Abundant DNase I-Sensitive Bacterial DNA in Healthy Porcine Lungs and Its Implications for the Lung Microbiome

Abstract

Human lungs are constantly exposed to bacteria in the environment, yet the prevailing dogma is that healthy lungs are sterile. DNA sequencing-based studies of pulmonary bacterial diversity challenge this notion. However, DNA-based microbial analysis currently fails to distinguish between DNA from live bacteria and that from bacteria that have been killed by lung immune mechanisms, potentially causing overestimation of bacterial abundance and diversity. We investigated whether bacterial DNA recovered from lungs represents live or dead bacteria in bronchoalveolar lavage (BAL) fluid and lung samples in young healthy pigs. Live bacterial DNA was DNase I resistant and became DNase I sensitive upon human antimicrobial-mediated killing in vitro. We determined live and total bacterial DNA loads in porcine BAL fluid and lung tissue by comparing DNase I-treated versus untreated samples. In contrast to the case for BAL fluid, we were unable to culture bacteria from most lung homogenates. Surprisingly, total bacterial DNA was abundant in both BAL fluid and lung homogenates. In BAL fluid, 63% was DNase I sensitive. In 6 out of 11 lung homogenates, all bacterial DNA was DNase I sensitive, suggesting a predominance of dead bacteria; in the remaining homogenates, 94% was DNase I sensitive, and bacterial diversity determined by 16S rRNA gene sequencing was similar in DNase I-treated and untreated samples. Healthy pig lungs are mostly sterile yet contain abundant DNase I-sensitive DNA from inhaled and aspirated bacteria killed by pulmonary host defense mechanisms. This approach and conceptual framework will improve analysis of the lung microbiome in disease.

Dead or Alive: Deoxyribonuclease I Sensitive Bacteria and Implications for the Sinus Microbiome

“…Of the limited number of studies performed to date that looked at live versus dead bacteria the majority was performed in isolated bacterial populations or environmental samples, which makes it difficult to compare. 25,26 However, Maurice et al 27 found that Ͼ17% of bacteria in human fecal samples were propidium iodide-sensitive, and Pezzulo et al 15 found that 63% of bacteria from porcine bronchoalveolar lavage fluid samples were DNase sensitive. These striking differences indicated that the fraction of dead bacteria observed in a given microbial community may vary greatly, depending on disease state, location, sampling method, and other factors yet to be elucidated.…”

“…Viability PCR depends on cell membrane impermeability to exclude a dye, such as propidium monoazide or propidium iodide, from gaining access to the nucleus. 15,25 Damaged or dead cells that have lost membrane integrity will take up the dye, which is then covalently cross-linked to the cell's DNA. PCR amplification of this bound DNA is blocked, and, therefore, downstream detection by sequencing is prevented.…”

“…26 As an alternative to these dye-based assays, two groups recently published studies that used the enzyme DNase to differentiate live from dead bacteria. 15,26 DNase is an endonuclease that nonspecifically cleaves both single-and double-stranded DNA. As with phorbol 12-myristate 13-acetate and propidium iodide, DNase can only act on accessible DNA, which is not protected within an intact cell membrane.…”

“…The samples were incubated at 37°C for 10 minutes to allow for DNase I-mediated degradation of free DNA, followed by 75°C for 10 minutes to deactivate the DNase I. 15 Samples were immediately transferred to bead beating tubes (PowerSoil DNA Kit; MoBio Labs, Carlsbad, CA), and any remaining solution was centrifuged at 21,000 rcf for 5 minutes to collect remaining cells and to ensure complete removal of the DNase buffers. DNA was then extracted per the manufacturer's standard protocol.…”

“…This last point is frequently overlooked but critical to our understanding of the airway microbiome because the functional difference between metabolically active, replicating organisms, and dead bacteria or cell-free DNA is enormous. 14,15 In this study, we compared the quantity and quality of bacterial DNA collected by using the three most common techniques: brush, tissue biopsy, and swab. We then developed a novel deoxyribonuclease I (DNase I) dependent technique to differentiate between DNA collected from live versus dead bacteria, and to determine what percentage of the bacteria in the sinuses is alive.…”