Peptide Nucleic Acid Fluorescence Research Articles

Polymorphonuclear leukocytes restrict growth of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients.

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures

Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used (‘Staphylococcus aureus/CNS’, ‘Enterococcus faecalis/OE’, ‘GNR Traffic Light’ and ‘Yeasts Traffic Light’ PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect

Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization – temperature, time and formamide concentration – in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.

Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization.

The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 104 CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens.

Detection of Dehalococcoides spp. by Peptide Nucleic Acid Fluorescent in situ Hybridization

Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites.

Rapid detection of Enterococcus spp. direct from blood culture bottles using Enterococcus QuickFISH Method: a multicenter investigation

The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively.

Clinical consequences of using PNA-FISH in Staphylococcal bacteraemia

To optimize patient treatment and rational use of antimicrobials, it is important to provide fast information on findings in blood-cultures (BCs). The purpose of this study was to evaluate the impact of using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) on positive BCs containing Gram-positive cocci in clusters to differentiate between Staphylococcus aureus (SA) and coagulase negative staphylococci (CoNS) on the prescribed antimicrobial therapy and on the number of contacts between microbiologist and clinician. All cases of positive BCs in our laboratory with SA or CoNS in the year 2011 were identified and the charts were reviewed retrospectively. The group of patients with BCs tested with PNA-FISH was compared to the group of patients with untested BCs. A total of 200 patients with SA and 725 patients with CoNS were included. The mean number of contacts was 0.82 when PNA-FISH showed CoNS and 1.39 when PNA-FISH was not done (p < 0.0001). More patients were recommended appropriate antimicrobial therapy for SA bacteraemia in the PNA-FISH group (98.0%) than in the non-PNA-FISH group (89.4 %) (p = 0.025). The percentage treated with dicloxacillin was 29.6 in the PNA-FISH group, and 8.2 in the non-PNA-FISH group (p = 0.0003). The use of PNA-FISH on BCs in this study was associated with more appropriate and narrow spectrum antimicrobial therapy for patients with SA in an area with low prevalence of methicillin-resistant SA, and a lower number of contacts between clinical microbiologist and clinician about BCs with CoNS as contaminants.

Inflammatory Patterns in Upper Airway Disease in the Same Geographical Area may Change over Time

Inflammatory patterns of nasal polyps (NPs) may vary. Changes over time have not been investigated so far. This study was designed to evaluate the inflammatory patterns of NPs in Thailand at two time points 12 years apart, explore differences in Staphylococcus aureus (SA) mucosal carriage rates over time, and the latter's relationship with the inflammatory patterns. Formalin-fixed nasal tissue was obtained from 89 (47 in 1999 and 42 in 2011) patients suffering from chronic rhinosinusitis with NPs (CRSwNPs). Tissues were evaluated for eosinophils, neutrophils, IgE(+) cells, IgE and macrophage mannose receptors, interleukin (IL)-5 and IL-17 cytokine profile, and the presence of SA, using automated immunohistochemistry and peptide nucleic acid-fluorescence in situ hybridization. We found a significant increase in the absolute values of eosinophils and IgE(+) cells in the 2011 CRSwNP tissue series compared with 1999 and a significant but smaller increase in neutrophils. Semiquantitative evaluation revealed significantly higher mean values of positive cells for all studied inflammatory markers in the 2011 group of patients, except for the high-affinity IgE receptor. This "eosinophilic shift" of inflammation was accompanied by higher SA carriage, as well as higher frequencies of SA invasion (54.8% versus 10.6%; p < 0.001) in the 2011 compared with 1999 subjects. Patients with asthma were more likely to have higher SA carriage rates compared with nonasthmatic patients. There was a shift from predominantly neutrophilic to eosinophilic CRSwNPs in Thai patients within 12 years, with an increase in various inflammatory markers including IgE, which is associated with an increase in intramucosal presence of SA.

Microbiological screening for earlier detection of central venous catheter-related bloodstream infections

Catheter-related bloodstream infections (CRBSIs) are currently detected with a reactive diagnostic policy, that is, application of tests to patients with clinically suspected CRBSI. The aim of our study was to evaluate whether CRBSIs could be anticipated in an earlier stage by microbiological screening using peptide nucleic acid fluorescence in situ hybridization (PNA FISH) with universal hybridization probes or acridine-orange leucocyte cytospin (AOLC) tests in haemodialysis and haematological patients with CVCs in situ compared with routine test. Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) and AOLC tests using blood samples from both CVC lines in patients undergoing haemodialysis were performed three times a week and from one CVC line in haematological patients were performed daily. Results were compared with those obtained from routinely performed CRBSI diagnostic tests. One hundred fifteen patients with 139 catheter periods were investigated. The mean observation time per catheter period was 25 days (IQR 13.5-43.5), resulting in 5615 CVC days with a total of 4839 tested blood samples. Five CRBSI cases were detected by routine measures resulting in a CRBSI rate of 0.9/1000 catheter days. Four of five CRBSIs could be anticipated by positive PNA FISH and AOLC tests 2-8 days before the diagnosis was established with routine measures. The proactive anticipative strategy using microscopic examination of CVC blood samples to anticipate CRBSI in an earlier stage compared with routine measures is a new diagnostic approach in patients with CVCs and a high risk of developing CRBSI.

