Metagenomic Analysis of the

Microbiome Associated with

Single-Use Glove Manufacturing

Barry Michaels1,
Jenna Brooks-McLaughlin2, Ryan McLaughlin2, Katherine
1 Sandoval3, Steve Ardagh4
2

barrymichaels@mindspring.com

4
3

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Safety Assurance for Food Contact
Single-Use Gloves

Multi-layered Full-field
Fingerprint Test Regime
& Glove Product Conduit

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 COVID-19 Flood of
• ~100 factories Malaysia, Thailand,
defective, often bad
China, Indonesia, Sri Lanka, India,
quality or used PPE Philippines, South Korea, Singapore,
repackaged w/ >70% Pakistan, Vietnam
Global Factories Southeast Asia

Single-Use w/ Poor Sanitation &

Polluted Water Sources
• Most chemically & microbially
contaminated in SE Asia

Gloves /
year >300 Inadequate Glove Worker
Health Standards
• Child & slave/oppressive labor
conditions

Billion (40
Billion US Lack of Glove • NSF Standards inadequate, not
employed & no exam glove target
Microbiological Standards
Food consensus

Industry) Glove Microbiome

Unknown & Use
• New & unused glove production
associated microbiome unknown-&
Associated w/ False Sense un-characterized
of Purity/Cleanliness

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 Project
Origins Project Objectives:
Enable Completion of Hazard Analysis

As a Public Benefit
Corporation:
Implementation Enhancement of Microbial Preventive Controls:
of a Fingerprinting
Program: Risk
Reduction & Understand Nature of Risks to Reduce:
Contamination
Reduce foodborne
Food worker

Target Hardening resulting in food

spoilage
outbreak potential
occupational skin
disease

Preventive
Bioterrorism Agent Detection Methodology
Controls Understand Critical Defense
Target hardening
Points (CDPs)
(Blockchain)
 Si ngle-Use Glove Manufacturing Microbiome and the
Detection of Microbes of Public Health Significance
[glove wearers (hand contact), patients (in Healthcare) & food (products)]

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 Background: Cross-Contamination (C-C) & Outbreaks of
Infectious Disease Attributed to Single-Use Gloves Are the:

Food Industry C-C Health Care C-C

• 16% of Foodborne outbreaks • Hospital acquired outbreaks

traced to glove cross-
CDC NEARS EHS-Net Reported C-10 Bare-
hand & C11 Gloved-hand as Cross- contamination (Maki et al.
Contamination Contributing Factors in 1990; Patterson et al. 1991;
Foodborne Illness Outbreaks (2000-2015)
40
Okuda et al. 1995; Piro et al.
2001)
Percent (%) of Outbreaks

35
30
25
20
• 1.7M infections & 99K
15
10
deaths/year (1 in 25
5 patients)
0
1995 2000 2005 2010 2015 2020 • Increasing antimicrobial
C11 Gloved-Hand C10 Bare-Hand
resistance (AMR)

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 Glove Manufacturing:
Technical & Human Components

From glove mold “inside” becomes Potential unsafe, unsanitary, and

“outside” and visa-versa unfair working conditions

Note: there
are 2
washing or
leaching
stages
(before &
after “Off
mold”)
where
significant
microbial
filth can be
introduced

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 Glove Microbial Hazard Origins: Wastewater
Recycling

Water: Zhang & Liu 2019

Approximation / Glove Production Process:
• Former cleaning>Former drying> (13 sub-processes)
• Coagulant dip>Coagulant drying >(Latex) (Nitrile) (PVC) dip>Wet gel
drying>Vulcanizing in oven>cooling & beading at rim
1
• >pre-cure leaching (hot/warm water tank series)
• >Polymer dip>Polymer drying Glove Chemical Leachate
• >Post-cure leaching (hot/warm water tank series) 2

• >Stripping from Former>Offline chlorination >

• Hot Air Drying, Packing & Packaging Aerobiology, Worker Handling
Note: while this a 96% nitrile glove study, data from
& Packaging Microbiome
the vinyl sample indicative of similar issues.
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 Study Goal #1
Glove Contamination Resulting in Food Spoilage*

(Spoilage Continuum from Blackburn 2006)

