Synanthropic spiders, including the global invasive noble false widow Steatoda nobilis, are reservoirs for medically important and antibiotic resistant bacteria

Abstract

The false widow spider Steatoda nobilis is associated with bites which develop bacterial infections that are sometimes unresponsive to antibiotics. These could be secondary infections derived from opportunistic bacteria on the skin or infections directly vectored by the spider. In this study, we investigated whether it is plausible for S. nobilis and other synanthropic European spiders to vector bacteria during a bite, by seeking to identify bacteria with pathogenic potential on the spiders. 11 genera of bacteria were identified through 16S rRNA sequencing from the body surfaces and chelicerae of S. nobilis, and two native spiders: Amaurobius similis and Eratigena atrica. Out of 22 bacterial species isolated from S. nobilis, 12 were related to human pathogenicity among which Staphylococcus epidermidis, Kluyvera intermedia, Rothia mucilaginosa and Pseudomonas putida are recognized as class 2 pathogens. The isolates varied in their antibiotic susceptibility: Pseudomonas putida, Staphylococcus capitis and Staphylococcus edaphicus showed the highest extent of resistance, to three antibiotics in total. On the other hand, all bacteria recovered from S. nobilis were susceptible to ciprofloxacin. Our study demonstrates that S. nobilis does carry opportunistic pathogenic bacteria on its body surfaces and chelicerae. Therefore, some post-bite infections could be the result of vector-borne bacterial zoonoses that may be antibiotic resistant.

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Synanthropic spiders,
including the global invasive noble
false widow Steatoda nobilis, are
reservoirs for medically important
and antibiotic resistant bacteria
John P. Dunbar1,5*, Neyaz A. Khan2,5, Cathy L. Abberton3, Pearce Brosnan3,
Jennifer Murphy3, Sam Afoullouss4, Vincent O’Flaherty2,3, Michel M. Dugon1 & Aoife Boyd2
The false widow spider Steatoda nobilis is associated with bites which develop bacterial infections
that are sometimes unresponsive to antibiotics. These could be secondary infections derived from
opportunistic bacteria on the skin or infections directly vectored by the spider. In this study, we
investigated whether it is plausible for S. nobilis and other synanthropic European spiders to vector
bacteria during a bite, by seeking to identify bacteria with pathogenic potential on the spiders.
11 genera of bacteria were identied through 16S rRNA sequencing from the body surfaces and
chelicerae of S. nobilis, and two native spiders: Amaurobius similis and Eratigena atrica. Out of 22
bacterial species isolated from S. nobilis, 12 were related to human pathogenicity among which
Staphylococcus epidermidis, Kluyvera intermedia, Rothia mucilaginosa and Pseudomonas putida are
recognized as class 2 pathogens. The isolates varied in their antibiotic susceptibility: Pseudomonas
putida, Staphylococcus capitis and Staphylococcus edaphicus showed the highest extent of resistance,
to three antibiotics in total. On the other hand, all bacteria recovered from S. nobilis were susceptible
to ciprooxacin. Our study demonstrates that S. nobilis does carry opportunistic pathogenic bacteria
on its body surfaces and chelicerae. Therefore, some post-bite infections could be the result of vector-
borne bacterial zoonoses that may be antibiotic resistant.
Bacterial infections represent a major threat to human health. For example, typhoidal Salmonella causes 27
million annual cases of typhoid fever resulting in 223,000 deaths1, and non-typhoidal Salmonella is responsible
for over 93 million cases of gastroenteritis leading to 155,000 annual deaths2. Bacterial infections contribute
signicantly to sepsis3 and in 2017, 49 million cases of sepsis resulted in 11 million deaths worldwide. Antibi-
otic resistance further increases the threat to human health with drug-resistant bacteria causing 700,000 annual
deaths worldwide4. According to the World Health Organization’s (WHO) global action plan on antimicrobial
resistance, it is essential that antibiotic resistance is tackled across every contact zone between humans and the
environment5. Contamination of human dwellings, and more specically food and water storage facilities, is a
major issue1. As such, identifying the source of contamination is crucial for reducing the spread of pathogens.
Synanthropic animals (wildlife associated with human habitats) can be major reservoirs and vectors of patho-
genic bacteria. Wild, domesticated and captive animals can be colonised by bacteria and act as reservoirs6,
transmitting pathogens through physical contact, including bites, stings and scratches7. For example, rats have
historically caused epidemics and rat-borne zoonotic pathogens are once again increasing across Europe8. How-
ever, some animal groups that can potentially spread pathogenic bacteria in and around human habitats are oen
overlooked. Recently, venomous snakes were identied as reservoirs for Salmonella with potential to contribute
to the health crisis through shedding contaminated faeces around homes and vectoring bacteria during bites1,9.
OPEN
Venom Systems & Proteomics Lab, School of Natural Sciences, Ryan Institute, National University of Ireland
Galway, Galway, Ireland. Discipline of Microbiology, School of Natural Sciences and Ryan Institute, National
University of Ireland Galway, Galway, Ireland.        
National University of Ireland Galway, Galway, Ireland.       
Natural Sciences and Ryan Institute, National University of Ireland Galway, Galway, Ireland.
