Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens? A clinical investigation using PCR and culture techniques.

Abstract

BACKGROUND: Non-union continues to be one of the orthopedist's greatest challenges. Despite effective culture methods, the detection of low-grade infection in patients with non-union following tibial fracture still presents a challenge. We investigated whether "aseptic" tibial non-union can be the result of an unrecognized infection. METHODS: A total of 23 patients with non-union following tibial shaft fractures without clinical signs of infection were investigated. Intraoperative biopsy samples obtained from the non-union site were examined by means of routine culture methods and by polymerase chain reaction (PCR) for the detection of 16 S ribosomal RNA (rRNA). Control subjects included 12 patients with tibial shaft fractures. RESULTS: 23 patients (8 women and 15 men; mean age: 47.4 years) were included into this study. Preoperative C-reactive protein levels (mean: 20.8 mg/l) and WBC counts (mean: 8,359/ul) in the study group were not significantly higher than in the control group. None of the samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were found by conventional culturing methods in only 1 case of an open fracture from the control group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue specimens from 2 patients (8.7%) with non-union. The analysis of these variable speciesspecific sequences enabled the identification of specific microorganisms (1x Methylobacterium species, 1x Staphylococcus species). Both PCR-positive patients were culture-negative. CONCLUSIONS: The combination of microbiological culture and broad-range PCR seems to substantially add to the number of microbiological diagnoses obtained and may improve the clinican's ability to tailor therapy to the individual patient's needs.

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Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens?
A clinical investigation using PCR and culture techniques
Journal of Orthopaedic Surgery and Research 2012, 7:20 doi:10.1186/1749-799X-7-20
Justus Gille (justus_gille@usa.net)
Steffen Wallstabe (steffenwallstabe@hotmail.com)
Arndt-Peter Schulz (aps77@web.de)
Andreas Paech (Andreas.Paech@uk-sh.de)
Ulf Gerlach (u.j.gerlach@buk-hamburg.de)
ISSN
1749-799X
Article type
Research article
Submission date
23 December 2010
Acceptance date
20 May 2012
Publication date
20 May 2012
Article URL
http://www.josr-online.com/content/7/1/20
This peer-reviewed article was published immediately upon acceptance. It can be downloaded,
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which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Is non-union of tibial shaft fractures due to
nonculturable bacterial pathogens? A clinical
investigation using PCR and culture techniques
Justus Gille1,*
Email: justus_gille@usa.net
Steffen Wallstabe2
Email: steffenwallstabe@hotmail.com
Arndt-Peter Schulz1
Email: aps77@web.de
Andreas Paech1
Email: Andreas.Paech@uk-sh.de
Ulf Gerlach2
Email: u.j.gerlach@buk-hamburg.de
1 Department of Trauma and Reconstructive Surgery, University of Schleswig-
Holstein, Campus Luebeck, Luebeck 23538, Germany
2 Department of Trauma Surgery and Sportsmedicine, BG-Traumahospital
Hamburg, Hamburg 21033, Germany
* Corresponding author. Department of Trauma and Reconstructive Surgery,
University of Schleswig-Holstein, Campus Luebeck, Luebeck 23538, Germany
Abstract
Background
Non-union continues to be one of the orthopedist’s greatest challenges. Despite effective
culture methods, the detection of low-grade infection in patients with non-union following
tibial fracture still presents a challenge. We investigated whether “aseptic” tibial non-union
can be the result of an unrecognized infection.
Methods
A total of 23 patients with non-union following tibial shaft fractures without clinical signs of
infection were investigated. Intraoperative biopsy samples obtained from the non-union site
were examined by means of routine culture methods and by polymerase chain reaction (PCR)
for the detection of 16 S ribosomal RNA (rRNA). Control subjects included 12 patients with
tibial shaft fractures.