*Spoilage Continuum from Blackburn 2006. IAFP 2021 Eagle Protect PBC 9
 Study Goal #2:
Identify Potential Contamination from
Foodborne Pathogens & Indicators (Outside Gloves)

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 Study Goal #3
Identify Agents/Occupational Skin Disease (OSD) &
Opportunistic Pathogens (Inside Gloves)

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 Study Goal #4
A) Who Are They, B) What Are They Doing & C) Where
did they come from ?
Single-Use Glove Manufacturing Microbiome

(Ecological factors driving microbial abundance & distribution from Dickey et al. 2021)
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 Culture & Enrichment Media 16S
Utilized:
Glove Rinse Fluid: PBS w/ Tween (0.1% & 0.5%)

Most Probable Number (MPN) Determination &

Enrichment:
• Tryptic Soy (TSB)
• Brain Heart Infusion (BHI)
• Fraser (FBB - Listeria selective)
• Enterobacteria Enrichment (EE)
• Actinomyces (Actino B)
• M-Green Yeast & Fungi
Shotgun WGS

Vigorous rinsing/stomachering of 1) insides & 2) outsides of gloves w/ 6 broth

enrichment followed by heat treatment (or not) to elicit spore forms as well as
aerobic/anaerobic incubation leads to a viability-based metagenomic analysis

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Glove Study 1) Understand Glove
Count Distribution &
2) Use 16S to
Determine If
Methodology Compare Early Work
(Berthelot et al. 2006
Diversity &
Richness Differs,
& Hughes et al. Inside vs. Outside
2013) (16 brands)
a) 26 glove brands
(25 nitrile & 1 vinyl) 4) Understand
Origin(s) of
3) Compare
b) Insides (I), Outsides (O), & Abundance &
Communities
Represented &
I + O combined Diversity Potential Hazards
Information, Shotgun (Food & Healthcare)
WGS vs. 16S (26 w/ venture into
c) n= 1,300 (50 gloves pools) brands)
& multiple sample pools Resistomes of
found microbes

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Microbial Densities (CFUs)Identified on Gloves via the Lens of
MPN TSB Sample Culture Methodology (repeated for other
enrichment broths utilized w/ DNA pooling)

250

200

150

100

50

Inside Aerobic Outside Aerobic Inside Anaerobic Outside Anerobic

Inside Aerobic HT Outside Aerobic HT Inside Anaerobic HT Outside Anerobic HT

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 Mean Bacterial Counts per Glove in Colony Forming Units
(CFU) w/ Samples A-Y Nitrile Glove Brands & Z Vinyl
Based on MPN Glove 400

Count Data : >0.5 350

million total CFU
300
subjected to
overlapping 250

enrichment media & 200

serial dilutions.
150
Positives Pooled from
ea. Glove Sample w/ 100

DNA Extracted 50
followed by 16S &
0
Shotgun sequencing

W
H

Q
A
B

M
N
C
D

R
S
T
U

Z-vinyl
E

Y
F
G

I
J

O
P
Inside /glove Outside /glove Mean count

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 Glove Count Distribution Modeling of Log 10 Results from
Current Study (2021) where Cox Box Plot indicates that
CFU numbers of 104 to 106 are possible per 100 count box

Glove Box (100 glove)

Count Distribution (Log-
normal) from 26 brands
(25 nitrile & 1 vinyl)
Wessa P. 2017. Maximum-likelihood
Normal Distribution Software (v1.2.1)
Office for Research Development &
Education, URL
http://www.wessa.net/rwasp_fitdistrpoiss
on.wasp/

Log10 CFU/100 glove box

Bacterial count ranged from 102 to ~3.5 x 104 CFU/100 glove box w/ geometric mean = 3.11 Log10.

