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Gram-positive bacteria isolated from the common bed bug, Cimex lectularius L.

Abstract--The common bed bug, Cimex lectularius L., is an obligate blood-feeding ectoparasite that readily feeds on humans, resulting in rashes, dermatitis, and psychological distress. Bed bug populations have increased exponentially in the last twenty years due to increased pesticide resistance and international travel. Despite their blood-feeding and ubiquity in human habitations, bed bugs are not vectors of any known pathogen. Recent isolates of antibiotic resistant bacteria from bed bugs in a homeless shelter prompted us to identify bacteria from bed bugs in low-income housing in Maryland and Colorado and from a laboratory colony, commonly known as the Harlan strain. Using standard bacteriological techniques and amplification of the 16s ribosomal RNA (rRNA) gene, four species of Gram-positive bacteria were identified from surface swabs and whole body homogenates of bed bugs. Staphylococcus arlettae and Staphylococcus epidermidis were identified from the Harlan strain; Micrococcus and Kocuria kristinae were identified from the Colorado samples. Micrococcus species have been previously identified from bed bugs, but this is the first report of K. kristinae, S. epidermidis, and S. arlettae from this insect. As an internal control for the 16s rRNA amplification, a sequence specific to Wolbachia, the bacterial endosymbiont commonly found in bed bugs, was amplified. Using these techniques, no bacteria from the gut contents were isolated. Bacteria found commonly on human skin are closely associated with bed bugs and do not pose a risk to human health. Despite their habitats and blood-feeding behavior, bed bugs from these sites were devoid of pathogenic aerobic bacteria. The mechanisms underlying bed bug resistance to exogenous bacterial infection merit further investigation.

INTRODUCTION

The common bed bug, Cimex lectularius L., is an obligate blood-feeding ectoparasite that readily feeds on humans. Bed bug bites can cause adverse reactions in the human host, including rashes, necrotic lesions, dermatitis, allergies, and psychological distress (Goddard and deShazo, 2009). Global bed bug infestations were common until the 1930s and 40s, when dichlorodiphenyltrichloroethane (DDT) was first used to control insect pests (Usinger, 1966). From the 1940s until 1972, when DDT was banned by the U.S. Environmental Protection Agency (EPA), bed bugs were virtually eradicated from human habitations in the developed world (Potter, 2011). Infestations of bed bugs have increased exponentially in the last twenty years in North America, Europe, and Australia, representing a significant financial, psychological, and public health burden (Krueger, 2000; Doggett et al., 2004). Their global resurgence is due to decreased indoor pesticide use, increased pesticide resistance, introductions from multiple global foci, and increased trade in secondhand furniture (King et al., 1989; Boase, 2001; Anderson and Leffler, 2008).

Because all five nymphal stages, males, and females feed on blood, the role of bed bugs in transmitting pathogens has been investigated for almost a century (Burton, 1963). Delaunay and colleagues (2011) reviewed literature from the past fifty years that evaluated bed bugs as vectors of 45 pathogens. Several bacterial genera have been isolated from the surface of C. lectularius, including Stenotrophomonas, Enterobacter, Bacillus, Staphylococcus, Arthrobacter, and Micrococcus (Reinhardt et al., 2005; Delaunay et al., 2011). Recently, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) were identified from bed bugs collected in homeless shelters (Lowe and Romney, 2011), which prompted us to undertake a bacterial survey of bed bugs from apartments. We isolated and identified bacteria from bed bugs from a laboratory colony and apartments in Maryland and Colorado.

MATERIALS AND METHODS

Live bed bugs

The "Harlan strain" of C. lectularius has been maintained in laboratory colony since 1973, when it was established from a natural population collected in Fort Dix, New Jersey. The colony was maintained at 28[degrees]C, at a photoperiod of 16: 8 hours of light to dark, and fed weekly on a human volunteer (SRI Human Subjects Committee Approval, Case 1199). Bed bugs were also collected by licensed pest control operators in an apartment in Baltimore, Maryland and from a couch in an apartment in Rifle, Colorado.

