research ▲ JOURNAL OF WOUND CARE VOL 18, NO 8, AUGUST 2009 317 R.D.Wolcott, MD, Medical Director, Southwest Regional Wound Care Centre, Lubbock, Texas, US; V. Gontcharova, 1 MS, Bioinformatician; Y. Sun, 1 PhD, Molecular Microbiologist; A. Zischakau, 1 MS, CLSp(MB), Molecular Diagnostic Technician; S.E. Dowd, 1 PhD, Director of Medical Biofilm Research; 1 Research and Testing Laboratory, Lubbock, Texas, US. Email: sdowd@ pathogenresearch.org Bacterial diversity in surgical site infections: not just aerobic cocci any more I t was generally thought that Gram-positive aer- obic cocci, particularly Staphylococci, are the pri- mary cause of surgical site infections (SSIs). 1,2 Indeed, the isolates most commonly identified in SSIs using culture-based methods are Staphy- lococcus aureus, coagulase-negative staphylococcus, Enterococcus spp. and Escherichia coli. 1-3 However, this does not take account of the fact that the biofilm phenotype, with its multispecies communities, is the natural state of existence for most types of bacterium, and that over 99% of bac- teria identified in every environment are organised in biofilm communities. 4 Indeed, it is now becom- ing accepted that not only are biofilms the preva- lent cause of chronic wound infection, 5 but also that culture methods cannot identify biofilms. 5-7 SSIs have many of the characteristics of chronic wounds specifically and of chronic infections in general. 5,6 Their management therefore requires an understanding of biofilm phenotype bacteria. 8 Few studies have used modern molecular meth- ods to evaluate the microbial diversity in SSIs. Previ- ous studies have relied on clinical and laboratory culture methods to evaluate which organisms can be isolated in pure culture from an SSI. As Staphylo- coccus, Streptococcus, Escherichia and Pseudomonas spp. grow easily in clinical and laboratory culture media and are easily isolated, it is not surprising that they are the microorganisms most frequently associated with SSIs. However, throughout the lit- erature it is stated that, on average, fewer than 5% of all bacteria can be easily grown in laboratory culture media. Therefore, 95% of all bacteria that might be associated with or causative factors for SSIs are never isolated and so have never been identified. This lim- its our ability to treat such infections. Molecular techniques have an advantage over culture methods as they do not rely on growing limited species of bacteria in the laboratory, but instead are able to identify all microorganisms contained within a sample on the basis of their genetic material. 9-12 Examples of molecular techniques that pose an alternative to traditional bacteriological analysis are bacterial tag-encoded FLX amplicon pyrosequenc- ing (bTEFAP) and quantitative polymerase chain reaction (qPCR). bTEFAP is a molecular technology that uses genetic information to identify all bacteria contained within a wound sample. qPCR is another mo lecular detection technique that enables us to detect the specific genetic signatures of bacteria and provide relative or even absolute quantification of that genetic material. ● Objective: To evaluate the microbial diversity in chronic surgical site infections (SSIs). ● Method: Bacterial populations in 23 chronic SSIs were identified using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), which is an universal bacterial identification method.These results were then validated using quantitative polymerase chain reaction (qPCR). ● Results: bTEFAP identified two previously uncharacterised Bacteroidales in all of the SSIs and showed that it was the predominant population in the majority of these chronic wounds. Other bacteria identified included Corynebacterium spp., Peptoniphilus spp., Staphylococcus spp., Staphylococcus aureus, Serratia marcescens, Prevotella spp. and Pseudomonas aeruginosa. Rarefaction analysis of the data indicated that, on average, six genera occurred in any given SSI, suggesting that such infections are multispecies. On average, over 60% of the bacteria evaluated in the SSIs were anaerobic bacilli.The previous literature indicates that aerobic cocci predominate in such wounds. ● Conclusion: This modern molecular survey indicates that our previous understanding of which bacteria cause SSIs may be faulty. The high prevalence of anaerobic bacilli and the overwhelming predominance of two previously uncharacterised Bacteroidales suggest that such bacteria may be a leading contributor to such infections. Further research on the identification and treatment of such bacteria are warranted. ● Declaration of interest: Scott E. Dowd is director of a clinical molecular diagnostic company. surgical site infection; biofilm; Staohylococcus aureus; bTEFAP; qPCR