Received 4 May 2016; accepted 12 June 2016; published 15 June 2016
Infectious spondylitis is difficult to diagnosis due to its latent symptoms and is caused by direct iatrogenic inoculation of methicillin-resistant Staphylococcus aureus (MRSA)-related thoracic spondylitis after cervical spine surgery  or distant infections of the genitourinary tract, skin and soft tissue, intravascular devices, gastrointestinal tract, respiratory tract, and oral cavity  . Pyogenic vertebral osteomyelitis has an overall mortality rate of up to 11%  . Greater than 60% of infectious spondylitis cases occur in males and are caused by bacteria  . The two major types of bacterial infectious spondylitis are tuberculosis, which is caused by Mycobacterium tuberculosis and is the most common cause of spinal infection (with a prevalence ranging from 9% - 46%), and non-tuberculosis, of which S. aureus is the predominant pathogen worldwide and accounts for 20% - 84% of cases  -  .
In pyogenic spondylodiscitis, the main underlying diseases in an aging group have been shown to be diabetes, malignant tumors and pyelonephritis and the pathogens have included Enterobacteriaceae (7% - 33%), such as Escherichia coli in Japan  , coagulase-negative staphylococci (CNS) (5% - 16%), such as Staphylococcus epidermis, and Streptococcus spp., such as viridian as well as group C Streptococcus and S. agalactiae  . Additionally, the pathogens differ between community-acquired (CA) and hospital-acquired (HA) infections. Gram-positive bacteria, including S. aureus, Streptococcus intermedius, CNS, and S. agalactiae, are responsible for CA infections (10/12, 83.3%), and MRSA and gram-negative bacteria are responsible for HA infections  .
In MRSA, CA-MRSA isolates carry the staphylococcal cassette chromosome mec (SCCmec) IV element, whereas HA-MRSA isolates consist of SCCmec II and III  . Furthermore, the major virulence factors of S. aureus include Panton-Valentine leukocidin (PVL), γ-hemolysin (Hlg), toxic shock syndrome toxin 1 (TSST-1), and exfoliatin A (ETA) and B (ETB). In Taiwan, multi-locus sequence typing (MLST) analysis of a nasal carriage and community-onset infection determined ST188 of the clonal complex 1 (CC1) as the predominant virulent clone of the MSSA isolate and ST59 as the common ST type for MSSA and MRSA  . In addition, major ST types for necrotizing fasciitis-associated CA and HA S. aureus are ST59 and ST239  . In this study, microbial associated infectious spondylitis and the characteristics and pathogenesis of six S. aureus isolates were investigated.
2. Materials and Methods
2.1 Study Design
This experiment was approved by the institutional review board (IRB) of the Chang Gung Memorial Hospital (CGMH, IRB 98-0675B), and informed consent was obtained from all of the patients who were hospitalized at the 1000-bed Chiayi CGMH in southern Taiwan from 2010 to 2012. In total, 43 cases of bacterial infectious spondylitis were enrolled, and the bacterial species were identified at the Department of Anatomic Pathology and Laboratory Medicine, Chang Gung Memorial Hospital, Chiayi Branch. Among these, six patients with infectious spondylitis infected by S. aureus were retrospectively analyzed for factors including age, gender, infection site; moreover, comorbid underlying chronic conditions, such as diabetes mellitus, hypertension, chronic liver disease, chronic renal insufficiency, chronic obstructive pulmonary disease, and malignancy, were investigated. Additionally, infectious pathogens, empiric antibiotics, the number of operative debridements and reconstructions, the duration of hospitalization, and the in-hospital mortality rate were evaluated.