Sample preparation

Bed bugs from each site were used to prepare four samples to isolate bacteria: a surface sample, a whole body homogenate, a gut sample, and a negative control. The surface sample was prepared by agitating single adult bed bugs in 100 [micro]L of sterile phosphate buffered saline (PBS) and spreading the rinse on individual Trypticase Soy Agar (TSA) plates. Whole bodies from each site were homogenized using microfuge tubes and a pestle in 100 [micro]L of sterile PBS, which was spread on TSA plates. Gut samples were prepared by sterilizing the surface of bed bugs with 90% isopropyl alcohol removing their heads, and expressing their gut contents into sterile PBS, which was spread on TSA plates. The negative control entailed sterilizing the surface of bed bugs with isopropyl alcohol, rinsing them with 100 [micro]L of sterile PBS, and spreading this rinse on TSA plates. A positive control was included by swabbing the skin of the human volunteer and immediately streaking a TSA plate. All plates were incubated at atmospheric oxygen levels at 37[degrees]C for 24 hours. Each colony was maintained in pure culture for the duration of the study. Three bed bugs (one from each environment) were analyzed in each experiment.

Identification of colonies

Standard microbiological protocols were performed without modification (Leboffe and Pierce, 2010): Simple stain, negative stain, Gram stain, endospore stain, capsule stain (Hiss Method), wet mount, aerotolerance, inoculation on eosin-methylene blue agar, catalase activity, oxidase activity (BBL DrySlide, Becton Dickinson Co.), gelatinase activity, and coagulase activity. A potassium hydroxide (KOH) assay was performed to support the results obtained by the Gram stain (Bourgault and Lamothe, 1988).

Polymerase Chain reaction (PCR)

The 16s ribosomal RNA (rRNA) of bacteria from each colony was amplified. An isolated colony was transferred to sterile Tryptic Soy Broth (TSB) and incubated with shaking at 37[degrees]C for 24 hours. Five hundred [micro]L of culture was transferred from TSB tubes into sterile microfuge tubes, and centrifuged at 13.3 x [10.sup.3] for 1 minute. The pellet was resuspended and boiled in 500 [micro]L of sterile water. After centrifugation at 13.3 x [10.sup.3] g, the supernatant was used as DNA template. Each reaction contained 5 [micro]L DNA template; 5 pMol each of 16s Forward and Reverse Primers (Table 1); 5 [micro]L Epicenter patented polymerase blend (Fail Safe Epicentre Technologies, Omaha, Nebraska); and 10 [micro]L Premix I (Fail Safe Epicentre Technologies, #FS99100). The samples were heated to 95[degrees]C for 5 minutes, followed by 35 cycles of [95[degrees]C for 1 minute, 50C for 30 seconds, and 72C for 90 seconds]. The internal technical control for the 16s rRNA PCR was a 136 basepair sequence for Wolbachia, an endosymbiont commonly found in bed bugs (Sakamoto and Rasgon, 2006: Sakamoto et al., 2006). Whole bed bugs from the Harlan strain were homogenized, and genomic DNA was extracted for use as template. Primer sequences are listed in Table 1. The PCR conditions to amplify the 16s rRNA from Wolbachia were: 94[degrees]C 1 minute, followed by 30 cycles of [94[degrees]C for 1 minute, 94[degrees]C for 30 seconds, 60[degrees]C for 45 seconds, and 72[degrees]C for 30 seconds] [15, 16]. Resulting PCR products were sequenced by Operon/Eurofin (Huntsville, Alabama) with 5 pMol of Bac8 Forward Primer (Table 1). Sequences were uploaded and analyzed by the Ribosomal Database Project (Michigan State University, East Lansing, MI).

RESULTS

Bacterial isolation from bed bugs

Samples taken from the Harlan bed bugs yielded three colonies with different morphologies, which were designated as Harlan surface (1), Harlan surface (2), and Harlan whole body homogenate. No bacterial growth was detected from the sample from the surface of bed bugs from Maryland. One colony was isolated from the Colorado whole body homogenate and one from the surface collection. Analysis of the whole bug homogenate from Maryland was discontinued because the colonies isolated following the initial sampling protocol were not of sufficient size. BIOLOG (Hawyard, California) is a phenotypic identification system that determines the ability of a bacterial isolate to perform a variety of biochemical reactions, based on characteristic metabolic profiles (Klingler et al., 1992; Miller and Rhoden, 1991). These results are summarized in Table 2.