These authors
contributed equally: John P. Dunbar and Neyaz A. Khan. *
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Moreover, a recent study demonstrated that bacteria can survive within the venom and venom glands of snakes
and spiders10.
Spiders occupy a varied range of synanthropic niches11. ey eat a diverse range of prey, with some capable
of catching and consuming large arthropods, sh, lizards, snakes, birds, rodents12–16 and medically important
pests, including mosquitos and house ies17–19. Some will readily feed on carrion20–22. Wild caught specimens of
Steatoda nobilis were observed by us feeding on dead prey for up to eight days in laboratory conditions (unpub-
lished data). e innate immune system of arthropods protects against pathogenic microbes23–25; however, once
dead, the microbes are free to thrive and multiply on the corpse of their host. It is inevitable that spiders will
encounter microbes through the environment or through feeding, especially on carrion. e potential therefore
exists for spiders to harbour virulent bacteria26,27 and they have been implicated in bite cases that subsequently
led to bacterial infections28.
e clinical manifestations arising from spider bites (araneism) are diverse29–33. For example, necrotic ara-
neism (necrosis resulting from spider bite) is most commonly documented from bites by members of the Lox-
osceles genus, though infrequently other species are involved26,30,34–37. Bacterial infection following a spider bite
could potentiate prolonged and debilitating pathologies26. Indeed, a study showed the presence of Clostridium
perfringens in the venom and on the chelicerae of Loxosceles intermedia. When C. perfringens was conjugated
with L. intermedia venom and injected into rabbits, their synergism increased the size of the dermonecrotic
lesion26. is synergistic activity however has not yet been proven in humans38. e implication of the spider
as the source of these bacterial infections remains controversial. Spiders generally avoid humans and bite only
as a defensive response to being trapped39. e spiders are then oen crushed, escape, or are captured using
non-sterile methods, and as a result, comprehensive microbiological analysis is not possible. A previous study
that identied bacteria on Tegenaria agrestis (hobo spiders) deemed them to be non-pathogenic40. e authors
argue that infections associated with spider bites are typically caused by bacterial species commonly found in
the environment and on human skin38,40. Moreover, spider venoms are considered a rich source of antibacterial
peptides41 leading to the proposal that these are sterile environments that neutralise bacteria. In this scenario,
infections are secondary to the spider bite itself9,10,38,40,42. We therefore face a conundrum in determining if infec-
tions are caused by opportunistic bacteria already present on the skin (secondary infections) or are vectored
directly from the bite via the chelicerae (vector-borne bacterial zoonoses).
e noble false widow spider, Steatoda nobilis, has expanded its range across Europe43, (including Ireland12,44,45
and the UK43), through Western Asia46,47, and the Americas43,48–53. is species is increasingly linked to medically
signicant bites to humans, especially in Ireland and the UK29,48,50,53,54. As range expansion continues, so will the
increase in bite cases43. Envenomation symptoms of S. nobilis bites include prolonged moderate to intense pain,
swelling and erythema, piloerection, diaphoresis, facial ushing, feverishness, vasodilation of blood capillaries,
and minor necrosis29. Two native species of spiders, Amaurobius similis and Eratigena atrica, are commonly
found in and around houses and gardens throughout Europe. While both species are capable of biting humans,
they are not a common source of complaint by the general public. Irish and British media regularly report on
alleged bites by S. nobilis and a BBC report attributes one death to bacterial infection resulting from the bite55. In
some media reports, the victims were said to be unresponsive to antibiotics, indicating a potential involvement
of antibiotic resistant bacteria. However, media reports typically lack conclusive evidence of spider bites. is led
Hambler to call for an urgent evaluation of the potential risk of bacterial transmission from bites by S. nobilis55.
In an unpublished case series involving conrmed S. nobilis bites currently being assessed by the authors, three
victims were treated for subsequent mild to debilitating bacterial infections, including cellulitis and dermatitis.
One victim required hospitalisation and an aggressive course of intravenous antibiotics.
False widow spiders (genus Steatoda), like the closely related black widow spiders (genus Latrodectus), can
occasionally subdue small vertebrates13,16,27,56,57 as they possess a fast-acting neurotoxic venom13,58–60. It is the
presence of α-latrotoxin that can induce neuromuscular paralysis and death in humans following envenomation
by Latrodectus species59. e venom protein composition of S. nobilis was recently characterised and revealed
that approximately two-thirds of the venom is composed of Latrodectus-like toxins, including the most powerful
toxin classes, i.e. α-latrotoxins, α-latroinsectotoxins, and δ-latrocrustotoxins58. Also present are the enzymes (e.g.
metalloproteases, serine proteases, and chitinases) that are thought to cause tissue damage and thereby facilitate
spread of venom toxins into the prey. In high concentrations, α-latrotoxin can cause localised cell death and,
when potentiated by the presence of enzymes, induce necrosis58,thus providing substrate that could facilitate
bacterial virulence26.
