Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing

Chronic wounds impact the health of over 8 million people in the United States each year, and the direct healthcare associated cost of these wounds is over 25 billion of dollars each year [1]. These chronic wounds include venous stasis ulcers, diabetic foot ulcers, decubitus ulcers, and non-healing surgical wounds. Medicine has worked to address the many barriers to healing including repetitive trauma, poor nutrition, impaired host defense, and infection. The presence of bacterial biofilms in chronic wounds has been established [2], and wound care clinicians attempt to manage these bacteria in chronic wounds [3]. In this study, we examine the bacterial components of the wound bioburden using two clinical laboratory tests: traditional aerobic culturing and recently developed culture-free sequencing of bacterial 16S DNA.

Previous studies have compared culturing to molecular methods [2, 4‐10], and we have performed studies to compare culturing to the molecular testing method we use in this study [2, 9]. These studies agree that molecular microbial tests are more sensitive than culture testing and hold promise for improving patient care. The current study adds to this body of work. The molecular test used in this study produces a relative abundance score, and we describe the relevance of this score and the correlation it has to culture results.

Providing more accurate information to the clinician via more sensitive testing is typically the goal of the clinical microbiology laboratory. More sensitive microbial testing, such as culture-free sequencing of bacterial DNA that is used in this study, can identify numerous microbial taxa in a single clinical sample. Unfortunately, the clinician can have difficulty discerning which bacteria could be most clinically relevant. Being able to correlate the results of this newer testing method with a familiar testing method (culturing) would be helpful for the clinician. Culture-free sequencing of bacterial DNA reveals qualitatively which bacteria are present, but it also reports a relative abundance score for each organism. Correlating this relative abundance score with the likelihood that the same bacterium would be detected by culture has not been performed until now. Correlating the results of molecular microbial diagnostic tests with the results from culturing will give clinicians insight into understanding these new tests and their clinical relevance. Improving clinicians’ ability to interpret test results will enable them to better customize their patient care and overcome each individual patient’s barriers to healing.

In this study, the chronic wounds from 51 subjects were sampled for study in accordance with Western Institutional Review Board protocol #20062347. All subjects signed informed consent documents. All samples were obtained at the Southwest Regional Wound Care Center (Lubbock, Texas) in 2009. Types of wounds included chronic wounds such as venous stasis ulcers, diabetic foot ulcers, pressure ulcers, and non-healing surgical wounds. Each sample of devitalized tissue was obtained using sharp debridement as part of standard of care. Each chronic wound site was sampled in duplicate for parallel testing. One portion was analyzed by culture in an academic, hospital-based microbiology laboratory while the other portion was analyzed by molecular methods at PathoGenius laboratory (Lubbock, Texas). Both labs are accredited by the College of American Pathologists.

Like previous studies, this study demonstrates that culture testing and molecular testing of wound bacteria produce differing and often conflicting results because molecular methods are more sensitive than culture methods [7‐10]. The current study demonstrates that it is important to consider the relative abundance of the bacteria that are detected by the molecular test used in this study and not only the qualitative results of the test. The relative abundance of the bacterial species within the sample helps to predict the likelihood that the species will be detected by culture. That is, bacteria with greater relative abundance within a wound sample are more likely to be cultured than other bacteria with less relative abundance. These findings are different from what has been previously reported in a study using a different molecular method of analysis (fluorescent in situ hybridization), which reported, “the absence of a correlation between the bacterial species Staphylococcus aureus and Pseudomonas aeruginosa detected in wound by culture and what is in fact present” [5].

In the current study, 51 samples were cultured, and 93 isolates were identified. One sample yielded two species from the same genus, so 92 genus-level taxa were identified in the 51 samples. An average of 1.8 ± 0.9 bacterial genera were identified in each sample (Table 1), representing 14 unique genera in total. Staphylococcus and Enterococcus comprised the majority of the detected genera; they were detected 28 and 21 times, respectively. When considering only the 92 detected genera, 16S DNA sequencing identified the same genus in the sample 78 times. Of those 78 times, the bacterial population was determined to be a dominant population (>10% of the wound microbiota relative abundance) 43 times, a major population (1-10% of the wound microbiota relative abundance) 18 times, and a minor population (<1% of the wound microbiota relative abundance) 17 times. Fourteen (14) times, the bacterial genus identified by culture was not detected by molecular testing. It is possible that some of the 14 bacterial genera that were identified by culture but not identified by 16S testing may have been misidentified by one of the testing methods (Table 2). Genetic testing is the gold standard for species identification, so if one of the tests misidentified an organism, it is most likely that culturing misidentified the organism. Another possibility is that deeper 16S DNA sequencing would have revealed the genera that were identified by culture.

