicrobial susceptibility testing. We also demonstrate the ability of the strain to trigger pneumonia in vivo, and to invade spleen tissue. Our data may have important implications in the clinic, as the oral microbial flora in patients with hematological malignancies could be a reservoir of pneumoniacausing agents.   Whether Paenibacillus sp. strain VT 400 is more prevalent in individuals with acute leukemia remains to be established. However, it is clear that the isolate may have direct clinical implications for patients with therapy-induced immunosuppression. We now intend to determine the prevalence of Paenibacillus sp. strain VT 400 among different groups of patients, as well as among patients beyond hematology and bone marrow transplantation units.

Availability of supporting data
The complete genome has been deposited in GenBank under the Accession No. LELF01000000. The type strain is deposited in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen under Accession Number DSM 100755.