Previous studies on Latrodectus hesperus demonstrated the potential for spiders to vector bacteria during
bite27. Chelicerae excised from 220 specimens recovered ve pathogenic antibiotic resistant bacterial species.
e microbial colonisers of S. nobilis chelicerae have never been investigated. Such a study would provide data
to (1) explain why bacterial infections are increasingly associated with bites by S. nobilis, (2) explain why some
patients are unresponsive to frontline antibiotics, and (3) determine if the etiological agent could be vectored
directly from the spider’s chelicerae or transferred from the body surface on to the area of the bite site. is could
have signicant implications for advising rst line medical sta who are treating bites by S. nobilis and help in
choosing appropriate care and treatment.
e main objectives of this study were to (1) characterise the microbiome of the non-native S. nobilis, along
with the native A. similis and E. atrica; (2) identify bacteria species residing on the body surface and chelicerae
of the spiders and (3) test the susceptibility of these bacteria to antibiotics.
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Results
Isolation and genus identication of bacteria on Amaurobius similis, Eratigena atrica and Ste-
atoda nobilis. In the rst stage of this study, we investigated the presence of bacteria on the bodies and
chelicerae of 3 spider species. 9 bacteria genera were recovered from A. similis, E. atrica and S. nobilis (Table1
and data not shown). 5 Salmonella, 1 Bacillus, 2 Staphylococcus, and 1 Escherichia species were recovered from
9 full body samples of A. similis, which included 3 Salmonella and a Staphylococcus sp. identied from bodies of
euthanised spiders. Salmonella and Bacillus spp. were also identied on the body of E. atrica and a Staphylococcus
sp. on the body of S. nobilis.
Of particular interest was the identication of 8 dierent bacteria genera on the chelicerae of these spiders
(Table1, Fig.1). Bacillus, Raoutella and Staphylococcus spp, were recovered from the chelicerae of both A. similis
and E. atrica, among which Staphylococcus spp. were predominant, occurring 7 and 9 times respectively, whereas
Paenibacillus spp. were predominant on the chelicerae of S. nobilis being present in 7 out of 8 samples. e second
most predominant genus was Bacillus which occurred in 4 samples from A. similis and E. atrica and in 3 samples
from S. nobilis. Pseudomonas spp. were recovered from A. similis and S. nobilis, Salmonella and Advenela spp.
each occurred once in A. similis and Yersinia occurred once in E. atrica.
Isolation and species identication of bacteria in the S. nobilis microbiota. To test the hypothesis
that spiders can carry pathogens and could play a role in infection following spider bites, bacteria were isolated
from the body and chelicerae of S. nobilis and the sequence of the full-length 16S rRNA gene was determined
Table 1. Bacteria genera identied on chelicerae of A. similis, E. atrica and S. nobilis. a 34 chelicerae were tested
from 3 spider species: A. similis—16; E. atrica—10; S. nobilis—8. b Number of times the genus was isolated from
a spider species.
Spider SpeciesaBacterial Genus Occurrenceb
A. similis Advenella 1
A. similis Bacillus 4
A. similis Pseudomonas 1
A. similis Raoultella 1
A. similis Salmonella 1
A. similis Staphylococcus 7
E. atrica Bacillus 4
E. atrica Raoultella 4
E. atrica Staphylococcus 9
E. atrica Yersinia 1
S. nobilis Bacillus 3
S. nobilis Paenibacillus 7
Figure1. Comparison of the bacterial community composition and relative abundances from body and
chelicerae surfaces of A. similis, E. atrica and S. nobilis. Photographs of the spider species are shown to scale.
Data of chelicerae isolates are displayed for A. similis and E. atrica, and combined data of both chelicerae and
body isolates are displayed for S. nobilis. e tables display the number of times isolates of each bacterial genus
was isolated from a spider species. e pie chart displays the relative abundance of the bacteria genera isolated.
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to identify individual isolates to species level. Streptococcus and Staphylococcus were targeted by using selective
CNA blood agar and Baird-Parker agar, respectively. Due to the increasing incidence of development in patients
of infection associated with S. nobilis bites this species was an ideal candidate for this study. 20 chelicerae, 15
full body (5 of which had been dead for 24–48h before sampling) and 2 “spider walk” samples of S. nobilis were
analysed.
Twenty-ve dierent bacterial isolates were cultured, and identied through 16S rRNA sequence analysis,
within the microbiota of S. nobilis: 17Gram-positive and 8Gram-negative bacteria. For the majority of sequences,
the percentage identity was > 99% with their respective most similar species (Table2). Among these, 100% iden-
tity was found for 3 sequences to Staphylococcus edaphicus, Staphylococcus warneri and Bacillus thuringiensis.
Two isolates displayed individual identity of 98% for Bacillus pumilus and Streptococcus anginosus, suggesting
the isolates to be closely related to these two species.
Five isolates showed haemolytic activity on blood agar: 4 Bacillus spp. and 1 Micrococcus sp. Twelve isolates
were related to human pathogenicity among which were 4 Staphylococcus spp., 3 Bacillus spp., and one each of
Rothia, Streptococcus, Dietzia, Pseudomonas and Kluyvera spp. e association with human pathogenicity for
each bacterial species was assessed using the bacterial metadatabase BacDive (Table2).
Bacteria were isolated by each of the 3 sampling methodologies: 2 species were isolated from the agar plate
with spider walks, 5 from the chelicerae and 18 from the full bodies (11 from dead spiders and 7 from live spi-
ders). e 2 bacterial species from the spider walks (Kluyvera intermedia and Staphylococcus epidermidis) were
dierent from the species found on other sites. e bacteria detected on the chelicerae were mostly distinct from
the bacterial community on the full body, except for Staphylococcus capitis which was present on both sites.
