Comprehensive Approach for Microbial Isolation from Hidradenitis Suppurativa Tunnels

Summary

We showcase a method to successfully isolate fastidious and anaerobic organisms from cutaneous sinus tracts (tunnels) in tissues excised from patients with Hidradenitis Suppurativa.

Abstract

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's dermal layers, causing significant discomfort, foul-smelling discharge, disfigurement, contractures, and scarring, which severely diminish the quality of life. HS is associated with alterations in the skin microbiome, impacting immune regulation and the skin's defense against harmful bacteria. Despite its prevalence, the contribution of the HS microbiome to disease pathology and the limited response to treatment remains largely unknown. To date, multiple 16S rRNA sequencing studies on HS tissue have only achieved genus-level granularity, identifying an increase in Gram-negative anaerobes and a decrease in skin commensals. A deeper understanding of microbial dysbiosis in individuals with HS is essential for optimizing treatment strategies. This requires a two-pronged approach to assessing the HS microbiome, including the isolation of bacterial species, which are often underutilized in translational studies focused on skin disorders. Isolating individual microorganisms from HS tissue is crucial for elucidating the role of bacteria in HS pathogenesis. Here, we highlight reproducible methods to successfully isolate anaerobic pathogens from HS tunnel tissue, providing the initial and most critical step in understanding bacterial role in HS. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address the complex microbial burden of this chronic condition.

Introduction

Hidradenitis Suppurativa (HS) is a common dermatologic condition characterized by nodules and abscesses that progress to sinus tracts (tunnels) formed within the skin's dermal and subcutaneous layers, causing significant pain, producing purulent discharge, disfigurement, and debilitating psychosocial sequelae^1,2. HS disproportionately affects women and individuals with skin of color, typically emerging in late adolescence or early adulthood². The condition's severe physical symptoms are compounded by its profound psychosocial impact, including depression, anxiety, and social isolation, which can severely diminish the quality of life³. HS tunnels formed at the advanced disease stage significantly decrease the odds of patients' response to currently approved biological therapy, and surgical excision remains the only treatment approach^4,5.

Multiple studies have characterized the microbiome associated with HS tunnels, showing a prevalence of anaerobic pathogens and a reduced abundance of cutaneous commensals^6,7,8, with recent studies identifying Porphyromonas spp. (type I) and Prevotella spp. (IV) in tunnels, among other genera⁹. Another study found variation in HS microbiome by the depth of the tissue sampling, confirming the uniqueness of microbial composition in the tunnel tissue¹⁰. In addition, dysbiosis has been shown to affect the immune response in HS, further implicating the role of microbes in the disease pathogenesis^11,12,13,14. Although prolonged systemic antibiotic treatment with clindamycin, rifampicin, and tetracyclines are in use and has shown a reduction of the number of draining tunnels in affected patients^15,16, the data on antibiotic-resistant anaerobic pathogens in HS remains unknown. Thus far, isolating bacterial strains from HS tunnels has not been reported, limiting studies on novel antimicrobial treatments and in-depth evaluation of pathogens' contribution to the disease pathogenesis of HS. Standardization of methods for pathogens isolation would not only facilitate true insights into the bacterial role in the pathogenesis of HS but also allow for translation toward more targeted and improved interventions.

Here, we highlight a method to successfully isolate microorganisms from HS tunnel tissue. Isolating individual bacterial species is a crucial, initial step in understanding their role in HS pathology. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address bacterial pathogens in this chronic condition.

Protocol

This protocol was approved by the Institutional Review Board (IRB) at the University of Miami (protocol #20200187). Informed consent is obtained from patients (n = 18, mean age ± standard deviation= 30.9 ± 9.4, 12 females, 6 males) diagnosed with HS tunnels and/or their legal guardian(s) prior to the procedure after previous discussion of research to allow for any concerns or questions to be addressed.

1. Patient sample collection

1. Prior to handling excised tissue, use strict sterile precautions to minimize the risk of contamination. The individual handling the tissue should wear a lab coat, face mask, and surgical cap and use sterile gloves and sterile instruments to handle the tissue.
2. For sterility control, submerge pre-autoclaved forceps in the anaerobic transport vial prior to processing the tissue (Table of Materials).
3. Collect skin tissue, surgically resected by means of a scalpel or CO₂ laser, from affected areas of patients diagnosed with HS tunnels and immediately place it in a sterile Petri dish using sterile forceps for further processing.
4. While at the surgery center or outpatient clinic office, identify tunnels within excised tissue by probing skin with pre-autoclaved forceps, as shown in Figure 1A.
5. Take 6 mm punch biopsies through the full-thickness skin of the identified tunnel immediately after tissue excision and immerse in the semi-solid buffered media with reducing agents in the anaerobic system transport glass vials to optimize the survival of anaerobic bacterial species for further analysis.
6. Transport the tunnel tissue in the anaerobic vials to the laboratory on ice.
7. Perform sample documentation as described below.
   1. On the day of the procedure, collect patient information, including demographics, age, self-reported ethnicity/race, and current medications, without any personal identifiers. The body sites of resected tissue accompany a body chart, as HS tunnels can occur at multiple body sites. Take photographs of excised tissue from both sides of the sample before and after tissue processing, documenting the location of the biopsy generated from a tunnel. On arrival at the laboratory, log the coded tissue samples without any personal identifiers in an electronic, secure database.