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Results
23 patients (8 women and 15 men; mean age: 47.4 years) were included into this study.
Preoperative C-reactive protein levels (mean: 20.8 mg/l) and WBC counts (mean: 8,359/µl)
in the study group were not significantly higher than in the control group. None of the
samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were
found by conventional culturing methods in only 1 case of an open fracture from the control
group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue
specimens from 2 patients (8.7%) with non-union. The analysis of these variable species-
specific sequences enabled the identification of specific microorganisms (1x
Methylobacterium species, 1x Staphylococcus species). Both PCR-positive patients were
culture-negative.
Conclusions
The combination of microbiological culture and broad-range PCR seems to substantially add
to the number of microbiological diagnoses obtained and may improve the clinican’s ability
to tailor therapy to the individual patient’s needs.
Keywords
Non-union, Tibial fracture, Low-grade infection, Molecular diagnosis, PCR technique
Article summary
We investigated whether “aseptic” tibial non-union is related to unrecognized infection. 23
patients (8 women and 15 men; mean age: 47.4 years) were included in the study. 16 S rRNA
was detected in 2 cases (8.7%) of non-union. PCR-positive patients were culture-negative in
both cases.
Introduction
Diaphyseal tibia fractures are the most common lower limb fractures worldwide [1]. Despite
advances in management, tibia fractures remain vulnerable to many complications, which
often require secondary surgery. Potential complications include delayed union, non-union,
malunion, compartment syndrome and infection [2]. A recent study on open and closed
diaphyseal tibia fractures treated by all modalities reported an overall revision rate of 22.4%
[3], which was often the result of non-unions.
In about 10% of cases the healing process is delayed [4]; for certain at-risk patients, it can
affect over 30% [5]. Established causes of delayed union and non-union of tibial fractures are
systemic deficits, e.g. advanced patient age [6] and diabetes mellitus [7]; prior local
impairment of the extremity, e.g. chronic impairment of the soft tissues or blood circulation
[8]; and characteristics involving the traumatic impact itself, e.g. fracture localization [9],
degree of soft tissue damage [10] and bacterial contamination [1,11].
Non-union, especially when infected, continues to be one of the greatest challenges in
orthopedic surgery. After open reduction and internal fixation of tibial shaft fractures, the rate

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of superficial infection is up to 22% and deep infections occur in up to 15% [2], the latter of
which can potentially lead to septic nonunion of the tibia. Clinical signs, laboratory
investigations of infection parameters and microbiological findings are often insufficient for
detecting infection. Distinguishing between infection and aseptic non-union is essential for
determining the proper clinical course of action [12]. The standard analyses for detecting
microorganisms – gram staining (for microscopic investigation) and culturing of tissue
biopsy specimens obtained during surgical revision – are reported to have poor sensitivity
[13]. The hypothesis is that evidence of bacterial infection is supported further by the
detection of bacterial DNA, which suggests bacterial persistence in the area of non-union
despite negative culture results. PCR targeting highly conserved regions of the bacterial
genome (e.g. the 16 S rRNA gene) has been used successfully to detect nonculturable
bacteria that cause a variety of infections, including septic arthritis [14] and meningitis [15].
The aim of this study was to investigate whether”aseptic” nonunion after tibial shaft fractures
is due to nonculturable bacteria by means of PCR amplification of 16 S rRNA genes and to
compare the efficiency of PCR with that of standard culture techniques.
Materials and methods
All patients participating in the present study were informed in detail about the surgical
technique and all alternative procedures with their respective advantages and disadvantages,
and all participants chose to undergo the index surgical procedure. All patients signed
informed consent forms to participate in the study. The study was performed in accordance
with the local ethical review board.
From November 2009 through March 2010, a consecutive series of 23 patients with non-
union following tibial shaft fractures were investigated. Normal healing was defined as union
within 4 months, delayed union as healing between 4 and 6 months and non-union was
defined as the absence of healing after 6 months [16]. Only patients without clinical signs of
infection were included in the study. Exemplary x-rays of one case from the treatment group
are shown in Figure 1. Control subjects included 12 patients undergoing open reduction and
internal fixation for acute tibial shaft fractures.
Figure 1 Consecutive x-rays of a 30-year-old male with tibial non-union and valgus
malalignment following stabilization with external fixator for 6 months (Figures a,b).
Postoperative findings after excision of necrotic tissue and re-osteosynthesis with a
multidirectional locking plate (tifix®-tibia-plate, Litos, Hamburg, Germany) in combination
with reconstruction of skeletal defects by implantation of autologous bone removed from the
iliac crest (Figures c,d). Conventional and molecular bacteria detection methods were both
negative
A stage-adapted treatment algorithm for tibial non-union has been established, as previously
published [16]. Based on contemporary evidence among the recent literature, we do not
routinely administer antibiotics beyond 5 days postoperatively [17]. We favor oral antibiotic
administration, because our experience has shown us that the route of antibiotic
administration (oral versus parenteral) does not affect the rate of disease remission if the
bacteria are sensitive to the antibiotic used.
All patients received standard preoperative care. Skin was decontaminated with Cutasept H
(Bode Chemie, Germany). Two grams of cefazolin (Basocef, Deltaselect GmbH, Germany)