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 Bacterial Count Distributions & Inadequate Standards
Based on Microbial Fingerprints Identified in this Study

NSF 2005 Standard: <1,000 CFU/glove

Food Gloves (no microbial taxa limitations)
Krammer & Assadian 2016: <10,000 CFU
Exam Gloves (no E. coli or S. aureus)

NSF 2005
Kramer &
Assadian 2016

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Metagenomic Procedures (concentration via filtration followed by typical workflow):
16S rDNA & Shotgun sequencing Illumina MiSeq Reagent V3 (600-Cycle) & Reagent V2 (300-Cycle)
Classifiers, aligners & profilers:
16S- V3-V4 QIIME2 v2019.4 & SILVA v123.
ITS1 (Fungi)- Vsearch & UNITE v02.02.2019
Shotgun WGS- Blast NT & Sourmash

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 2019 Identified Possible Glove Two-sidedness Community Diversity Differences:
Inside to Outside of Gloves w/ Shotgun WGS (inside = user safety; outside = food or patient safety)
100
Only 20% Staphylococcus petrasii subsp. pragens is
Paen ibacillus yonginensis
Paenibacillus timonensis 2301 032
Correspondence Paen ibacillus thermophilus
Paen ibacillus puldeungensis
Paenibacillus polymyxa TD9 4
90
between Inside & Paen ibacillus pini JCM 1 6418
Paenibacillus phoenicis
Paen ibacillus lentus
Outside of Glove Paen ibacillus filicis
Paenibacillus fae cis
Paen ibacillus cookii
80 Paen ibacillus chiben sis
Paenibacillus barengoltzii NBRC 1 0121 5
43 CFU/ Paen ibacillaceae
Oceanobacillus oncorhynchi subsp. incaldanens is
Natranae robaculum magadiens e
glove Lactobacillus futsaii JCM 17 355
Jeotgalibacillus malaysie nsis
70 Fontibacillus
114 CFU/ Bacillus tropicus
Bacillus timone nsis

glove Bacillus s ubtilis YF001

Bacillus paranthracis
Bacillus paramycoides
60 Bacillus oryz ite rrae
Bacillus oceanisediminis 2 691
Bacillus mobilis
Bacillus megaterium WSH-002
Bacillus manliponen sis
Bacillus infantis N RRL B-14 911
Bacillus filamen tosus
50 Bacillus cytotoxicus NVH 391-98
Bacillus ciccens is
Bacillus cereus W
Bacillus atrophaeus UCMB-5137
Bacillus aryabhattai B8W22
Sporolactobacillus nakayamae subs p. racemicus
40 Paenibacillus xylanilyticus
Paen ibacillus translucen s
Paen ibacillus puris patii
Paenibacillus pinihumi DSM 23905 = JCM 1641 9
Paen ibacillus dongdonen sis
Paen ibacillus bovis
30 Paenibacillus barcinonensis
Paen ibacillus amylolyticus
Paen ibacillus
Lys inibacillus composti
Halolactibacillus miurensis
Bacillus z hangz houensis
Bacillus xiame nen sis
20 Bacillus s wezeyi
Bacillus s tratos phe ricus LAMA 585
Bacillus s porothermodurans
Bacillus s onorensis N BRC 10 1234 = K CTC 1391 8
Bacillus s olimangrovi
Bacillus s olani
10 Bacillus niabens is
Bacillus maritimus
Bacillus marisflavi
Bacillus malikii
Bacillus lichen iformis WX -0 2
Bacillus kore ensis
Bacillus idrie nsis
0 Bacillus haynesii
Bacillus halotolerans
Inside Outside Bacillus crescens
Bacillus capparidis

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 Glove Two-Sidedness: 16S Chao1 vs. PD (Faith) Richness Diversity Plot
Species Richness vs.
Phylogenetic Diversity
175
(Inside from Outside) w/
Simpson & Shannon alpha
150
diversity outcomes
16S Amplicon sequencing
study: Insides & outsides 125

Species Richness (Chao1)

(sampled independently)
from 15 brands of Nitrile & 100
1 brand of Vinyl gloves.
Significant differences in
brands. Overall identified 75

differences in phylogenetic
diversity and species 50
richness (mainly in beta
diversity, contributed by the
divergence in conditions 25

during manufacture
(forming, vulcanization 0
process, leach tank 0 2 4 6 8 10 12 14
temperatures, chemical Phylogenetic Diversity (Faith)
residues & cleanliness) Species Rich Out Species Rich In

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 Alpha Diversity Variability Insides to Outsides of Gloves
(conflicting by brands w/ trend toward slight overall increase)