2. HS skin processing

1. Set up and plating
   1. Wear a lab coat and gloves, and tie hair to avoid contamination. Pre-warm blood agar plates, including Laked Brucella Blood agar with Kanamycin and Vancomycin (LKV) agar, Trypticase Soy agar (TSA II) 5% Sheep Blood (SB) agar, Brain Heart Infusion (BHI), and Phenylethyl alcohol agar (PEA) with 5% SB agar to 37 °C in an incubator for 10 min. Clean the biosafety cabinet with 70% ethanol prior to processing of tissue.
   2. Once the agar plates are warmed, label the base or side of the plate with the coded sample name and the date. Place autoclaved forceps, scalpels, pre-sterilized inoculating loops, and agar plates in the biosafety cabinet.
   3. Remove the 6 mm punch biopsy from the anaerobic transport vial with sterile forceps by submerging the forceps in the media. Quickly chop tissue into small pieces, approximately 1 mm² each, using a sterile scalpel. Place tissue in a sterile microcentrifuge tube containing 500 µL of Reinforced Clostridial medium (RCM) and briefly vortex.
      NOTE: Homogenization was not utilized because the samples would be exposed to the aerobic environment for an extended period, which could have led to additional aeration during the process and potential loss of anaerobic bacteria.
   4. Using a new sterile loop, perform repeated quadrant streaking of the tissue suspension containing bacterial organisms onto the TSA II 5% SB, LKV, PEA with 5% SB, and BHI agar plates.
   5. Using the remaining chopped tissue suspension, make glycerol stocks by adding 20% v/v sterile glycerol and 80% v/v tissue suspension in a cryotube. Store glycerol stocks at 80 °C. In the case of limited viability of isolated bacteria, tissue stocks can be used to repeat the isolation.
   6. Place all plates in the incubator at 37 °C in an anaerobic chamber enclosed with a CO₂ pack. Check the plates every 2-3 days, expecting growth of anaerobes 7-14 days post plating. Document colony morphologies by photographing plates.
2. Tissue banking
   1. Use an 8 mm punch biopsy to collect additional full-thickness skin samples through the tunnel and away from the tunnel for Formalin Fixed Paraffin Embedded (FFPE) sample and histological evaluation, as a larger tissue sample is more likely to capture the full tunnel.
   2. Preserve additional 6 mm punch biopsies for DNA (snap frozen), RNA (in RNA later), and protein isolation (snap frozen).
3. Colony maintenance
   1. Once colonies are observed on the original plates, photograph plates and take note of distinct colony morphology¹⁷. In general, anticipate 10-15 different colony morphologies (see Figure 1).
   2. Prepare pre-warmed LKV, BHI, PEA with 5% SB and TSA II 5% SB agar plates (Table of Materials). Label each plate label with the sample ID, location of the biopsy, and date.
   3. Using a sterile pipette tip, pick up a single colony and streak it in a zig-zag motion on the same type of plate on which it was detected. Then, using the same pipette tip with the colony as a template for a 16s rDNA PCR amplification, follow the steps below.
   4. Repeat step 2.3.3 using the colonies selected from all types of agar plates with a new sterile pipette tip and a different singular colony of the distinct morphology from the original plate. Again, immediately after streaking, use the pipette tip with the remaining colony to submerge it in a dedicated PCR plate well, as this will provide a template for PCR amplification.
   5. Repeat steps 2.3.3-2.3.4 for each distinct colony until all colonies with specific morphologies in all four types of agar plates have been streaked and corresponding PCR wells have been inoculated simultaneously.
   6. Store the newly plated agar plates in the incubator at 37 °C in an anaerobic chamber enclosed with a CO₂ pack. Once the colonies grow, ensure the purity of the isolated colony before preserving bacterial stocks (see below). If the plated colonies lack uniformity, repeat step 2.3.3. Perform the re-plating process every 4-5 days, depending on colony growth, until the colonies are uniform in appearance.
4. Identification of bacterial species by 16s rDNA colony amplicon sequencing
   1. Prepare a PCR plate with a master mix composed of 2.5 µL of 10 µM 16S rDNA V1-V3 forward (5' AGAGTTTGATTCCTGGCTCAG-3') and reverse primers (5' AGAGTTTGATTCCTGGCTCAG-3'; 10 µM), 12.5 µL of 2x Master Mix polymerase and 10 µL of microbial DNA-free water per well for a total volume of 25 µL per colony.
   2. Calculate the volume of a PCR master mix required for amplification from all selected colonies, negative control (no template), and positive control - any laboratory strain can be used; we commonly use Staphylococcus aureus USA300.
   3. Bring a PCR plate to the biosafety cabinet, covering wells to avoid contamination. Use a single colony from a sterile pipette tip for plating. Immediately resuspend the colony with rigorous swirling into the designated well with the prefilled master mix. Use the same pipette tip for both subculturing and sampling for PCR.
   4. Repeat step 2.4.3 for all singular HS colonies and positive control.
   5. Run PCR with the following protocol: 95 °C for 5 min for initial denaturation required to lyse the selected colony, followed by 40 cycles of 95 °C for 15 s, 54 °C for 1 min, use of the final 4 °C cycle is optional. A thermocycler may also be used with the same program settings.
   6. Run gel electrophoresis with amplified qPCR products to verify the size of the amplicon, which is expected to be 311 bp (Figure 2). Purify successfully amplified 16s rDNA fragment with the PCR purification kit as per the manufacturer's instructions.
   7. Send the purified PCR product for 16S ribosomal RNA Sanger sequencing using 16S rDNA V1-V3 forward and reverse primers.
5. Bacterial stocks
   NOTE: The fastidious anaerobes are known for their limited viability on the agar plates.
   1. In order to assure strain preservation prior to obtaining sequencing results, which will confirm species identity, generate agar plates from a single colony and confirm purity to generate stocks. Generate stocks directly from the agar plate in parallel with the optimization of the growth conditions in the liquid media.
   2. To prepare the stock from the plate generated in step 2.3, use a 6 mm sterile inoculating loop to pick up as many uniform colonies as possible. Then, vigorously resuspend bacteria in 800 µL of RCM with 20% sterile glycerol in cryotubes and store them at -80 °C to create a bacterial stock.
      NOTE: This step assures timely preservation of fastidious pathogens prior to obtaining characterization by sequencing data.
   3. Perform optimization of the growth in liquid culture and generation of stocks from a single colony once the classification of the strain is confirmed.