Biofilm formation with mixed cultures of Pseudomonas aeruginosa/Escherichia coli on silicone using artificial urine to mimic urinary catheters

In this study, single and dual species biofilms of Pseudomonas aeruginosa and Escherichia coli, two common bacteria associated with urinary tract infections, were formed in silicon coupons immersed in artificial urine medium. In single species experiments, E. coli appeared to form biofilms more easily than P. aeruginosa. In mixed biofilms, both species apparently benefited from the presence of the other, as the average Log total cells cm−2 of mixed biofilms (7.29 cells cm−2) was higher than obtained for single cultures (6.99 cells cm−2). However, the use of selective media seemed to indicate that P. aeruginosa was the only microorganism to benefit in mixed biofilms (Log 7 CFU of P. aeruginosa cm−2, compared to Log 6 CFU cm−2 obtained in pure cultures). Peptide nucleic acid-fluorescence in situ hybridization combined with confocal laser scanning microscopy confirmed that E. coli was indeed being outnumbered by P. aeruginosa at 48 h. Whereas E. coli is the main causative agent of catheter-associated urinary tract infections, the results from this study indicate that the reason for the higher prevalence of this microorganism is not related to an enhanced ability to form biofilm and outcompete other species that may also be present, but rather to a better ability to form single-species biofilms possibly due to a more frequent access to the catheter surface.

Correction: Discriminating Multi-Species Populations in Biofilms with Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH)

Correction: Discriminating Multi-Species Populations in Biofilms with Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH)

Interactions between Lactobacillus crispatus and Bacterial Vaginosis (BV)-Associated Bacterial Species in Initial Attachment and Biofilm Formation

Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder.

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

BackgroundBacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.ResultsTherefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.ConclusionsThis quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples

Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) probe. The probe (Lac663) was tested on 36 strains belonging to different Lactobacillus species and on 20 strains of other bacterial species. The sensitivity and specificity of the method were 100% (95% confidence interval (CI), 88.0 to 100.0%) and 95.0% (95% CI, 73.1 to 99.7%), respectively. Additionally, we tested the applicability of the method on milk samples added with Lactobacillus strains at probiotic range concentrations and other taxonomically related bacteria, as well as pathogenic bacteria. The Lac663 probe bound exclusively to Lactobacillus strains and the described PNA-FISH method was capable of directly quantifying Lactobacillus spp. in concentrations at which these potential probiotic bacteria are considered to have an effective benefit on human health.

Interstitial telomeric sequences are not preferentially involved in the chromosome damage induced by the methylating compound streptozotocin in chinese hamster cells

The effect of the methylating compound streptozotocin (STZ) on interstitial telomeric sequences (ITSs) was investigated in Chinese hamster ovary (CHO) cells by using peptide nucleic acid-fluorescence in situ hybridization with a pantelomeric probe. Cells were exposed to increasing concentrations of STZ, and chromosomal aberrations were analyzed at the first mitosis after treatment. The frequency of chromosomal aberrations directly involving ITSs increased in STZ-treated cells by a factor of 2.6 (2 mM) and 3.6 (4 mM) when compared with the frequency of these aberrations in control cells (P < 0.05). However, no significant differences were found between control and exposed cells in the percentage of aberrations directly involving ITSs, demonstrating that these repeat regions were not preferentially involved in the chromosome damage induced by STZ. In addition, STZ did not alter telomerase activity, suggesting that this enzyme may not be involved in the induction of chromosomal aberrations by this compound.

Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples

Several methods for the rapid and specific detection of Salmonella in food samples have been described. Here, we compare 4 of those methods in terms of assay time, procedure complexity, detection limit, sensitivity, specificity and accuracy. Milk, eggs and mayonnaise samples were artificially contaminated with Salmonella enterica serovar Enteritidis cell concentrations ranging from 1×10−2 to 1×102CFU per 25g or ml of food. Samples were then pre-enriched and analyzed by either: i) real-time PCR, using the iQ-Check Salmonella kit; ii) immunocapture, using the RapidChek SELECT Salmonella; iii) a peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method and iv) the traditional bacteriological method ISO 6579:2002. All methods were able to detect Salmonella in the different types of food matrixes and presented a similar detection level of 1CFU per 25g or ml of food sample. The immunocapture and the PNA FISH methods proved to be very reliable, as their results were 100% in agreement with the ISO method. However, real-time PCR presented a significant number of false positives, which resulted in a specificity of 55.6% (CI 95%, 31.3–77.6) and an accuracy of 82.2% (CI 95%, 63.2–91.4) for this method. Sensitivity was 100% since no false negative results were observed. In conclusion, the implementation of these molecular techniques, mainly the immunocapture and PNA-FISH methods, provides a reliable and less time-consuming alternative for the detection of Salmonella spp. in food samples.