Simpson Alpha Diversity Shannon Alpha Diversity

5

0.98
4.5

Pairwise Comparison 0.93 4

of Alpha Diversity
(Richness &
Evenness) in 3.5
Microbiota Data for
Consecutively 0.88
Collected Samples 3
from the Same Glove
Brands (Inside to
Outside) 0.83 2.5

2
Inside Outside
0.78
Inside Outside A-I C-I E-I F-I G-I
A-I C-I E-I F-I G-I H-I H-I J-I U-I V-I W-I
J-I U-I W-I X-I Y-I Z-I X-i Y-I Z-I
Beta Diversity (similarity (or not) between multiple communities & samples):
Ordination Multivariate Analysis-Principal Coordinates Analysis (16S)
Vector arrows show inside of glove to outside of glove for same brand with cluster
A, an 8 glove inside cluster having B, C, D & E as mixed mainly outside clusters.
Note: corroborating results obtained from MDS, UMAP & NMDS Ordination

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Microbial Life Identified on Single-Use Gloves
Phylogenetic Tree Depiction
(16S = yellow highlighted; Shotgun WGS = arrows)
(figure from: Zhu et al. Phylogenomics of 10,575 genomes (2019)

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 Taxonomic Representation of Actinobacteria,
Firmicutes, Bacteroidetes & Proteobacteria
Taxonomic with Shotgun WGS & 16S Amplicon
Sequencing
Representation 100

percent (%) of Phyla 90

Percent (%) of Taxonomic Groups Identified

Identified on Gloves 80

70
Common to
60
Shotgun & 16S 50
Sequencing 40
(99.97% & 87.7% of 30

total abundance 20

Shotgun & 16S 10

respectively) 0
Phylum Class Order Family Genus

Shotgun WGS 16S Amplicon

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Word Cloud Assembled Genera & species Based on Frequency of
Occurrence (Shotgun) in Glove Samples
[method from Bilen et al. 2018 (human microbiome))]

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Extremophiles
Found on
Gloves-
Origins &
Lifestyles
(16S & Shotgun):
• Extremophiles:
• Hot springs
• Serpentine
formations
• Hydrothermal &
geothermal vents
• Brine & hypersaline
• Soda-lake taxa
• Acid mine drainage
• Seafloor sediment
• Anoxic
environments
• Desert & arid
environs
(figure from Merino et al. 2019) 2
7
 Microbes Found on Gloves- Origins & Lifestyles: Agricultural
Microbiota: livestock animals & fish (internal & external), Plant
phyllosphere & rhizosphere
(figure from Ikeda-Ohtsubo et al. 2018)

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Glove Microbes Identified-Origins
& Lifestyles:
Some of Most Polluted
Waterways Sources in SE Asia
(abundantly contaminated
wastewater effluents)
• Poor water quality start
• Factory wastewater
• Sewage & septic failures
• Livestock & plant wastes
• Fertilizer run-off
• Dense feed lot waste
• Forest & wildlife wastes
• Household waste
• Urban run-off pollution
(figure from 2018 AFP PHOTO/Timur
Matahari)

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Identified Chemical Degradation
Cycle Species on Gloves:
• Polymers- polyethylene, PVC, natural • Herbicides
rubber isoprene
• Quinclorac
• Nitriles-degrading from acrylic fibers,
plastics & nitrile rubber • Thiobencarb
• acrylonitrile into acrylamide & • 2,4-D (2,4-
acrylic acid Dichlorophenoxyacetic acid)
• free cyanide (CN-) & cyanates • pentachlorophenol types
• Styrene degrading into nitrile • phenoxyalkanoic acid types
• Vinyl Glove Plasticizers • Insecticides
• phthalates (PAEs) • chlororganic pesticides
• Phenols • hexachlorocyclohexane
• chlorinated phenols • Textile dyes & colorants
• polychlorinated biphenyl • Environmental toxic chemicals
(PCBs) • methyl tert-butyl ether (MTBE)
• Benzoates & • tert-amyl methyl ether (TAME)
Dihydroxybenzoates
• hexabromocyclododecane
• Polycyclic aromatic hydrocarbons (HBCD) flame retardant
(PAHs)
• dibenzo-p-dioxin or
• BTEX compounds (benzene, dibenzofuran
toluene, ethylbenzene &
xylene) • trichloroethane (TCE)
• naphthalene, • Alcohols (ethanol, methanol,1-
propanol, tert-butyl etc.)
• carbon tetrachloride
• acetone • Heavy metal tolerance
carrying Multidrug Resistance
• Chlorinated ethenes (CEs) (MDR) Genes
• cis-1,2-dichloroethylene
(cDCE)