Dierences in microbiota were also observed between bodies of live and dead spiders, with a wider variety of
genera isolated from dead specimens, and primarily Bacillus spp. from live specimens.
Anti-bacterial inactivity of S. nobilis venom. To investigate the hypothesis that bacteria can be trans-
ferred from the chelicerae into the host during the bite without being killed by the venom, S. nobilis venom
was tested for its antibacterial property through Minimum Inhibitory Concentration (MIC) and agar diu-
sion assays. MIC assays were performed by testing diluted venom against pathogenic strains of Escherichia coli,
Table 2. Bacteria isolated from S. nobilis. a 37 samples tested from S. nobilis- 20 chelicerae, 15 full body
(5 euthanised) and 2 spiders walk. C chelicerae; FB full body; D dead; SW spider walk. b Pathogenicity was
dened based on bacterial metadatabase BacDive (https ://bacdi ve.dsmz.de/). “+” indicates bacterial species is
associated with opportunistic infections due to underlying acute or chronic health conditions. “−” indicates no
known association of bacterial species with infection. c (1) & (2) indicate dierent strains of same species based
on diering antibiotic susceptibility (Table3). d 98% sequence identity to S. anginosus. e 98% sequence identity
to B. pumilis.
Bacterial species SourceaGrowth on baird parker Haemolytic Pathogenicb
Pseudomonas azotoformans C − − −
Pseudomonas peli C − − −
Rothia mucilaginosa C − − +
Staphylococcus capitis (2)cC + − +
Streptococcus sp.dC − − +
Bacillus aerius FB + + −
Bacillus altitudinis FB + + −
Bacillus licheniformis FB + − +
Bacillus mycoides (1) FB + − −
Bacillus mycoides (2) FB + + −
Bacillus thuring iensis FB + − +
Micrococcus endophyticus FB − − −
Bacillus sp.eFB-D + + +
Dietzia timorensis FB-D + − +
Micrococcus luteus FB-D − + −
Paenibacillus mobilis FB-D − − −
Pseudomonas putida FB-D − − +
Rothia amarae FB-D + − −
Serratia fonticola (1) FB-D + − −
Serratia fonticola (2) FB-D + − −
Staphylococcus capitis (1) FB-D + − +
Staphylococcus edaphicus FB-D + − −
Staphylococcus warneri FB-D + − +
Kluyvera intermedia SW − − +
Staphylococcus epidermidis SW + − +
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Methicillin Resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes. Aer incubation for 24h in liq-
uid media the absorbance at OD590 for growth of each pathogen in the presence of the highest concentration of
venom (1:100) was 0.61 ± 0.09 (E. coli), 0.68 ± 0.06 (MRSA) and 0.34 ± 0.09 (L. monocytogenes), which was very
similar to growth in the absence of venom (0.61 ± 0.01, 0.57 ± 0.06 and 0.35 ± 0.03, respectively). It was neces-
sary for the venom to be diluted in the MIC assay due to limited venom availability. e agar diusion assay was
therefore deployed to assess the antibacterial activity of pure venom, as smaller volumes were sucient with this
method. Furthermore, we investigated the antibacterial ability of the venom against bacteria which are part of
the spider microbiota. Two isolates recovered from S. nobilis chelicerae were the target bacteria in the assay: the
Gram-negative Pseudomonas azotoformans and the Gram-positive S. capitis. 0.5µl undiluted venom was applied
to solid agar media spread with the bacteria. Alternatively, spiders bit the agar plate of bacteria directly. Aer
24h no zone of bacterial clearance was observed on any of the culture plates (data not shown), indicating that
the pure undiluted venom did not inhibit the growth of either species. ese data demonstrate that the venom
did not inhibit growth of either spider commensal bacteria or human pathogens, indicating that bacteria could
survive in spider venom during transfer from the chelicerae to the host during a spider bite.
Antibiotic susceptibility testing of strains isolated from S. nobilis. Antibiotic susceptibility test-
ing was performed by disk diusion assays in accordance with the CLSI standards to determine the range of
antibiotic resistance of the bacteria residing on S. nobilis and to determine which antibiotics would be the most
eective in treating infection caused by those pathogens following a spider bite. e 25 isolates were tested
against 9 antibiotics of 8 dierent classes, consisting of 8 broad spectrum antibiotics and 1 antibiotic with greater
ecacy against Gram-negative bacteria (Colistin B) (Table3). Of these 25 isolates, 10 are species listed in CLSI
guidelines, namely—Staphylococcus (5), Pseudomonas (3), Streptococcus sp. and K. intermedia. For these isolates,
resistance and susceptibility were inferred from their EUCAST breakpoints for each antibiotic. For the rest, lack
of, or a minimal (≤ 8mm), zone of clearance around the antibiotic disk was considered as resistant. Resistance to
each antibiotic was displayed by at least one isolate, except for ciprooxacin (Fig.2, Table3). Only one isolate was
resistant to chloramphenicol (S. edaphicus) or tetracycline [S. capitis (2)], whereas 9 isolates resisted nalidixic
acid and 6 were erythromycin resistant (Fig.2, Table3). 76% of isolates were resistant to at least one antibiotic
and some isolates were multidrug-resistant. Pseudomonas putida, S. capitis (2) and S. edaphicus were notable for
resistance to 3 antibiotics. All Staphylococcus isolates showed resistance to gentamicin and nalidixic acid, with
the exception of S. capitis (1) for gentamicin and Staphylococcus warneri for nalidixic acid. Dietzia timorensis,
Rothia amarae and the Streptococcus sp. isolate had identical resistance proles with resistance to nalidixic acid
and colistin only. ese data demonstrate that there is a broad range of antibiotic resistance activity amongst bac-
teria residing on S. nobilis and the choice of antibiotic treatment for infected bites requires careful consideration.