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were administered for perioperative prophylaxis subsequent to taking the biopsies.
Intraoperative biopsy samples (at least 3, each measuring 1 cm3) obtained from the non-union
were all divided into 2 portions. Specimens were placed into separate sterile tubes without
additional substrates. Specimens obtained for PCR were stored at −70°C. Samples were then
examined by means of routine culture methods and by polymerase chain reaction (PCR) for
the detection of 16 S ribosomal RNA (rRNA) in the laboratory. These procedures permitted
the independent examination and interpretation of the results. Histopathologic findings were
not recorded because of poor sensitivity, especially in cases of low-grade infection [18,19].
Bacterial isolation and standard culture methods
All specimens were incubated in brain-heart infusion broth (bioMérieux, Nuertingen,
Germany), TVLS medium [20], a medium described by Lodenkaemper and Stinen [21], on
Columbia blood plates (aerobic 5% CO2), and then on Brucella agar plates (anaerobic)
(bioMérieux, Nuertingen, Germany), as previously described [12]. Samples were incubated
for 14 days. Susceptibility testing was performed according to the German Institutional
Standard Nr. 58940 [22].
DNA isolation
Tissue samples were immediately stored at −70°C, and DNA was purified from homogenized
specimens after proteinase K digestion and column extraction with the NucleoSpin DNA kit
(Macherey-Nagel, Dueren, Germany). All DNA procedures before and after PCR were
performed in separate designated rooms with separate pipetting devices to avoid
contamination of the samples with foreign DNA. Master-mixture water controls were used
for every sample that was processed.
PCR amplification
The sequences of the universal primers (16rRNA gene) and primers of the control gene
(glyceraldehydes-3-phosphate dehydrogenase; GAPDH) are indicated in Table 1.
Oligonucleotides used in this study were provided by Eurofins MWG Operon, Ebersberg,
Germany. DNA was amplified in a 25µL-reaction mixture consisting of ready-to-go PCR
beads (up to 23µL; Amersham Pharmacia Biotech, Muenchen, Germany), 0.5µL of each
primer (100 pmol/mL), and 1µL of the sample. After amplification, 5µL of the amplified
product was analyzed in a 2% agarose gel. Amplification products were sequenced by
SeqLab and then analyzed using the National Center for Biotechnology Investigation Blast
database [23,24].
Table 1 Nucleotide sequences of primers used to determine agents responsible for non-
union following tibial shaft fractures
Primer
Sequence
16S rRNA

Forward
5′-AGAGTTTGATCCTGGCTCAG-3′
Reverse
5′-CCCACTGCTGCCCGTAG-3′
GAPDH

Forward
5′-TCTGCCCCCTCTGCTGATGCCCCC-3′
Reverse
5′-CCATCACGCCACAGTTTCCCGGAG-3′
Note. GAPDH
Glyceraldehydes-3-phosphate dehydrogenase