Simple and negative stains indicated that all colonies from surface collections and whole body homogenates were cocci in shape. All isolates were determined to be Gram positive by Gram stains and confirmed by KOH tests. Additionally, none of the isolates possessed capsules or endospores. Harlan surface (1), (2), Harlan whole body homogenate, and Colorado surface isolates were determined to be facultative anaerobes, and the isolates from the whole body homogenate and surface swab from the Colorado strain were obligate aerobes. The isolates were tested for the presence of the enzymes catalase, oxidase, gelatinase, coagulase, urease, and amylase. All of the isolates possessed catalase and did not produce oxidase or amylase. Only the Harlan surface (1) and (2) isolates produced coagulase, and only the Maryland whole body homogenate and Colorado surface isolates produced urease.

Isolates from Harlan surface (1), (2), Harlan whole body homogenate, and Colorado surface grew on a Mannitol Salt Agar (MSA) plate, but only Harlan surface (1) and (2) fermented mannitol. No isolates grew on Eosin Methylene Blue (EMB) agar plates, and only the isolate from the Harlan whole body homogenate was capable of partially hemolyzing red blood cells (alpha hemolysis). All other isolates were incapable (gamma hemolysis).

Only the isolate from the Colorado surface sample grew on the citrate agar slant, indicating that it is capable of utilizing citrate as a sole carbon source. Triple Iron Sugar (TSI) test results suggested that none of the samples fermented lactose or sucrose and did not reduce sulfur. Sulfur Indole Motility (SIM) medium indicated that samples Harlan surface (1), (2), and whole body homogenates of bed bugs from Maryland and Colorado did not reduce sulfur, produce indole, and were not motile. The sample from the surface of a bed bug from Colorado reduced sulfur, but did not produce indole, and was not motile. Isolates from the Maryland whole body homogenate and the Colorado surface sample were able to ferment mannitol, sucrose, and glucose, while the isolate from the Colorado whole body homogenate was only able to ferment sucrose. The isolate from the Colorado whole body homogenate was also the only isolate capable of mixed acid fermentation and 2,3-butanediol production based on the Methyl Red/ Voges Proskauer (MR/VP) tests, and all isolates except for those from the Colorado whole body homogenate and the Colorado surface were capable of nitrate reduction.

Polymerase chain reaction

After morphological examination and biochemical tests, each colony was identified by16s rRNA amplification and sequence analysis (Table 3). After amplification, the DNA was sequenced (Eurofins MWG Operon, Huntsville, Alabama) and uploaded to the Ribosomal Database project (http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp, East Lansing, MI).

The samples isolated from Colorado surface were identified as Kocuria kristinae and the Colorado whole body homogenate was determined to be Micrococcus. Subsequent BIOLOG analysis was unable to identify bacteria from the Maryland whole body homogenate and a Harlan whole body isolate. Two isolates from Harlan surface samples were identified as Staphylococcus arlettae. 16s rRNA amplification identified Wolbachia in all bed bugs. Samples from human skin revealed Gram-positive cocci; these colonies were identified by sequence analysis to be in the genus Kocuria.

DISCUSSION

Bacteria were initially cultivated on TSA plates in an aerobic environment. TSA is a rich, undefined medium that allows for the growth of many different bacteria, but some bacteria on or in bed bugs might not have found this medium hospitable. We then employed selective/differential media after obtaining initial isolates on TSA. We maintained cultures at 37[degrees]C to allow for the growth of most mesophiles, which flourish at a range of 20-45[degrees]C, and because bacteria that are obligate commensals of humans would grow best at this temperature. Using these standard bacteriological techniques, bacteria from gut contents were not isolated. In addition, by cultivating all the samples in an aerobic environment, we selected for obligate or facultative aerobes and against obligate anaerobes. Historically, only TSA and aerobic incubation have yielded bacteria from bed bugs, but we are using Anaerobic Transfer Medium (ATM) and anaerobic conditions in our next experiments.

