Methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA, respectively) can cause non-tuberculosis infectious spondylitis. In 43 cases of bacterial infectious spondylitis, Mycobacterium tuberculosis and S. aureus were the two major causative pathogens. MRSA caused more anterior operations and thoracic infections, while MSSA caused more posterior infections and lumbar infections. Differences between six S. aureus isolates from infectious spondylitis were characterized. MLST and staphylococcal cassette chromosome mec (SCC mec) analysis identified MSSA ST959 and ST30 isolates, MRSA ST239/SCC mec IIIA isolates 2 and 3, ST59/SCC mec IIIA-like isolate 6, and ST30/SCCmec IV isolate 5. While all of the isolates were resistant to penicillin and ampicillin, the MRSA isolates were more resistant than the MSSA isolates. Carbapenem-resistant MRSA ST239/SCC mec IIIA and ST59/SCC mec IIIA-like isolates of the agr1 type were also resistant to clindamycin and erythromycin. Leukocidin genes ( pvl or lukED ) and hemolysin genes ( hla , hld and hlg ) were present in all of the isolates. All six isolates caused more necrosis than apoptosis in the human alveolar basal epithelial cell line A549; however, ST59/SCC mec IIIA-like isolate 6, ST30/ SCC mec IV isolate 5 with pvl genes, and MSSA ST30 isolates with tst caused greater than 40% cell death after the 4-h incubation. Regardless of the MRSA isolate and its SCC mec type or the MSSA isolate, the infectious spondylitis-associated S. aureus isolates differed genetically, and the pvl and tst genes may be important genes for cell necrosis.
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 [
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 [
In MRSA, CA-MRSA isolates carry the staphylococcal cassette chromosome mec (SCCmec) IV element, whereas HA-MRSA isolates consist of SCCmec II and III [
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.
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 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) [
SCCmec types I-IV were identified by multiplex PCR amplification [
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.
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 (
Bacterial species | Number (%) | OP | Sex | Age | location | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | P | A + P | F | M | Range | Mean | L | T | L + S | L + T | |||||
M. tuberculosis | 13 (30.2) | 10 | 1 | 2 | 4 | 9 | 34 - 83 | 66.6 | 7 | 4 | 1 | 1 | |||
S. aureus | 18 (41.9) | 14 | 4 | 7 | 11 | 29 - 81 | 58.3 | 7 | 9 | 1 | 1 | ||||
MRSA | 10 (23.3) | 9 | 1 | 4 | 6 | 43 - 80 | 59.5 | 3 | 7 | ||||||
MSSA | 8 (18.6) | 5 | 3 | 3 | 5 | 29 - 81 | 56.7 | 4 | 2 | 1 | 1 | ||||
Others | 12 (27.9) | 7 | 5 | 6 | 6 | 33 - 83 | 61.8 | 11 | 1 | ||||||
Total | 43 (100) | 31 | 10 | 2 | 17 | 26 | 29 - 83 | 61.7 | 25 | 14 | 2 | 2 | |||
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.
All six S. aureus isolates were resistant to PEN and AMP (
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) (
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 (
Isolate | SCCmec type | C (II, III) | D (I, II, IV) | E (III) | F (III) | H (IIIA) | Susceptibility testinga | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P | AMP | OX | CF | TE | OT | CC | E | SXT | ETP | IMP | MEM | |||||||
1 | + | R | R | S | S | R | R | I | I | S | S (0.125) | S (0.016) | S (0.094) | |||||
2 | IIIA | + | + | + | + | R | R | R | R | R | R | R | R | R | R (>32) | R (>32) | I (12) | |
3 | IIIA | + | + | + | R | R | R | R | R | R | R | R | R | R (>32) | R (>32) | I (12) | ||
4 | R | R | S | S | R | R | S | S | S | S (0.125) | S (0.023) | S (0.094) | ||||||
5 | IV | + | + | R | R | R | I | S | S | I | I | S | I (4) | S (0.064) | S (2) | |||
6 | IIIA-like | + | R | R | R | S | S | S | R | R | S | R (>32) | S (2) | S (6) |
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).
Isolate | Plasmid (kb) | Pulsotype | ST type | Haemolysis | Leukocidin genes | Hemolysin genesa | tst | agr type | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
pvl | lukED | hla | hlb | hld | hlg | hlg-2 | agr 1 | agr 2 | agr 3 | agr 4 | |||||||
1 | 50 | III | 959 | α | + | + | + | + | + | ||||||||
2 | <6.6 | II | 239 | γ | + | + | + | + | + | ||||||||
3 | <6.6 | IIA | 239 | γ | + | + | + | + | + | ||||||||
4 | 50 | IIIA | 15 | γ | + | + | + | + | + | ||||||||
5 | None | I | 30 | α | + | + | + | + | + | + | |||||||
6 | None | IV | 59 | β | + | + | + | + | + | + | |||||||
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
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 (
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 [
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
Strain | Apoptosis (%) | Necrosis | Total cell death | Ratio of necrosis over total apoptosis | ||
---|---|---|---|---|---|---|
Early | Late | Total | ||||
E. coli pir116 | 1.59 (32.3%) | 3.33 (67.7%) | 4.92 | 2.75 | 7.67 | 0.53 |
S. aureus ATCC 25923 | 2.46 (30.9%) | 5.51 (69.1%) | 7.97 | 2.69 | 10.66 | 0.34 |
No. 1 | 1.28 (38.0%) | 2.09 (62.0%) | 3.37 | 6.39 | 9.76 | 1.90 |
No. 2 | 0.66 (22.0%) | 2.33 (78.0%) | 2.99 | 3.80 | 6.79 | 1.27 |
No. 3 | 1.13 (26.3%) | 3.17 (73.7%) | 4.30 | 6.76 | 11.06 | 1.57 |
No. 4 | 1.38 (30.0%) | 2.88 (70.0%) | 4.26 | 14.65 | 18.91 | 3.44 |
No. 5 | 1.08 (25.9%) | 3.08 (74.1%) | 4.16 | 23.80 | 27.96 | 5.72 |
No. 6 | 1.40 (24.0%) | 4.44 (76.0%) | 5.84 | 16.55 | 22.39 | 2.84 |
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 [
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)] [
In general, almost all of the S. aureus isolates lacked the toxin genes tst, eta and etb (
In the present study, all six clinical isolates caused more necrosis than apoptosis (
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.).
Tsung-Jen Huang,Chi-Han Lee,Meng-Huang Wu,Yen-Yao Li,Tsung Han Yang,Chin-Chang Cheng,Ching-Yu Lee,Chih-Cheng Lu,Chishih Chu, (2016) Infectious Spondylitis-Associated Staphylococcus aureus with Virulence Gene pvl or tst Causes More Necrosis than Apoptosis in Human Alveolar Basal Epithelial Cell Line A549. Advances in Microbiology,06,479-488. doi: 10.4236/aim.2016.67047