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Amplification of the GAPDH control gene was performed using real-time PCR (Light Cycler
Detection System; Roche Molecular Biochemicals, Mannheim, Germany) with the FastStart
DNA Master SYBR kit (Roche Molecular Biochemicals). The PCR protocol included the
following work stages: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 5 s,
and 72°C for 10s. In the dissociation protocol, single peaks were confirmed to exclude
nonspecific amplification.
Statistical analysis
Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS
15.0, Chicago, IL, USA) for descriptive statistics with a level of significance set at p < 0.05.
The non-parametric Wilcoxon’s signed rank test was used to analyze the data.
Results
The experimental group of patients with tibial non-union consisted of 8 women and 15 men,
with a mean age of 47.4 years (range: 20–82 years). The mean age of the control group was
49.3 years (range: 17–73 years), including 2 females and 10 males.
The mean interval of the fracture to the index operation due to non-union was 10.2 months
(range: 6–34 months). At the time of the initial trauma, 8 cases (34.8%) were rated as open
fractures due to severe soft tissue injuries according to the classification by Gustilo et al. [25].
Four open fractures (33.3%) were counted in the control group. Up to 8 revision surgeries
(mean: 1.9; range: 1–8) had been performed in 18 cases (78.3%) prior to the index operation.
For all patients in the experimental group, clinical pathological data – especially C-reactive
protein levels (mean: 20.8 mg/l; range: 1.6–92.7) and WBC counts (8,359/µl; range: 4,360–
15,130) were not significantly higher compared to the control group (CRP: mean: 25.7 mg/l;
range: 2.9–133, WBC: mean: 11,234/µl; range: 6,110–17,210).
None of the non-union culture samples showed evidence of microorganism growth. Bacterial
isolates were found by conventional culturing methods in only 1 case of an open fracture
from the control group (Figure 2). The isolated pathogens were Streptococcus suis and
Enterococcus species. Tissue from the fracture gap in this case after initial débridement
yielded negative results by PCR.
Figure 2 38-year-old male with an open tibial fracture (control group). This figure
demonstrates the findings before the initial treatment. The isolated pathogens in culture were
Streptococcus suis and Enterococcus species. Tissue from the fracture gap after initial
débridement yielded negative results by PCR
16 S rRNA was detected in tissue specimens from 2 cases (8.7%) of non-union. Further
analysis of these variable species-specific sequences enabled the identification of specific
microorganisms; in one case Methylobacterium species and in the other sample
Staphylococcus species were identified. The PCR-positive patients were both culture-
negative.

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Discussion
To the best of our knowledge, this is the first study on conventional and molecular bacteria
detection methods in non-unions following tibia shaft fractures to validate the hypothesis that
non-culturable microorganisms are a potential source of non-union.
Diaphyseal tibia fractures are the most common lower limb fractures worldwide [1]. There is
controversy among the literature regarding the best way to manage open tibial shaft fractures.
Recently, a metaanalysis of randomized prospective studies was performed comparing
external fixators and unreamed IM nails. There was no statistically significant difference
between the two methods of stabilization with respect to union, delayed union, deep infection
and chronic osteomyelitis [26]. Non-union continues to be one of the orthopedist’s greatest
challenges, especially in its septic form.
As for many other infectious processes, early detection can often alter the natural course of
the disease and ultimately improve long-term outcomes for patients [27]. There might be
clinical signs highly suggestive of infection, but diagnosis can be a difficult task, particularly
in the case of late and/or chronic infections [28].
Laboratory markers such as C-reactive protein, erythrocyte sedimentation rate, and white
blood count are sensitive markers of inflammation and plausible infection, but they are
unable to localize the exact site of infection and they are associated with low specificity [29].
In this series, no significant difference was obvious between the treatment and control group
according to inflammatory laboratory markers, although the mean values in both groups
diverged from the norm. Elevations in patients from the control group might have been due to
trauma, which often leads to increased levels of laboratory markers in the absence of
infection [30,31].
The standard analyses for detecting microorganisms – gram staining (for microscopic
investigation) and culturing of tissue biopsy specimens obtained during surgical revision –
are reported to have poor sensitivity [12]. The sensitivity seems to be related to the amount of
biopsies taken [32]. Antibiotics administered for perioperative prophylaxis, as well as
extended transportation time, inadequate quantities of vital bacteria and preservation of
specimens before processing may all lead to negative culture results [33].
Although there is a large body of evidence on wound bacteriology after open fractures [25]
with positive culture results in up to 83% [34], the literature regarding infection in closed
fracture gaps that affect healing is lacking [35]. In our series, none of the closed fractures in
the control group were culture-positive.
Many molecular tools for bacterial DNA detection from clinical samples have been
developed. One of the most significant contributions thus far has been amplification-based
techniques (PCR), since some studies have confirmed its excellent sensitivity and specificity
[27]. Moreover, the PCR technique takes less than 5 hours to complete, which is significantly
shorter than the couple of days required for routine cultures. In a prospective study
comparing PCR and culture techniques in the diagnosis of prosthetic joint infection, Gallo et
al. demonstrated a significantly higher sensitivity, accuracy and negative predictive value for
PCR versus culture [13]. There was 83% concordance between the results of intraoperative
culture and PCR detection of causative bacteria [13]. This is in accordance with Hoeffel et