2.2. Identification and Genetic Analysis of Staphylococcus aureus Isolates
Six infectious spondylitis-associated S. aureus isolates were identified in the hospital laboratory and the university. The bacteria were first analyzed by coagulase testing and Gram staining. Furthermore, S. aureus was identified by PCR amplification of the S. aureus-specific clfA, 16S rRNA, and nuc genes, as previously described   . The genotype of each isolate was determined by SmaI-digested PFGE analysis, according to a previously reported method  . Briefly, whole-cell embedded agarose plugs were digested with the restriction endonuclease, SmaI (New England Biolabs, Ipswich, MA, USA). The DNA fragments were resolved by a CHEF DR-III apparatus (Bio-Rad, Hercules, CA, USA). The isolates were defined as a subgenotype for those with ≤ 3 fragment differences and as a genotype for those with more than 3 fragment differences. The MLST types for each isolate were determined according to the method described by Enright et al.  and by analysis of MLST databases (http://saureus.mlst.net).
2.3. Antimicrobial Susceptibility Testing
The susceptibility of the six S. aureus isolates to the following antimicrobials was examined: AMP (10 μg), CEF (30 μg), CIP (5 μg), CLI (2 μg), ertapenem (10 μg), ERY (15 μg), IPM (10 μg), MEM (10 μg), OXA (1 μg), oxytetracycline (30 μg), PEN (10 units), TET (30 μg), and SXT (1.25 μg for trimethoprim and 23.75 μg for sulfamethoxazole) (Becton Dickinson, Spark, MD, USA). Susceptibility analysis was performed using the disc diffusion method and the guidelines of Clinical and Laboratory Standards Institute (CLSI)  . The S. aureus isolates BCRC10781 and BCRC15211 were used as OX-susceptible and resistant reference strains, respectively.
2.4. PCR Identification of SCCmec Types and Genes for the Virulence Factors PVL, Hlg, TSST-1, ETA, and ETB
SCCmec types I-IV were identified by multiplex PCR amplification   . If not groupable into types I-IV, isolates were grouped into SCCmec type V or VT (or VII) by the PCR detection of ccrC (ccr5) homologues  -  . Genes for virulence factors PVL, Hlg, TSST-1, ETA, and ETB were identified by simplex and multiplex PCR amplifications using primers described elsewhere   . Accessory gene regulator (agr) typing was also performed, as described previously  .
2.5. Cytotoxicity of Clinical S. aureus Isolates against Cell Line A549
Human alveolar basal epithelial cell line A549 was routinely cultured in RPMI-1640 medium with 10% FBS. After the transfer of 5 × 105 A549 cells into each well of a 24-well plate (Becton, Dickinson and Company), the isolate (1 × 107 cfu) was added to reach MOI = 20. The mixtures were incubated for 1 or 4 h at 37˚C and then washed twice with PBS. The cells were treated with 0.25% trypsin/0.53 mM EDTA for 5 min until cell detachment, and then 800 µl of RPMI-1640 medium with 10% FBS was added. The cells were then pelleted at 3000 rpm for 10 min. After washing with PBS, the cells were stained with propidium iodide (PI) and annexin V using an Annexin V-FITC Apoptosis Detection Kit (Cat. No. AVK250, Strong Biotech Corporation, Taiwan) for 10 - 15 min in the dark. The fluorescence of PI and annexin V in the cells was measured by flow cytometry, and the data were analyzed using WinMDI software.
3.1. Clinical Manifestation and Bacterial Species
S. aureus-associated infectious spondylitis was only observed in male patients with an infection in the lumbar spine (five patients) and thoracic spine (one patient) With the exception of one patient without any underlying disease, diabetes mellitus and renal disease were found in four of the patients, followed by hepatitis C infection and cellulitis in three patients, and gastric ulcer, septic shock, and arterial disease in two patients. S. aureus infection was not correlated with the duration of hospitalization, previous amputation or mortality. With a change in the antibiotic use, patient 3, who had eight underlying diseases, died. Patients 2 and 5, who had MRSA infections, received two and four spine debridements and reconstructions, respectively.
Among 43 bacterial infectious spondylitis, M. tuberculosis and S. aureus accounted for 30.2% (13 cases) and 41.9% (19 cases), respectively (Table 1). The remaining species included coagulase-negative Staphylococcus,
Table 1. Retrospective analysis of 43 cases of bacterial infectious spondylitis.
