OBJECTIVE: To retrospectively analyze the concordance of bacterial culture between bone tissue and deep soft tissue in diabetic foot osteomyelitis (DFO) patients and clinical characteristics of patients.
METHODS: This study collected samples from 155 patients with suspected DFO (who required amputation after clinical evaluation). Bacterial culture and drug susceptibility tests were performed on the patients\' deep soft tissue and bone tissue, and the consistency between the two was compared. In addition, the differences among DFO patients with different degrees of infection were compared classified by the PEDIS classifications.
RESULTS: Among the 155 patients diagnosed with DFO, the positive rate of bone culture was 78.7% (122/155). This study cultured 162 strains, including 73 Gram-positive bacteria, 83 Gram-negative bacteria, and 6 fungi. Staphylococcus aureus (33 strains) was the most common bacteria. The overall agreement between bone culture and tissue culture was 42.8%, with Staphylococcus aureus and Enterobacteria having the best (64.3%) and least agreements (27.3%), respectively. The drug sensitivity results in bone culture showed that Staphylococcus aureus was the main Gram-positive bacteria. The bacteria were sensitive to linezolid and vancomycin. Proteus mirabilis was the main Gram-negative bacteria. These were more sensitive than biapenem and piperacillin/tazobactam. Fungi were more sensitive to voriconazole and itraconazole.
CONCLUSIONS: The culture results of deep soft tissues near the bone cannot accurately represent the true pathogen of DFO. For DFO patients, bone culture should be taken as much as possible, and appropriate antibiotics should be selected according to the drug susceptibility results.

BACKGROUND: Diabetic foot osteomyelitis (DFO) is a dreaded complication as both diagnosis and treatment of the condition is laborious. However, for proper decision on antibiotics in medical management of DFO, accurate determination of microbes is necessary to narrow the spectrum of coverage and to reduce adverse effects of long-term administration of antibiotics. With differing pattern of use of antimicrobials and their resistance pattern in different countries, it is empirical to determine the microbiological characteristics of bone cultures in DFO from a referral institute in South India.
METHODS: This study was a retrospective chart review of all cases of proven DFO who had consented for debridement and bone culture or those who underwent amputation. Both deep soft tissue (DST) and bone cultures were obtained for aerobic bacteria. Clinical characteristics and site(s) of DFO were recorded. Investigations for peripheral artery disease were performed if clinically indicated.
RESULTS: In all, 105 patients with DFO were reviewed. Mean age was 62 years and 70% were men. Of those who were screened, 57% had evidence of peripheral arterial disease by arterial doppler. 46% of bone culture samples were sterile. Gram- negative organisms were more common (58%). Following staphylococcus, pseudomonas was the second common isolate. Of total staphylococcal isolates 37% were MRSA and 33% of klebsiella isolates were ESBL producing. Concordance rate between DST and bone cultures was 66%. 90% were mono-bacterial isolates. The commonest site of involvement of DFO was terminal phalanges of toes rather than base of 1st metatarsal.
CONCLUSIONS: Widespread use of antibiotics, tropical climate and route of entry of organisms causing DFO differed in our cohort of patients. Further studies from different regions of world would shed light onto different pattern of microbes causing DFO.

Medical literature offers no clear treatment guidelines when performing amputations for gangrene of the forefoot despite a high percentage that suffer poor outcome due to infection. Gas gangrene and wet gangrene are often preceded by dry stable gangrene. This is a retrospective review of consecutive patients who underwent forefoot amputation and bone biopsy as treatment of forefoot gangrene by a single surgeon. Procedures performed included digital, ray, or transmetatarsal amputation with bone biopsy sent for both culture and histopathologic evaluation. One hundred patients (35 females, 65 males) met inclusion criteria. Mean follow-up was 9.6 months. Mean age was 63.5 years old. Forty-six out of 100 (46%) had elective amputation while 54/100 (54%) were emergent for acute infection. Vascular intervention was performed in 52/100 (52%). Seventy-eight out of 100 (78%) had histopathologic diagnosis of acute osteomyelitis while 82/100 (82%) had positive bone culture. Patients with acute infection had worse outcomes, with higher rates of more proximal amputation and delayed wound healing. We found that 79.7% of patients who underwent forefoot amputation due to gangrene had underlying osteomyelitis. We also found that those with acute infection during the time of amputation had poorer postamputation outcomes such as delayed wound healing, revision surgery, and high rates of more proximal amputation. Therefore, it may imply that earlier amputation of stable gangrene prior to becoming acutely infected may decrease the occurrence of osteomyelitis and avoid some of the preventable postamputation complications. Further studies are warranted.