Discussion
e role of spiders in bacterial transmission has generated much debate27,33,38,40. In recent years, increasing
media reports from Ireland and the UK29,33,55 claim that victims of the noble false widow spider Steatoda nobilis
frequently suer debilitating and sometimes fatal bacterial infections55. While these reports are largely unsub-
stantiated, there have been no studies carried out to validate the true risk of bacterial infections associated with
this recently established spider.
In the rst part of this study, the microbiomes from S. nobilis (8 chelicerae (C), 1 full body (FB)), A. similis
(16 C, 6 FB) and E. atrica (10 C, 2 FB) were partially characterised, revealing diverse bacterial compositions of
9 dierent genera, most of which were detected on the chelicerae (Table1). All these bacterial genera contain
some species that are associated with human pathogenicity. Since S. nobilis is associated with bites that lead to
infections, we focused the next part of this study on the bacteria present on body and chelicerae of S. nobilis (20
C, 15 FB, 2 spider walks (Table2) and identied the bacteria to species level. In this subsequent investigation, 10
genera were recovered from body and chelicerae of S. nobilis, four in common with those found in the rst part of
this investigation. Testing the larger sample size of S. nobilis and identifying these isolates to species level allowed
us to determine their potential for pathogenicity. e bacteria identied are members of microbiota of animals/
humans and/or found in environmental settings. Eleven species are related to human pathogenicity (Table2)
and are recognised as opportunistic bacteria, among which S. epidermidis, K. intermedia, R. mucilaginosa and
P. putida are designated as class 2 pathogens. We observed dierences between bacterial communities on dead
and living spiders. Bacillus spp. were abundant on living spiders, as found in a previous study40. However, for
dead specimens only one Bacillus isolate was identied. e diversity of genera was greater on dead spiders and
included Dietzia and Serratia spp. is may be explained by the occurrence of saprophytes, such as S. fonticola, P.
putida and M. luteus, which thrive on corpses and could outcompete other bacterial species resulting in remodel-
ling of the microbiota diversity and abundance63. Dierent bacterial communities were observed between sites
of the living spiders. In contrast to full body sites, Bacillus spp. were not recovered from chelicerae or spider
walks, indicating they probably reside more abundantly on body parts such as the abdomen. From the spider
walk only Kluyvera and Staphylococcus spp. were isolated, possibly due to the low sample size. e chelicerae
had the most diverse communities, including Pseudomonas, Rothia, Streptococcus, and Staphylococcus spp. is
is possibly explained through direct exposure of the chelicerae to, and penetrating into, dead prey, in addition
to contact with their legs/feet during grooming.
Staphylococcus spp. were recovered from S. nobilis, A. similis and E. atrica, of which four species recovered
from S. nobilis were identied. Among them, S. epidermidis is a known human pathogen and responsible for
severe illnesses, including bacteraemia, urinary tract infections, endocarditis, septicaemia and nosocomial sepsis
originating from medical devices such as catheters and central lines61. Other Staphylococcus species identied can
be opportunistic human pathogens, i.e. can cause severe infection in a host with a weakened immune system, an
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altered microbiota (such as a disrupted gut microbiota), or breached integumentary barriers, and are considered
as typical components of the skin microbiome64–66.
Pseudomonas are ubiquitous in the environment and some species are associated with human infections.
Pseudomonas spp. were recovered from A. similis and S. nobilis of which three species were identied, and of these
one is related to human pathology. P. putida can cause bacteraemia, skin, so tissue, and urinary tract infections,
localised infections, pneumonia, peritonitis, septic arthritis, meningitis, and septicaemia67,68.
Table 3. Antibiotic susceptibility of 25 bacterial isolates from S. nobilis. Values shown are the average of three
independent experiments performed in duplicate. SD for each value is ≤ 2mm. a Antibiotic abbreviations and
classes: CIP ciprooxacin (ouroquinolone), CN Gentamicin (aminoglycoside), AML amoxycillin (penicillin),
E erythromycin (macrolide), C chloramphenicol, TE tetracycline, FOX cefoxitin (cephalosporin), NAL
nalidixic acid (ouroquinolone), CT colistin (polymyxin). b Amount of antibiotic in disk. c Bold bordered boxes
indicate resistance. d -; no zone of clearance. e Bolded species listed in CLSI guidelines. Blue digits indicate
susceptibility and red digits indicate resistance, according to CLSI guidelines; Black digits indicate antibiotic not
recommended/applicable for respective species in CLSI guidelines.