A: anterior; P: posterior; L: lumbar; T: thoracic; L + S: lumbar and S segment.
E. corrodens, E. coli, K. pneumonia, Micrococcus spp., Peptostreptococcus spp., Prevotella spp., Proteus mirabilis and S. enterica serogroup D. Anterior operations (72.1%) were more prevalent than posterior operations (23.3%). M. tuberculosis and MRSA caused more anterior operations, while MSSA caused more posterior operations. Infection sites differed among species that mainly infected a single site in the lumbar (58.1%) or thoracic (32.6%) spine. More lumbar infections were observed for other species (91.7%, 11/12), followed by MSSA (75%), M. tuberculosis (69.2%), and MRSA (30%). Infectious spondylitis occurred more in males than in females (1.5:1), with a ratio of 2.25 for M. tuberculosis, 1.5 for MRSA (1.5:1) and 1.67 for MSSA, but was equally distributed for other species.
3.2. Antimicrobial Resistance and SCCmec Type
All six S. aureus isolates were resistant to PEN and AMP (Table 2). Among the 5 MRSA isolates, SCCmec type IIIA isolates 2 and 3 were resistant to all 11 antimicrobials examined, while the MRSA SCCmec type IIIA-like isolate 6 was only resistant to 7 of the tested antimicrobials and lacked PCR products for C and F of the normal SCCmec IIIA type. Additionally, SCCmec type IV isolate 5 was less resistant than the other three isolates. While the MRSA SCCmec type IIIA and IIIA-like isolates were resistant to at least one of the two carbapenems (ertapenem and IMP), the SCCmec type IV isolate appeared to have a reduced susceptibility to ETP but was susceptible to two other carbapenems (IMP and MEM).
3.3. Genomic and Genetic Variations
MLST analysis revealed that MSSA isolates 1 and 4 belonged to ST959 and ST15, respectively, and four MRSA isolates belonged to ST239 (isolates 2 and 3), ST30 (isolate 5) and ST59 (isolate 6) (Table 3). In addition to being identical in ST239 and antimicrobial patterns, pulsotype analysis demonstrated that isolates 2 and 3 differed with respect to pulsotypes (Figure 1(a)). Plasmid analysis revealed that isolates 1 to 4 carried one plasmid of 50-kb or a less than 6.6-kb plasmid, and isolates 5 and 6 lacked a plasmid (Figure 1(b)). The agr type analysis indicated that all MRSA SCCmec type IIIA and IIIA-like isolates belonged to agr 1, and the other isolates belonged to a separate agr type.
3.4. Virulence Gene Analysis
Among the virulence genes examined, all of the isolates carried leukocidin genes; lukED was identified in isolates 1 to 4, and pvl was observed in isolates 5 and 6 (Table 3). The hemolysin genes hla, hld and hlg encoding α, δ, and γ-hemolysin, respectively, were identified in all of the isolates, while β-hemolysin was only identified in isolate 6 (Table 3). Within the two hlg types, five isolates carried hlg-2. The exfoliative toxin genes eta and etb were not found, while the toxic shock syndrome toxin gene, tst, was only observed in MRSA isolate 5.
Table 2. SCCmec type and antimicrobial susceptibility of S. aureus isolates.
aP: penicillin, AMP: ampicillin, OX: oxacillin, CF: cephalothin, TE: tetracyclin, OT: oxytetracycline, CC: clindamycin E: erythromycin, SXT: trimethoprim/ sulfamethoxazole, ETP: ertapenem, IMP: imipenem and MEM: meropenem. The susceptibility to ETP, IMP, and MEM was determined by Disc diffusion and minimum inhibitory concentration (MIC) methods; the number in the parenthesis is the MIC value (μg/mL).
Table 3. MLST and toxin gene analysis of Staphylococcus aureus isolates.
ahla, hlb, hld, and hlg (or hlg-2): hemolysin genes α, β, δ and γ, respectively; tst: toxic shock syndrome toxin 1; agr: accessory gene regulator. eta and etb was not found in any isolate.