Several different bioreactors have been developed to study bone biology. Keeping a bone viable for long-term studies is still a challenge. We have developed an ex-vivo bone bioreactor that can keep the ex-vivo live bone viable for more than 4 weeks. Keeping a bone viable for over a month can be used as an alternative model for in-vivo experiments in animals. We hypothesize that the perfusion flow and mechanical load on the bone provide a real-time environment for the bone to survive. Cancellous bones were harvested from the bovine metatarsals and were placed in the dynamic culture with cyclic loading at regular intervals. After a period of week 4, the bone cores were retrieved from the bioreactor and tested for viability using calcein-AM and ethidium homodimer -1 fluorescent dyes and were compared with the cores that were placed in static culture with and without any loads on them and Day 0 bone core that acted as a positive control. The bone blocks were then fixed in 10% formalin, and bone mineral density was evaluated using a DXA scanner before staining them for H&E to study the morphological changes. Results revealed that the bone cultured in the bioreactor was viable as compared to the one in the static culture with and without constant load. Bone cores cultured in our ex-vivo bioreactor system also maintained their morphology and no statistical difference was found in the bone mineral density compared to positive controls and the statistical difference was found when compared with the cores cultured in static culture. This tool can be used to study bone biology for various applications such as bone ingrowth studies, to study the effect of drugs, hormones, or any growth factors, and much more.

Microbiological cultures of per-wound bone biopsies have shown a lack of correlation and a high rate of false-negative results when compared with bone biopsy cultures in diabetic foot osteomyelitis. The selection of samples from the area of active osteomyelitis, which contains a complete census of the microorganisms responsible for the infection, is essential to properly guide antimicrobial treatment. We aimed to comparatively evaluate the quantitative and qualitative cultures taken from different areas, in metatarsal heads resected for osteomyelitis. For this purpose, we consecutively selected 13 metatarsal heads from 12 outpatients with plantar ulcers admitted to our diabetic foot unit. Metatarsal heads were divided transversally into 3 portions: plantar (A), central (B), and dorsal (C), and the 39 resulting samples were cultured. Qualitative and quantitative microbiological analysis was performed, and the isolated species and bacterial load, total and species specific, were compared between the 3 metatarsal bone segments. The primary outcome of the study was the bacterial diversity detected in the different bone sections. Cultures were positive in 12 of the 13 included metatarsal heads (92%). A total of 34 organisms were isolated from all specimens. Ten of the 12 cultures (83%) were polymicrobial. Ten of the 13 metatarsal heads (77%) had identical microbiological results in each of the 3 bone sections. The largest number of microorganisms was found in the central section. The overall concordance between sections was 91%. The predominant microorganisms were coagulase-negative staphylococci (41%). Statistical differences were not found in the bioburden between sections (range 3.25-3.41 log10 colony-forming unit/g for all sections; P = .511). The results of our study suggest that microorganisms exhibit a high tendency to spread along the metatarsal bone and that the degree of progression along the bone is species dependent. The central portions of metatarsal bones tend to accumulate a higher diversity of species. Thus, we recommend this area of bone for targeted biopsy in patients with suspected osteomyelitis.

Guidelines suggest culturing clinically uninfected bone at the margin after surgical resection for osteomyelitis, but little published evidence supports this procedure. To investigate whether culturing marginal bone after completing resection of infected bone affected antibiotic use or further surgical intervention, we collected data on sequential patients undergoing amputation for a foot infection at our tertiary care hospital between January 2014 and May 2015. We recorded patient age, sex, presence of diabetes mellitus, level of amputation, whether marginal bone was sent for culture, microbiology of any marginal bone specimens, type and duration of antibiotic therapy, and any further surgical resection. Among 132 patients, the mean age was 71.9 years, 103 (78.0%) were male, and 79 (59.8%) had diabetes. Treating surgeons sent marginal bone in 58 (43.9%) of these patients, 50 (86.2%) of which were culture positive. Patients with a positive bone culture were significantly more likely to undergo further surgical intervention (20.0% vs 6.1%, p = .047). For patients with diabetes, compared with those without, surgeons did not send marginal bone for culture more often (46% vs 42%, p = .72), nor did they undertake further surgical interventions more frequently (13.4% vs 10.1%, p = .89). Our results suggest that the clinicians used the marginal bone culture findings to make clinical decisions but do not clarify if there is a benefit to performing this procedure. Although patients whose proximal bone specimens were culture positive were more likely to undergo a surgical intervention, the reasons for, and benefit of, this additional surgery were unclear.