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Heavy Metal Tolerance Reported in Glove Sample Microbial
Strains w/ Links to Antibiotic Resistance Genes
(figure: Valsamatzi-Panagiotou et al. 2020; table modified from: Baker-Austin et al. 2006)

Occurrence of Heavy Metal Tolerant Strains

Pb
B
Se
Mo
Cr
Ni
Cd
Hg
Ag
Co
Mn
Zn
N,O,P A H J
Cu
Glove Sample or Pool ID

Resistance mechanism Metal ions Antibiotics

Reduction in permeability As, Cu, Zn, Mn, Co, Ag Cip, Chlor, ß-lactams
Drug and metal alteration As, Hg ß-lactams, Chlor
Drug and metal efflux Cu, Co, Zn, Cd, Ni, As Chlor, ß-lactams
Alteration of cellular target(s) Hg, Zn, Cu Cip, ß-lactams, Rif
Abbreviations: Chlor, chloramphenicol; Cip, ciprofloxacin; Rif, rifampicin
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 Antibiotic Resistance
Genes Identified to 14
Key Antibiotic Classes: 13
(Shotgun sequence data search via

Percent of Total Antibiotic Resistant Genes in Samples

AGR-ANNOT; CARD; NCBI BARRG; 12
ResFinder; VFDB) 11 mmpL3

10 emrE
Antibiotics Resistance Genes in Sequences:
drra
9
LmrS
Penam class:
8
Methicillin (MecI) mprF
Carbapenem (BlaZ) 7 ileS
FosB
Rifamycin class: 6
Rifampin (rpoB2,rpoB) CAT
5
Aminoglycoside class: ampC
4
Kanamycin/Colistin etc. (LmrS, drra, mprF) BlaZ
3 rpoB
Macrolide class: 2 rpoB2
Erythromycin etc. (emrE, mmpL3)
MecI
Phenicol class: 1
Chloramphenicol (CAT) 0
A B E F G H J U V W X Y Z
Fosfomycin class: (FosB)
Glove Sample ID
Mupirocin class: (ileS)

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 Kingdom Fungi Represented on Gloves as per ITS1 ID
• Key Distinctive Characteristics: Ascomycota:
• Directly on hyphae Ascomycota: Pezizomycotina
• Inside sporangia Saccharomycotina
• Fruiting bodies
Neocallimastigomycota

Yeast Saccharomyces sp.

Orpinomyces sp.
Mucoromycota
Basidomycota

Penicillium hyphae
Orbiliales, Cladosprium (5 spp.),
Pseudorobillarda, Exophiala,
Chromocleista, Penicillium (13
spp.), Aspergillus, Ochrolechia,
Leveilula, Phyllactinia, Acidea,
Rhizopus sp. sporangia Phialocephala, Oidiodendron,
Claussenomyces, Plectosphaerella,
Knoxdaviesia, Epichloe
6 Genera: Bannoa, Tomentella, Cystogaricus, Inocybe, Dermocybe, Lacrymaria
Microorganisms Causing Spoilage Identified on Gloves