Steinhaus (1941) identified four new species of bacteria from adult bed bugs, including Bacterium tegumenticola from the integument, Corynebacterium paurometabolum from the ovaries, and one species each of Micrococcus and Sarcina. Another commensal bacterium of arthropods, Candidatus Michloria mitochondrii, was identified from bed bugs collected from ships (Richard et al., 2009). Stenotrophomonas maltophilia, Enterobacter hormaechei, Bacillus licheniformis, Staphylococcus saprophyticus, Arthrobacter sp., Micrococcus luteus, and three species of fungi were isolated from harborages or surfaces of bed bugs (Reinhardt et al., 2005). In contrast, over 20 species of bacteria have been isolated from the cockroaches Periplaneta americana and Blatella germanica (Pal et al., 2005; Zarchi and Vatani, 2009). The relative paucity of bacteria identified from an obligate blood-feeding ectoparasite, especially from the gut contents, suggests some mechanism of resistance or immunity. One hypothesis in favor of selection for resistance against bacterial infection in bed bugs is predicated upon their unique mating strategy, known as traumatic insemination. Male bed bugs penetrate the female cuticle and ejaculate into an organ known as the spermalege (Reinhardt et al., 2003). The spermalege has been shown empirically to defend the female against infection by exogenous pathogens that might be introduced during traumatic insemination (Reinhardt et al., 2003). Male bed bugs also attempt to penetrate other males and nymphs (Ryne, 2009; Harraca et al., 2010). In addition, bacteriolytic lysozymes in the male seminal fluid might function to protect the spermatozoa or the female from bacterial infection (Otti et al., 2009). Innate immunity might confer a selective advantage in an insect that routinely introduces surface bacteria into the hemocoel of both sexes and immature stages by traumatic insemination.

Bacterial endosymbionts of the genus Wolbachia have been identified repeatedly from bed bugs (Hypsa and Aksoy, 1997; Rasgon and Scott, 2004: Sakamoto and Rasgon, 2006; Sakamoto et al., 2006). As a positive technical control, we attempted to amplify the 16s RNA of Wolbachia. A 136 bp fragment was present after gel electrophoresis of PCR products, and the sequence of the PCR product was specific to Wolbachia, demonstrating that while not cultured from the bed bugs sampled, Wolbachia was still detectable.

Kocuria kristinae, a facultative anaerobic, Gram-positive bacterium was isolated from the surface of a bed bug from Colorado. This bacterium has been identified from the Oriental eye fly, Siphunculina funicola, in Thailand (Chansang et al., 2010). This is the first report of K. kristinae from the bed bug. The genus Kocuria is commonly found in the environment; it is part of the flora of the skin and mouths of healthy humans (Szczerba, 2003). All three of the colonies isolated from human skin were Kocuria. Pathogenic infections are rare but have been associated with immunocompromised hosts, with only 15 documented cases from 1995 to 2010 (Dunn et al., 2011).

Staphylococcus arlettae was isolated twice from the surfaces of colony bed bugs. S. arlettae is a non-motile, Gram-positive coccus commonly isolated from human and animal skin and mucous membranes. S. arlettae appears to be highly salt tolerant and has been isolated from fermented ham (Tu et al., 2010) and salted cod (Vilhelmsson et al., 1997). S. arlettae has also shown promise in the biodegradation of azo dyes from the effluent of a textile factory (Elisangela et al., 2008). This is the first report of this nonpathogenic bacterium from any insect.

Staphylococcus epidermidis was isolated from the whole body homogenate of a colony bed bug. S. epidermidis is a non-motile, Gram-positive coccus that is part of the normal resident human skin microflora (Kong, 2011). S. epidermidis is often cultured from healthy human skin and may protect against infection by pathogenic bacteria (Iwase et al., 2010). S. epidermidis is typically not pathogenic, although persons who are immuno-compromised or who have catheters or surgical prostheses are at risk for infection (Fey and Olson, 2010). S. epidermidis has been isolated from the cockroach Diploptera punctata in human habitations (Tatfeng et al., 2005), but this is the first report of S. epidermidis from the bed bug.

A Micrococcus species was isolated from whole body homogenate from Colorado. Micrococcus luteus has been reported previously from bed bugs (Reinhardt et al., 2005). Micrococcus species are Gram-positive, oxidase-positive bacteria that are part of the normal human skin Nora (Roth and James, 1988). Micrococcus is not considered pathogenic but has been associated with catheter-related bacteremia in cancer patients who may have been immunocompromised (Ramos et al., 2009).