The diagnosis of osteomyelitis (OM) is a challenging but critical pathology to uncover in patients with concomitant Charcot neuro-osteoarthropathy (CN). The reference standard to diagnose OM is bone biopsy for histopathologic and microbiologic examination. The presence of CN, however, can have a negative effect on the accuracy of either method to identify OM. The aim of the present study was to examine the concordance between bone pathology and bone cultures in the presence of CN in the diagnosis of OM. A total of 286 patients with diabetes mellitus (DM) and CN were identified retrospectively, with 48 patients identified with OM. OM was confirmed by radiographs, magnetic resonance imaging, erythrocyte sedimentation rate, and C-reactive protein, positive probe-to-bone test results, and intraoperative inspection. Seventy matched pairs of bone pathology and cultures with complete data were compared and analyzed. Statistical analysis included concordance, positive predictive value, negative predictive value, sensitivity, specificity, and kappa coefficient. Concordance between bone pathology and bone culture was 41.4%, with agreement in 29 of 70 paired specimens. The diagnostic test accuracy of histopathologic examination to diagnose OM in CN bone in our study was 51.4%. The diagnostic test accuracy of microbiologic examination to diagnose OM in CN bone was 50%. The positive predictive value was 72.2%. The negative predictive value was 44.1%. The sensitivity was 57.8%. The specificity was 60.0%. The kappa coefficient was 0.165. The reference standard method of histopathologic and microbiologic examination of bone specimens has little concordance and can lead to inaccurate or inconclusive information. The low sensitivity and specificity demonstrated in the present study does not support the use of the current reference standard method of bone biopsy for histologic and microbiologic diagnosis of OM when CN is present. Thus, a diagnosis of OM in patients with CN should only be considered in the presence of strong clinical, laboratory, and imaging correlates.

In fetal rat bones in culture the divalent cation ionophore A23187 inhibited in a dose-dependent manner both the release of proteoglycans and the subsequent degradation of cartilage induced by retinoic acid, indicating that calcium was involved in its action. A23187 had to be present continuously to manifest its inhibitory effect; retrieval of the ionophore abolished the suppression, demonstrating that the effect was reversible and not due to toxicity. A23187 at 1.0 μM, which completely blocked the retinoic acid-induced cartilage resorption, markedly suppressed3H-leucine,3H-mannose and3H-thymidine incorporation in control and retinoic acid-treated cultures. Reduced3H-thymidine incorporation did not appear to be responsible for the inhibition by A23187 of retinoic acid-induced cartilage resorption because inhibitors of DNA synthesis did not affect the retinoic acid response. In the presence of retinoic acid the ionophore at 0.3 μM had no effect on the incorporation of3H-leucine and3H-mannose, but suppressed the retinoic acid-induced proteoglycan release. This suggests that reduced protein and glycoprotein synthesis were not the main causes for the inhibitory effect of A23187. In conclusion, retinoic acid-induced cartilage degradation required calcium at some crucial points.

In explanted humeri of late fetal rats, retinoic acid was found to induce the release of proteoglycan followed by cartilage resorption. Tissue breakdown, which was demonstrated by losses of DNA, RNA, and protein, coincided with the appearance of necrotic cells. In control humeri chondrocytes were the main cell type, but in humeri treated for 4 days with retinoic acid the surviving cells were chondroblastlike. Sensitivity of proteoglycan release and tissue breakdown to retinoic acid decreased with age.The proteinase inhibitors cysteine, Trasylol, and soya and lima bean trypsin inhibitors did not antagonize the effects of retinoic acid. Phenylmethanesulfonyl fluoride suppressed cartilage resorption more effectively than proteoglycan release, while pepstatin merely suppressed cartilage resorption. The inhibition by EDTA of both the release of proteoglycan and cartilage resorption induced by retinoic acid was dose dependent. Zn2+ abolished these effects, whereas Mn2+ only relieved the release of proteoglycan induced by retinoic acid; this indicates that these two effects of retinoic acid are not necessarily linked.In view of our recent demonstration that the release of proteoglycan induced by retinoic acid requires RNA and protein synthesis, we suggest that the degradation of proteoglycans in response to retinoic acid is dependent upon continued synthesis of metalloproteinases.

Hidden infections in a reconstructive surgery program are frequently underestimated.
A retrospective study was undertaken of 1,891 civilian war-wounded patients from Iraq, Syria, Yemen and Gaza treated in Amman from August 2006 to January 2016. One thousand three hundred and fifty-three underwent surgical interventions for previous bone injury and had systematic bone cultures.
Among patients (167) without any clinical, biological or radiological signs of infection, 46% demonstrated infection based on bone cultures. We conclude that bone culture should become a prerequisite for any reconstruction in such contexts.