M 1 2 3 4 5-1 5-2 6 M M1 1 2 3 4 5-1 5-2 6 M2
Figure 1. Genetic analysis of six spondylitis-associated S. aureus isolates. (a) Pulsed-field gel electrophoresis (PFGE) of smaI- digested macro-fragments of the six isolates 1 - 6. M: λ chromosomal DNA marker. (b) Plasmid analysis of six isolates 1 - 6. M1: S. choleraesuis OU7526, which carries the 50-kb and 90-kb plasmids. M2: S. choleraesuis OU7085, which carries the 6-kb and 50-kb plasmids.
3.5. Clinical S. aureus Associated the Cell Death Types of Cell Line A549
Infectious spondylitis can result from distant infections, such as respiratory tract. Therefore, we used the human alveolar basal epithelial cell line A549 to investigate the cytotoxicity of S. aureus to epithelial cells. Although six S. aureus isolates resulted in different cell death rates of cell line A549 by necrosis, early and late apoptosis, all clinical isolates caused cell death, with over a 40% cell death rate for isolates 4, 5, and 6 at the 4-h incubation point, whereas the rate of cell death did not differ among the bacteria at the 1-h incubation point (Figure 2). While isolates 2 and 3 resulted in the lowest cell death rates (similar to that caused by E. coli pir116), the remaining four isolates showed increased necrosis compared with the control S. aureus ATCC25923. Analysis of the cell death types between 1 hour and 4 hours demonstrated that isolates 4 - 6 caused more cell death than did other isolates, and all six clinical isolates increased necrosis more than late apoptosis, with a ratio larger than 1.27 compared with E. coli pir116 (0.53) and S. aureus ATCC 25923 (0.34) (Table 4).
The prevalence of spinal infection varies and is dependent on the bacterial species, patients and investigators. Spondylodiscitis is observed at equal rates between CA and HA infections and is frequently associated with lumbosacral infection (72.8%), followed by thoracic and cervical tract infections  . The major pathogens are S. aureus (28%, 43.1%; 8 MRSA cases), coagulase-negative staphylococci (CNS) (8%, 12.3%), and Pseudomonas aeruginosa (8%, 12.3%). Another study of spinal infection cases has shown them to be caused by S. aureus (43.1% MRSA cases), gram-negative cocci, Mycobacterium tuberculosis, coagulase-negative Staphylococcus (12.3%), and P. aeruginosa (12.3%)  . In bacteremic vertebral osteomyelitis, S. aureus (82%) is the predominant pathogen for CA infections  . In the present study, S. aureus and M. tuberculosis were the major pathogens to cause infectious spondylitis and caused more infections in males; furthermore, M. tuberculosis and MSSA preferred to infect lumbar sites, whereas MRSA caused more thoracic infections (Table 1). Apparently, MRSA and MSSA differ in causing spinal infections.
In a study of patients with mycotic aneurysm and/or infectious spondylitis, Salmonella (18%, 34,6%) and Klebsiella pneumoniae (6%, 11.6%) of gram-negative bacteria (28%, 53.8%) and S. aureus (6%, 11.6%) and Viridans streptococcus (5%, 9.6%) of gram-positive bacteria were the major pathogens in 52 cases of mycotic
Figure 2. The percentage of A459 cell death types caused by S. aureus isolates at two periods.
Table 4. The ratio of A459 cell death types caused by S. aureus isolates between two periods.
aneurysm without infective spondylitis, in contrast with four Salmonella infections, one Streptococcus pyogenes infection and one S. aureus infection in six cases of spontaneous infectious spondylitis and mycotic aneurysm  . S. aureus infection significantly increased infectious complications, such as psoas, paravertebral abscesses and epidural abscesses in patients with vertebral osteomyelitis  . Additionally, MRSA caused more frequent persistent bacteremia and relapse compared with MSSA in hematogenous vertebral osteomyelitis  . Our MSSA isolates were ST959 and ST15, which differed from the major virulent clone ST188 of CC1 of MSSA  . In MRSA, the ST239 and ST59/SCCmec IIIA isolates and ST30/SCCmec IV isolates are responsible for infectious spondylitis (Table 2 and Table 3) and necrotizing fasciitis-associated S. aureus infection  .