Bacterial species
Zone of clearance (mm)
CIPaCN AMLE CTEFOX NALCT
5 µgb10 µg10 µg15 µg30 µg30 µg30 µg30 µg50 µg
Bacillus aerius 30 20 31 26 21 27 27 19 13
Bacillus altitudinis23 18 31 26 20 25 26 19 13
Bacillus licheniformis31 21 13 8c16 28 31 18 11
Bacillus mycoides (1)281813212021242
11
1
Bacillus mycoides (2)292310232125292
21
2
Bacillus sp.32 20 28 27 21 25 22 19 11
Bacillus thuringiensis17 17 8232324152
11
1
Dietzia timorensis 36 17 20 19 26 14 10 -d-
Kluyvera intermediae36 24 21 -27 28 24 27 22
Micrococcus aloeverae 24 20 29 21 34 32 32 -20
Micrococcus endophyticus 32 19 40 10 16 21 39 10 16
Paenibacillus mobilis30 23 29 22 19 26 72
81
5
Pseudomonas azotoformans 32 23 31 21 21 27 -24 16
Pseudomonas peli 27 17 11 -24 18 72
11
4
Pseudomonas putida 32 20 --
15 19 -11 15
Rothia amarae 19 16 26 27 26 22 22
--
Rothia mucilaginosa 12 16 26 27 26 27 28 -11
Serratia fonticola (1)2419--
14 22 22 23 15
Serratia fonticola (2)31307 -26 25 28 29 14
Staphylococcus capitis (1)32 24 32 23 28 27 25 -15
Staphylococcus capitis (2)29 19 32 24 21 12 28 -20
Staphylococcus edaphicus24 18 30 26 10 30 30 -13
Staphylococcus epidermidis 27 21 38 24 19 24 31 -9
Staphylococcus warneri30 21 36 22 25 23 28 10 10
Streptococcus sp.22 16 34 33 24 28 26
--
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Two species of Rothia were recovered from S. nobilis, of which one is related to human pathology. R. muci-
laginosa is a common constituent of the oral and upper respiratory microbiota. It is commonly associated with
teeth and gum disease, but is now considered an emerging opportunistic pathogen, especially in immunocom-
promised patients associated with endocarditis, pneumonia, arthritis, meningitis, skin and so-tissue infections,
prosthetic joint infections, and endophthalmitis. For example, it was isolated from ve cancer patients who
developed bacteraemia69,70.
e native spiders A. similis and E. atrica, are common synanthropic spiders throughout Europe and neither
species is considered to pose a threat to the general public. Bites are not thought to be common and therefore
the risk of transmission resulting in infection is likely to be low40. Some bacteria genera from T. agre stis that were
reported previously, were also identied in our study, e.g. Bacillus, Paenibacillus, Pseudomonas and Staphylococ-
cus, and of those isolates identied to species level, Bacillus thuringiensis is present in both datasets. is species
is now recognised as pathogenic, and our study shows it can display antibiotic resistance. In our study aer
allowing S. nobilis to walk on petri dishes with BHI agar, we recovered K. intermedia and S. epidermidis, indicat-
ing that spiders have potential to shed bacteria on the surfaces they touch. Following the rapid expansion of T.
agrestis in North America, local media reported that the species was responsible for envenomation-led necrotic
lesions. is claim has since been debunked, as with other spiders such as yellow sac spiders from the genus
Cheiracanthium30. However, the claims that S. nobilis may be involved in severe envenomation events in Ireland
and the UK appear to have some merit. As the geographical range of S. nobilis expands and their overall density
increases in heavily urbanised areas, envenomations are becoming a more common occurrence. As a result, the
transmission of pathogenic microbes during a bite event is now a cause for concern. e role of bacterial araneism
is controversial; however, it is accepted that it is experimentally plausible for spiders to vector bacteria, and a
conrmed infection vectored directly from a spider bite is discussed in the literature28,38. We demonstrated here
that (1) a wide range of bacteria ubiquitous in the environment are carried on spider chelicerae and exoskeleton
(Fig.1), and (2) some are potentially pathogenic and involved in a wide range of clinical manifestations. In total,
11 species of potentially pathogenic bacteria were isolated from bodies or chelicerae of S. nobilis. We believe
this clearly demonstrates the potential for bacteria to be vectored during bites and that it is just as likely that
infections arise zoonotically as from commensal bacteria present on the skin (as is the current consensus)9,10,38.
In the case of S. nobilis, vectored infection may be facilitated by the venom’s ability to kill localised skin
cells58, potentially disrupt normal immune response30, and provide substrate for bacteria to thrive. Moreover, S.
nobilis typically bite humans when accidently trapped or squashed between the skin and clothing/bed sheets29,54.
erefore, the site around the bite could be contaminated by bacteria present on either the chelicerae or the
body of the spider. Previous studies reveal spider venoms as rich sources of antibacterial peptides41 that could
neutralise bacteria in paralyzed prey38,71. However, recent advances in venomics studies conrms that spider
venoms are not sterile and should be viewed as microenvironments9. e results here demonstrate that S. nobilis
venom has no inhibitory eect on bacterial growth, suggesting that the venom is unlikely to eliminate bacteria
from the chelicerae.
