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 iso....

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, ....

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

List of materials used in this article

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