Isolation of markers and identification methods for infectious spondylitis may provide useful information for treatment. Based on an analysis of percutaneous endoscopic discectomy and drainage (PEDD) and computed tomography (CT)-guided biopsies, PEDD identified more causal pathogens than CT-guided biopsy [90% (18/20) vs. 47% (15/32)]  . However, PEDD biopsy and CT-guided biopsies revealed an equal prevalence [50% (9/18) vs. 44.4% (8/18)] to identify S. aureus infections. Furthermore, cytokines are also possible markers for diagnosis. For example, IL-17 plays an important role in the inflammatory process in ankylosing spondylitis  . To predict the clinical outcome of patients with bacterial pyogenic vertebral osteomyelitis, an erythrocyte sedimentation rate (ESR) over 55 mm/h and a C-reactive protein (CRP) value of 2.75 mg/dL at the fourth week are useful markers  . As an important regulatory gene, agr (and its protein) regulates the expression of cell surface and extracellular virulence factors   ; most human MRSA isolates are of the agr 1 type  . In this study, agr1 was found in MRSA SCCmec IIIA MDR isolates 2, 3, and 6 with higher resistance to carbapenem, CC and ERY than other agr types and SCCmec IV MRSA (Table 2), demonstrating that MRSA SCCmec IIIA isolates may be more difficult to treat. However, with suitable antibiotic treatment, patients can completely recover from pyogenic thoracic spondylodiscitis with an epidural abscess caused by S. aureus infection  .
In general, almost all of the S. aureus isolates lacked the toxin genes tst, eta and etb (Table 1 and Table 3). However, two important virulence genes that were identified in all of the isolates were leukocidin genes, which encode bi-component toxins including PVL (LukS-PV + LukF-PV), LukM (LukM + LukF’-PV) and LukED (LukE + LukD)   . Here, we determined that the ST959, ST239, and ST15 isolates carried genes pvl and a plasmid, whereas the ST30 and ST59 isolates carried genes lukED and hlg-2 or hlg and lacked a plasmid and the hemolysin gene (Table 3). Additionally, PVL, staphylococcal protein A and coagulase inhibited proliferation and induced apoptosis of osteoblasts  . A recent finding has demonstrated that α-toxin (Hla) is an important virulence factor that causes membrane damage and induces apoptosis of human peripheral blood mononuclear cells (PBMCs) during USA300 infection  .
In the present study, all six clinical isolates caused more necrosis than apoptosis (Table 4). Regardless of MRSA or MSSA, SCCmec type, and hemolysin genes, isolates 4, 5, and 6 caused more cell death of the human alveolar basal epithelial cell line A549, with a ratio of necrosis over late apoptosis equaling more than 2.84. Isolates 5 and 6 were SCCmec IV ST30 and SCCmec IIIA-like MRSAs that both carry the PVL gene, not LukED, whereas isolate 4 was MSSA and the only strain to carry tst. Therefore, the hemolysin genes, PVL, and tst may be important virulence factors for S. aureus to cause spondylitis.
In conclusion, M. tuberculosis and S. aureus were the major pathogens to cause infectious spondylitis. MRSA and MSSA differed with respect to the infection sites. All six S. aureus strains caused more necrosis than apoptosis. Regardless of MRSA and MSSA, the strains with pvl or tst caused more cell necrosis.
The authors thank the National Science Council, Executive Yuan, Taiwan, for financially supporting this research under contracts NSC 100-2320-B-415-002 (C.C.), NSC 98-2314-B-182-011-MY3 and 98-2314-B-182A- 096-MY3 (both to T.J.).