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al., who reported a PCR sensitivity and specificity of 71% and 49%, respectively, and a
positive predictive value of 22% and a negative predictive value of 7%, when compared with
culture methods [36]. The authors concluded that the PCR methods should not serve as
screening tests for musculoskeletal infections, but they could be useful to confirm infections,
especially after initiating antibiotic treatment. Shortcomings of the PCR technique compared
to routine cultures are higher costs, false-positive results and problems with interpretation
[27].
In our series, 16 S rRNA was detected in tissue specimens from 2 cases (8.7%) of non-union.
In contrast to our results, Szczèsny et al. report the presence of bacteria in the callus of closed
fractured bones in up to 42% [37]. Both viable bacteria and their DNA were detected.
Interestingly, the majority of isolates were not detected in the fracture gap tissue but in the
subcutaneous tissue and muscles. In patients with nonalignment, S. epidermidis and aureus
were detected in 4 out of 24 patients, whereas in the delayed healing group bacterial isolates
were found in 15 out of 43 patients [37]. The reasons for the diversity between their results
and ours remain unclear, although one can pinpoint the fact that in the present study, tissue
from non-unions was investigated. In addition, the results of 16 S PCR are known to be very
susceptible for contamination. Our tests were performed under sterile conditions in a
laboratory room designated for RNA isolation and identification only. Sodium chloride
solution used for tissue samples was checked for the presence of 16 S rRNA and was found
to be negative for all samples. Further, most authors consider the positive cultures of deep
tissue specimens to be contamination from external sources [38]. Contamination of
specimens could be precluded from being the source of detected isolates.
There are two limitations that need to be acknowledged and addressed regarding the present
study. The first limitation concerns the heterogeneous patient population, which reflects the
situation of patients with tibial non-union. The second limitation involves the extent to which
the findings can be generalized beyond the cases studied. The number of cases is too small
for broad generalizations. However, these limitations can be seen as fruitful avenues for
future research along the same lines.
This is the first study comparing routine cultures and molecular bacterial DNA detection in
tibial non-union. In summary, nonculturable pathogens seem to play a causative role in tibial
non-unions. The combination of microbiological culture and broad-range PCR seems to
substantially add to the number of microbiological diagnoses obtained and may improve the
clinican’s ability to tailor therapy to the individual patient’s needs.
Competing interests
The authors declare that they have no competing interests. None of the authors received
financial support.
Authors’ contributions
1. Conception and design of the study: JG, UG 2, Analysis and interpretation of data: SW,
AP, AS, JG 3, Collection and assembly of data: SW, AP, UG, AS, JG 4, Drafting of the
article: JG, UG. All authors read and approved the final manuscript.

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Acknowledgements
The authors thank Jan Rupp, PhD, for his support and the opportunity to perform the PCR in
his laboratory.
We gratefully acknowledge the skilled technical assistance of Ms. Angela Gravenhorst and
Mr. Stefan Bark.
None of the authors received financial support.
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Figure 1

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Figure 2