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Pathogens Foodborne (Based on NCBI BLAST nt 600 bp “Best-
hit”) Dermal pathogens an overlap w/ many additional species;
Opportunistic pathogens-too numerous to list)
• Bacillus anthracis str. A16
• Bacillus anthracis str. Turkey32
• Bacillus anthracis str. Vollum
• Bacillus anthracis str. Western North
• Bacillus cereus 03BB102
• Bacillus cereus ATCC 14579
• Bacillus cereus B4264
• Bacillus cereus biovar anthracis str. CI
• Bacillus cereus D17
• Bacillus cereus D17 (1651-00)
• Bacillus cereus E33L
• Bacillus cytotoxicus NVH 391-98
• Bacillus mycoides Rock3-17
• Bacillus paramycoides
• Bacillus paranthracis
• Bacillus tropicus
• Listeria monocytogenes XFL0605
America USA6153 • Staphylococcus aureus subsp. aureus
• Enterococcus faecalis
• Vagococcus teuberi
• Citrobacter freundii UCI 32
• Escherichia coli ZH193
• Salmonella enterica subsp. enterica serovar Senftenberg str.
361154004
• Pseudomonas aeruginosa
• Cladosporium cladosporioides
• Penicillium griseofulvum
• Penicillium marinum
• Penicillium thymicola
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Results: Studies 2 & 3 16S Amplicon
& Shotgun WGS Metagenomics
• Diversity Overall: >900 unique taxa, 229 genera and 245 species (+ >150 subsp., strains. The taxa
represented 21 phyla from, Archaea (2), Bacteria (14), Fungi (4) & Viruses (1).
• Dominant Phyla (Represented in Both 16S & Shotgun WGS:
Shotgun Genera:
• Actinobacteria: 7 Genera [Flaviflexus, Cellulomonas, Curtobacterium, Clavibacter,
Leucobacter, Mirococcus, Cutibacterium]
• Bacteroidetes: 1 Genus: [Prevotella]
• Proteobacteria: 17 Genera [ά: Paracoccus, Agrobacterium, β: Achromobacter, Alcaligenes,
Bordetella, Acidovorax, Diaphorobacter γ: Aeromonas, Citrobacter, Escherichia, Raoultella,
Salmonella, Alcanivorax, Acinetobacteria, Pseudomonas, Stenotrophomonas, Xanthomonas
• Firmicutes: 25 Genera [Bacillus {49}, Paenibacillus {19}, Anoxybacillus, Exiguobacterium,
Virgibacillus, Listeria, Lysinibacillus, Fontibacillus, Brevibacillus, Thermobacillus,
Jeotgalibacillus,Kurthia, Planococcus, Solibacillus, Ureibacillus, Sporolactobacillus,
Jeotagalicoccus, Staphylococcus {6}, Streptococcus, Enterococcus, Vagocococcus,
Lactobacillus, Clostridium, Ruminiclostridium, Natranaerobaculum]
• Domain Eukarya: Fungi- 26 Genera [Orpinomyces, Bannoa, Tomentella, Cystogaricus,
Inocybe, Dermocybe, Lacrymaria, Rhizopus, Saccharomyces, Orbiliales, Cladosprium {5},
Pseudorobillarda, Exophiala, Chromocleista, Penicillium {13}, Aspergillus, Ochrolechia,
Leveilula, Phyllactinia, Acidea, Phialocephala, Oidiodendron, Claussenomyces,
Plectosphaerella, Knoxdaviesia, Epichloe

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Significance of Study: (Food/Healthcare first in-depth study, environment
selects a resistome w/ frank & opportunistic pathogens presenting hazards to
wearer, patient & food. Contamination spoilage, food waste & bioterrorism issues.

Detailed SUG glove microbial testing has been limited to date (Deep sampling & Identification). Testing conducted by
earlier infection control researchers identified numerous microorganisms our studies confirmed are commonly present.
Paired w/ Count Distributions developed hazards are presented in both food & healthcare industries.

SUG manufacturing factories are often sited on contaminated & polluted waterways with conditions required for
production selecting microbial taxa for adaptation to degrading harsh chemicals and heavy metal tolerance. The
associated Microbiome develops an “Antibiotic Resistome” as evidenced by the resistance gene identified in glove
shotgun sequences.

In examining the microbial population diversity for insides and outsides of gloves a two-sidedness of SUGs was identified
(based on beta diversity regression methodology). This study identifies hazards for wearers due to dermal pathogens as
well as patients or food being handled. This is a result of negative aspects of the surviving flora as toxin and virulence
factor genes were also identified. Considering the hazards presented appropriate microbial standards seem to be lacking.

In addition to frank & opportunistic pathogens the glove manufacturing microbiome is also joined by a wide range of
spoilage genera and species. The concept of glove contamination impacting down stream food waste has not been
factored into issues of global food scarcity & sustainability.

Several pathogenic bacteria having Bioterrorism potential were identified using methods developed to study viable glove
populations. The Detection Methodology developed needs to be further refined along with systemic improvement in
glove manufacturing standards.

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