Since the development of penicillin and subsequent antibiotics in the 1940s, there has been a rise in antibiotic
resistant bacteria72 which currently kill over 700,000 people annually 4. erefore, it is important to determine
how antibiotic resistant bacteria move through the environment and establish contact zones between humans
and the environment5. Pathogenic bacteria recovered from the chelicerae of black widow spiders27 included mul-
tiple antibiotic resistant strains, with uoroquinolones and aminoglycosides recommended as the most ecient
antibiotics for treating infections arising from black widow bites. Out of three conrmed bite cases by S. nobilis
that resulted in dermatitis (data unpublished), one of the victims was unresponsive to antibiotic treatment. We
tested the susceptibility of 25 bacteria recovered from S. nobilis against nine antibiotics used by front line medical
Figure2. Antibiotic resistance prole of the bacterial community isolated from body and chelicerae of S.
nobilis. (A) Number of bacterial isolates resistant to each antibiotic. (B) Number of isolates showing resistance to
0, 1, 2 and 3 dierent antibiotics.
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sta and 19 antibiotic-resistant strains were identied (Table3). e most resistant isolates were P. putida, which
showed resistance to three broad range antibiotics (amoxicillin, erythromycin and cefoxitin), S. capitis (2) which
also showed resistance to three completely dierent class of antibiotics (gentamicin, tetracycline and nalidixic
acid) and S. edaphicus which showed resistance to gentamicin, chloramphenicol and nalidixic acid. S. capitis
and S. edaphicus are the only isolates in this study to show resistance against tetracycline and chloramphenicol,
respectively. In terms of resistance shown by the recovered isolates to each antibiotic (Fig.2A), 9 of the isolates
showed resistance to nalidixic acid followed by erythromycin (6), cefoxitin (5), gentamicin and amoxycillin (4),
colistin (3), tetracycline (1) and chloramphenicol (1). All bacteria recovered from S. nobilis were susceptible to
ciprooxacin. An abundance of multidrug-resistant isolates were identied with 3 isolates resistant to 3 dierent
antibiotics and 7 isolates resistant to 2 antibiotics (Fig.2B). ese data support the fundamental need to identify
bacteria from spider bite victims. Additionally, there is a need for catalogues of the microbiota of spiders and
cross-reference databanks with pathogenicity and antibiotic-resistance to better inform appropriate treatment
for infections associated with spider bites.
Conclusion
Our study demonstrates that the non-native S. nobilis and two native spider species, A. similis and E. atrica,
carry opportunistic pathogenic bacteria on their body surfaces and chelicerae. Bacteria may be vectored directly
from the spider, and as a result, post-bite infections may be the result of vector-borne bacterial zoonoses. Some
of the bacteria carried by spiders are multidrug-resistant. Furthermore, our results showed that the venom of S.
nobilis has no inhibitory eects against bacterial growth, indicating that it is most likely not a barrier to bacte-
rial infection resulting from a spider bite. We believe this study provides a baseline for future research targeting
synanthropic spider species to determine bacterial compositions and develop a database of bacterial species
isolated from spiders, and to determine links to human disease.
Methods
Spider and venom collection. Specimens of Amaurobius similis, Eratigena atrica, and Steatoda nobilis
were collected in Ireland, from garden walls and park railings in Lucan, Co. Dublin, Edgeworthstown, Co. Long-
ford, Galway city, Co. Galway and Ferrybank, Co Waterford. Specimens were collected using sterile forceps,
placed immediately into sterile tubes, and transported to the lab. Species identities were conrmed using identi-
cation guides specic to S. nobilis12 and Collins Field Guide for all other spiders62.
Using aseptic techniques, the specimens were dispatched, and the chelicerae were either clipped or swabbed.
For whole body cultures, spiders were either submerged in media or swabbed. For surface colonisation analysis,
spiders walked directly on Brain Heart Infusion (BHI) agar. e most common method for euthanising arthro-
pods is dispatchment. A select number of spiders were euthanised using CO2, and immediately processed to
determine if bacteria was recoverable by this alternate method.
For venom extractions, S. nobilis specimens were anesthetized using CO2 for 2min and venom was extracted
by electrostimulation with repeated pulses delivered at 15–20V. Venom droplets were collected from the venom
pores located on the outer subterminal part of the chelicerae using 5µl microcapillary tubes modied with a
tapered end for maximum eciency. Venom from approximately 100 specimens was pooled and then ash-frozen
in liquid nitrogen and stored at −80°C.
Preliminary testing for microbiomes from A. similis, E. atrica, and S. nobilis and 16S rRNA
gene amplication, sequencing, and analysis. Whole bodies or chelicerae from three species of spi-
ders: A. similis, E. atrica, and S. nobilis were transferred into 750μl (10% dilution) of Luria Bertani (LB) broth,
Nutrient broth (NB), Tryptic Soy broth (TSB), MRS broth and BHI broth, and incubated at both 37°C and 10°C.
Whole culture from each spider or chelicerae were pelleted, DNA was extracted collectively from each sample
using the QIAGEN Dneasy Blood & Tissue Kit and V3-V4 region of 16S rRNA was amplied using 341F 5′-CCT
ACG GGA GGC AGCAG-3′73, and 806R 5′-GGA CTA CHVGGG TWT CTAAT-3′74. e amplied product was
then sent to GATC Biotech for sanger sequencing. A BLAST search was carried out with the obtained sequence
using the NCBI rRNA/ITS database (https ://blast .ncbi.nlm.nih.gov/Blast .cgi).