16S sequencing methods resulted in an average of 2411 sequences per sample, with the average read length for all samples being 466 bases. From these sequences, 753 bacterial taxa (genera) were detected (average number per sample, 14.8 ± 7.5), and these 753 positive results were found within 145 unique genera (Figure 1, Table 1, and Additional file 1). Forty-seven percent (47%; 68/145) of the unique genera were aerotolerant and potentially culturable using aerobic culture methods. Sixty-six percent (66% ; 497/753) of the positive results were attributed to the 68 aerotolerant genera (average per sample 9.8 ± 5.3), and these were the bacteria considered in most of the analyses. Sequence analysis identified 18% (91/497) of the bacteria as dominant components of the population, 24% (120/497) as major components to the population, and 58% (286/497) as minor components of the population. Aerobic culturing detected 15.7% (78/497) of the potentially culturable organisms. Cultures identified dominant genera 47% (43/91) of the time, major genera 15% (18/120) of the time, and minor genera 5.9% (17/286) of the time (Figure 2).

It can be argued that the 58% (286/497) of results attributed to bacteria that occurred with less than 1% relative abundance in their samples were environmental contaminants within the wounds. If this is the case, it is important to note that 18% (17/92) of the bacteria that were isolated by culture were in this group, so culturing also detects bacteria that may be environmental contaminants. Another 15% (14/92) of the cultured bacteria were not detected at all using molecular methods, which could be because of very low levels of the bacteria within the wound. In total, one-third (31/92) of the genera detected by culture were either not detected using 16S sequencing or detected to be present at less than 1% of the relative abundance within the sample. The clinical usefulness of attempting to manage bacteria present at such low abundance within a sample is questionable. The molecular testing results suggest that culture testing may be insufficiently sensitive in some cases or overly sensitive in other cases. Therefore, careful clinical interpretation of culture results is necessary [11, 12].

Some bacteria were more commonly detected by culture than others (Table 3). In some instances, this variation in detection can be attributed to the relative abundance of the bacteria in the original sample (Figures 2, 3, & 4), but detection also depends upon the type of bacteria. For example, culturing identified Staphylococcus in 61% (28/46) of the samples that were positive by molecular testing (average relative abundance of 28% per sample), but Corynebacterium was only identified by culture in 16% (6/38) of the samples that were positive by molecular testing (average relative abundance of 24% per sample). Both the organism and its relative abundance affect the ability to detect the organism by culture.

Many of the bacteria that were identified by culture contributed to a large portion of the relative abundance of bacteria in the samples, but about half of the bacteria that were determined to comprise a dominant portion of the microbiota were not detected by culture (Figure 2). Some of the uncultured bacteria were obligate anaerobes (Figure 1 & Table 3), and the culture of obligate anaerobes was not attempted in this study because anaerobic cultures were not part of the patients’ standard of care. Obligate anaerobic bacteria were frequently identified by molecular testing, but they comprised an average of only 15.5% of the relative abundance in each sample. Previous studies have established that obligate anaerobes are important but difficult to detect in chronic wounds [2, 13]. In this study, molecular testing identified an average of 84.5% (±27.5%) of the relative abundance of the bacteria as aerotolerant, but many of these bacteria were facultative anaerobes and may have been growing in anaerobic conditions.

In general, the likelihood of an organism being cultured was positively related to its relative abundance (Figure 3). This trend was evident when all aerotolerant genera were examined together (OR = 1.05, 95% CI 1.04-1.07). Culturing detected 54.9% of the relative abundance (as determined by 16S sequencing) of the potentially culturable bacteria, and the remaining 45.1% of the relative abundance went undetected by culturing.

Similar results were identified at the species level: species with a higher relative abundance in the sample as determined by molecular testing were more likely to be cultured (Figure 4). Three species were examined. The trend was significantly positive for Pseudomonas aeruginosa (OR = 1.17; 95% CI, 1.08-1.31) and Staphylococcus aureus (OR = 1.08; 95% CI, 1.03-1.15), and approached significance for Enterococcus faecalis (OR = 1.05; 95% CI, 0.98-1.16).