Results

In this study, we describe a protocol for the isolation and characterization of anaerobic bacteria from HS tunnels. This protocol is novel and notable for creating the potential to test the function and virulence of these bacteria using in vitro, ex vivo, and in vivo skin infection models to increase our understanding of the microbial contribution to the pathogenesis of HS. First, we identified the tunnels from resected skin by probing them with sterile forceps (Figure 1A

Discussion

In this study, we present a novel method for isolating and maintaining bacteria colonizing HS tunnels. This reproducible method will not only allow in depth characterizing of strains present in these pathologic structures, but it will also enable subsequent functional studies exploring the role of specific microbes in the pathogenesis of HS. The protocol's critical steps include tissue collection and transport in anaerobic media, which facilitates the preservation of viable fastidious microorganisms from HS tunnels. ...

Materials

Name	Company	Catalog Number	Comments
6mm punch biospy	INTEGRA	33-36	Other suppliers can be used
8mm punch biospy	INTEGRA	33-37	Other suppliers can be used
Agarose	Sigma Aldrich	A9539	Other suppliers can be used
Anaerobic Chambers	BD	260672
Anaerobic Transport Media	Anaerobic Systems	AS-911
Brain heart Infusion Agar	Anaerobic Systems	AS-6426
CO2 gaspak	BD	260678
Difco Reinforced Clostridial Medium	BD	218081
Glycerol	SIGMA	G5516-1L	Other suppliers can be used
Hard shell PCR plates	BIO-RAD	HSP9601	Other suppliers can be used
Incubator	VWR	Symphony	Any callibrated incubator can be used
Inoculation loops	VWR	76544-926	Other suppliers can be used
LKV agar	HARDY Diagnostics	A60
Microbial DNA-Free Water	Qiagen	338132
Nunc CryoTube	Thermo scientific	377267	Other suppliers can be used
PCR (CFX Connect Real Time System)	BIO-RAD	CFX Connect Optics Module	Regular Themocycler can be used
PEA agar	HARDY Diagnostics	A93
Q5 High Fidelity 2X Master Mix	BioLabs	M0492S
QIAquick PCR Purification Kit	QIAGEN	28104
Reinforced clostridia media	BD	218081
Thin Forceps	Millipore Sigma	F4017	Other suppliers can be used
Trypticase Soy Agar (TSA II) with 5% sheep blood	Thermo scientific	221261