Impact of a rapid peptide nucleic acid fluorescence in situ hybridization assay on treatment of Candida infections

The impact of a rapid peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assay with an antimicrobial stewardship intervention on the treatment of Candida infections was studied. The utility of implementing the PNA FISH assay with an antimicrobial stewardship intervention in hospitalized patients with candidemia was evaluated by measuring the median time to Candida species identification, time to targeted therapy, and clinical outcomes, including time to culture clearance, hospital length of stay, and hospital mortality. Secondary objectives included determining the cost-effectiveness of the PNA FISH assay by assessing estimated antifungal drug costs (as average wholesale price) before (June 26, 2009-September 19, 2010) and after (September 20, 2010-June 13, 2011) test implementation and confirming test accuracy. For both groups, laboratory personnel notified the physician of the results of Gram's stain from blood culture. Time to targeted therapy significantly decreased after the implementation of the PNA FISH assay (p = 0.0016). The postimplementation group had a higher rate of culture clearance (p = 0.01). Median time to species identification was 0.2 day with the PNA FISH test versus 4 days with routine methods (p < 0.001). Accounting for the cost of the test itself and the cases in which patients were switched to more-expensive therapy on the basis of the test, we estimated that the PNA FISH test resulted in savings of approximately $415 per patient. Implementing a PNA FISH test to identify Candida species from yeast-positive blood cultures in conjunction with a pharmacy-driven antimicrobial stewardship protocol decreased the time to targeted antifungal therapy and the time to culture clearance.

The microorganisms in chronically infected end-stage and non-end-stage cystic fibrosis patients

Patients suffering from cystic fibrosis (CF) develop chronic lung infections because of highly viscous mucus, where bacteria can form biofilms. In this study, we investigated the microorganisms present in the lungs of end-stage and non-end-stage patients using standard culturing techniques and molecular methods. Tissue and sputum samples (n=34) from explanted lungs of five end-stage patients were examined along with routine expectorates (n=15) from 13 patients with non-end-stage CF, representing earlier stages of chronic lung infections. Previously, using peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH), we have shown that Pseudomonas aeruginosa was the sole pathogen in end-stage CF lungs (Pediatr Pulmonol 2009, 44: 547). In this study, this tendency was supported by the results of real-time PCR, confirming previous results obtained by standard culturing and 16S rRNA gene analysis (J Clin Microbiol 2011, 49: 4352). Conversely, the non-end-stage patients were found to harbor several species by culturing. PNA FISH confirmed heterogeneous microbiota and showed that the bacteria were located in monospecies aggregates with no apparent physical interaction between the different microcolonies. In conclusion, standard culturing identifies the dominating pathogens, which seem to reside in monospecies microcolonies. The possibility of signaling between the distinct microcolonies still has to be verified and elucidated.

Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer

Genomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex. We aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility. To this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects. We demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common spontaneous chromosome fragility pattern. A quantitative fluorescence in situ hybridization analysis demonstrates that familial cases display a significant decrease in the telomeric peptide nucleic acid-fluorescence in situ hybridization signal intensity in the metaphase chromosome. Moreover, three copies of the hTERT gene were found only in familial cases, although the result was not statistically significant. These results contribute in defining familial thyroid cancer as a clinical entity characterized by an altered telomere stability, which may be associated with the predisposition to develop the familial form of thyroid cancer.

Pathology and Biofilm Formation in a Porcine Model of Staphylococcal Osteomyelitis

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), NCTC-8325-4 (a laboratory strain of human origin; n = 3 animals) and UAMS-1 (a human osteomyelitis isolate; n = 3 animals). Two pigs were sham inoculated with saline. At 11 or 15 days post infection the animals were scanned by computed tomography before being killed and subjected to necropsy examination. Osteomyelitis lesions were present in the right hind limb of all pigs inoculated with strain S54F9 and in one pig inoculated with strain NCTC-8325-4. Microscopically, there was extensive loss of bone tissue with surrounding granulation tissue. Sequestrated bone trabeculae were intermingled with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore dependent on the strain of bacteria inoculated and on the formation of a biofilm.