Bacterial isolation from S. nobilis and 16S rRNA gene amplication, sequencing and analy-
sis. For isolating surface bacteria, S. nobilis spiders were washed individually with 5ml BHI broth for 5min.
Some spiders were washed immediately aer dispatchment, while others were processed 24–48h aer death.
e wash media was then incubated at 37°C overnight. For isolating bacteria from chelicerae, clipped chelicerae
from each individual spider were inoculated into BHI broth and incubated at 37°C. Aer 24h incubation, the
cultures were diluted and plated on BHI agar and incubated 48h to 72h at 37°C. Selective media, Baird-Parker
agar and TS-blood agar supplemented with colistin and nalidixic acid, were also inoculated with overnight
cultures and incubated 48h to 72h at 37°C. Colonies with dierent morphologies were selected for further
analysis.
e 16S rRNA gene was amplied using Taq polymerase (Bioline) and universal primers, 27F (5′-AGA GTT
TGA TCA TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) using Colony PCR75. e PCR prod-
uct was puried using the Wizard SV Gel and PCR Clean-Up System (Promega) and sequenced using primers,
27F 1492R (Eurons Genomics, Germany).
A BLAST search was carried out with the obtained sequence using the NCBI rRNA/ITS database (https ://
blast .ncbi.nlm.nih.gov/Blast .cgi). Closest bacterial species were identied using Blast tree view produced by
Blast pairwise alignment.
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Inhibitory eects of S. nobilis venom against pathogens. Antibacterial activity of S. nobilis venom
was assessed by agar diusion assay and Minimum Inhibitory Concentration (MIC) assay. Agar diusion assay
was carried out against S. capitis (2) and P. azotoformans (isolated from S. nobilis chelicerae). Spiders were stimu-
lated to aseptically bite Mueller–Hinton agar spread with 100μl of adjusted overnight bacterial culture (0.8
OD590). We could observe the fangs penetrating the agar in a biting motion and also observe venom being
expelled from the fangs. e restraining of the spider was enough to stimulate the bite and therefore no other
manual stimulation was required. In addition, 0.5μl neat venom was spotted onto the agar plate containing bac-
teria. Plates were incubated for 24h at 37°C and then assessed for zones of bacterial clearance.
e average volume of venom that each spider produces is approximately 0.22µl (with a maximum of approxi-
mately 0.6µl). Due to the limited amount of venom available, MICs were carried out by diluting the samples to
achieve usable volumes. e MICs were performed against clinical isolates E. coli DSM10973, MRSA BH1CC and
L. monocytogenes EGD-e. An overnight culture was adjusted with Muller-Hinton broth to an inoculum density
of 1 × 106cfuml−1. Starting with a 1:100 dilution of the venom, twofold serial dilutions of the venom, was tested
against all the pathogens in a nal inoculum of 5 × 105cfuml−1. Aer 24h incubation at 37°C, absorbance at
590nm was measured using a microplate reader (Tecan) with Magellan soware.
Antibiotic susceptibility testing. Disk diusion assays were carried out to determine antibiotic sus-
ceptibility. Experiments were conducted according to the Clinical and Laboratory Standards Institute (CLSI)
guidelines76. 6mm disks preloaded with each antibiotic (Oxoid) were placed onto Mueller–Hinton agar plates
that had been spread with 100µl overnight bacterial culture (1 × 108cfuml−1). Plates were incubated at 37°C
for 18h and the clear zone around each disk was measured using a ruler and interpreted according to European
Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints77 Bacterial spread plates without
antibiotic disks were used as negative control and the bacteria grew as a lawn each time. ree independent
experiments were performed in duplicate.
Ethical statement. No ethical approval was required to work with spiders. e three bite victims have
provided the authors with written consent to use their case history and other relevant details to produce manu-
scripts intended for publication in scientic journals. ey are aware that such publications may be available to
the public both in print and on the Internet.
Data availability
e datasets generated and analysed during the current study are available from the corresponding author on
reasonable request.
Received: 14 May 2020; Accepted: 17 November 2020
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Acknowledgements
e authors thank Eoin MacLoughlin for assistance during eld work. We thank Maria Condon for access
to property for sampling. e authors also thank Dr Maria Barrett for assistance with preparing samples for
sequencing. We are also very grateful to the two anonymous referees whose critical review of this manuscript
was extremely valuable. is project was funded through the Irish Research Council under a Government of
Ireland Postgraduate Scholarship held by Neyaz Khan (GOIPG/2017/910), the NUI Galway Ryan Award for
Innovation held by Michel Dugon and a NUI Galway College of Science PhD scholarship held by John Dunbar.
Author contributions
Conceived and designed the experiments: M.M.D., J.P.D., V.O.F., C.L.A., A.B., N.A.K. Performed the experiments:
J.P.D., N.A.K., C.L.A., P.B., J.M., S.A. Analysed the data: A.B., V.O.F., M.M.D., J.P.D., N.A.K., C.L.A. Wrote the
paper: J.P.D., N.A.K. Sourcing spiders and venoms: J.P.D. All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to J.P.D.
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