Exhaustively culturing all of the bacteria within a clinical sample has proven difficult [8, 10, 14]. Detecting the presence of anaerobes can be especially cumbersome, and anaerobes routinely are not identified, even though anaerobes are common contributors to the sample [8, 13, 14]. Additionally, many chronic infections include biofilms with dormant bacteria (viable but non-culturable [VBNC]), which are inherently difficult to culture [2, 15]. Culturing from a mixed sample of bacteria, which is common in clinical wound samples, results in culture bias where some bacteria are positively selected because they grow quickly and robustly in the culture media, but fastidious organisms can be negatively selected and may grow more slowly or not at all. Therefore, using standard culture results to extrapolate the relative or absolute quantity of a bacterial species that was present within the original sample should not be performed because this method is unreliable (Figure 2).

Molecular methods can overcome some of cultures’ shortcomings. Molecular testing can detect biofilm bacteria, anaerobes, and VBNC bacteria regardless of how well the bacteria may or may not grow in culture [10, 12]. Molecular methods can be designed to examine these organisms quantitatively, and, unlike culture testing, the presence of antibiotics within the sample does not interfere with the testing sensitivity. Molecular methods can elucidate the presence of bacteria regardless of whether or not the bacteria can be cultured.

Forty-six (46) culture isolates were cross-checked for identification using molecular methods, and discrepancies were observed in 20% (9/46) of the isolates (Table 2): 4 genus-level discrepancies and 5 species-level discrepancies. Of the 46 isolates, 43 were identified to species and 3 were only identified to genus. The 5 species-level discrepancies were arguably clinically insignificant. The four genus-level discrepancies were more clinically significant. Twice, biochemical testing determined isolates were staphylococci, but molecular testing determined the isolates were enterococci. Once, P. aeruginosa was identified by culture, but Salmonella enterica was identified by molecular testing. As mentioned previously, identification of bacteria by genotype is considered superior to the identification of bacteria by phenotype, so culture testing reported significant misidentifications of bacterial taxa 7% (3/46) of the time.

Limitations of this study include the small size of the sample population, the lack of anaerobic bacterial cultures, and the difficulty of replicating this study at other laboratories. Only 51 subjects were examined in this study, which was large enough to recognize statistically significant trends, but studies that analyze more samples should be performed. Anaerobic cultures were not obtained as this was a standard of care study. Many wound care specialists do not routinely request anaerobic cultures because the cost is significant, and organisms are rarely recovered. The molecular methods employed by in this study are available in very few clinical testing laboratories because of the high initial set-up costs and the large amount of data analysis that is required, which makes repeating this study at other institutions difficult.

The authors have performed previous studies to examine the clinical outcomes of patients when using molecular microbial diagnostic testing and have reported improvement in patient outcomes when using molecular testing when compared to when culture testing was used [16, 17]. The current study may help to explain why this improvement was observed. Culture-free 16S DNA sequencing detects more of the bacterial components of the wound microbiota, and it is able to determine the relative abundance scores of the bacteria comprising the polymicrobial infection. The increased sensitivity and the relative abundance scores are helpful in guiding clinical decision making. It is appropriate to perform prospective studies that compare culture and molecular methods of bacterial identification and their impact on clinical decision making and patient outcome.

This study suggests that bacteria with higher relative abundance scores (as determined by molecular testing) are more likely to be detected by culture, and bacteria with lower relative abundance scores are less likely to be detected by culture. Culture testing is not as sensitive as molecular testing, so culturing often fails to detect bacteria, even bacteria that are present in a high relative abundance. Bacteria that are present in high relative abundance are likely of high clinical relevance, including the bacteria that are not detected by culture. By considering the relative abundance scores when interpreting molecular results, clinicians can gain a clearer picture as to which bacteria may be most clinically relevant.

Prospective studies comparing patient and wound outcomes when using culture testing versus molecular microbial testing have not yet been performed, but retrospective studies suggest molecular microbial testing can improve patient outcomes [16, 17].

DDR is a Medical Laboratory Scientist and a Clinical Pathology resident. SBC is a Ph.D. biologist and statistician. YS is a Ph.D. molecular scientist. EJR is a Ph.D. computer scientist and bioinformatics researcher. RDW is a medical doctor and key opinion leader in the wound care community. He focuses his efforts on improving patient care by developing and using biofilm-based wound care practices.