Bed bug populations have increased exponentially worldwide, and this increase coupled with their intimate association with humans resurrects the question of their role in pathogen transmission. When Romero (2009) led bed bugs Borrelia hermsii, an etiological agent of relapsing lever, he found that spirochetes persisted in the gut for no more than one day. A microbial survey of bed bugs identified fungi and bacteria from harborages, abdomens, and the male reproductive organ, but found no microbes on the proboscis (Reinhardt et al., 2005). A recent article described bed bugs as "vectors" of MRSA and VRE (Lowe and Romney, 2011). However, these bacteria were cultured from whole homogenates of bed bugs, and standard vector competence trials were not conducted. Our isolation of Kocuria from human skin and from bed bugs--and similar results with S. epidermidis, S. arlettae, and Micrococcus--indicate that these insects support the microflora commonly associated with human skin. This likely explains the results of Lowe and Romney (2011), who identified antibiotic-resistant bacteria from bed bugs collected from a homeless shelter. To date, the role of bed bugs as vectors of any bacterial pathogen remains entirely speculative.

Aerobic bacteria associated with the surface and whole bodies of bed bugs were identified, but a similar systematic study of protozoa, fungi, and viruses would add substantially to our understanding of the natural microbial diversity of these insects.

ACKNOWLEDGMENTS

We thank Mr. Louis Sorkin of the American Museum of Natural History, New York, for providing us the Harlan strain of bed bugs. We thank Mr. Art Hall, Mr. Zachary Drake, Mr. John Hail, and colleagues at Pure Environmental Solutions for providing the bed bugs from Maryland and Colorado.

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CHELSEA COCKBURN (1), MATT AMOROSO (1), MARC CARPENTER (1), BRITTANY JOHNSON (1), RACHEL McNEIVE (1), ADAM MILLER (1), A. ELIZABETH NICHOLS (1), AVON LEA RIOTTO (1), AMANDA RZEPKOWZSKI (1), CRYSTAL MICHELLE SCOTT CROSHAW (1), KYLE SEIFERT (1) AND RAJEEV VAIDYANATHAN (1,2,3)

(1) Department of Biology, James Madison University, Harrisonburg, Virginia, 22807. U.S.A.

(2) Center for Immunology and Infectious Disease, SRI International, Harrisonburg, Virginia, 22802. U.S.A.

(3) Corresponding author. SRI International, 140 Research Drive, Harrisonburg, Virginia, 22802. Phone: 540-438-6620. Email: Rajeev.vaidyanathan@sri.com
Table 1. Primers used in this study.

          Description                       Sequence

Bac8 forward primer 5 pMol        5'-AGAGTTTGATCCTGGCTCAG-3'
16s reverse primer (Univ1492r)    5'-GGTTACCTTGTTACGACTT-3'
Wolbachia forward 0.4 [micro]M    5'-AGTCATCATGGCCTTTATGGA-3'
Wolbachia reverse 0.4 [micro]M    5'-TCATGTACTCGAGTTGCAGAGT-3'

Table 2. Results of stains, aerotolerance, and biochemical assays.
OA = obligate aerobe; FAN = facultative anaerobe;
ND = not done; GH = gamma hemolysis; AH = alpha hemolysis;
NA = unable to determine because of lack of growth.

                      Harlan    Harlan      Harlan      Maryland
                      surface   surface   whole body   whole body
                        (1)       (2)     homogenate   homogenate

Shape                  Cocci     Cocci      Cocci        Cocci

Gram                     +         +          +            +

KOH *                    -         -          -            -

Capsule                  -                    -            -

Endospore                -         -          -            -

Aerotolerance           OA        OA         FAN          FAN

Catalase                 +         +          +            +

Oxidase                  -         -          -            -

Gelatinase               -         -          -            -

Coagulase                +         +          -            -

Urease                   -         -          -            +

Amylase                  -        ND          -            -

Mannitol salts agar
(growth/mannitol
fermentation)           +/+       +/+        +/-          +/-

Eosin methylene        -/NA      -/NA        -/NA         -/NA
blue agar

Blood agar              GH        GH          AH           GH

Citrate                  -         -          -            -

Lactose                  +         +          -            +
fermentation (TSI)

Sucrose                  +         -          +            +
fermentation (TSI)

Sulfur                   -         -          -            -
reduction (TSI)

Sulfur                   -         -          -            -
reduction (SIM)

Indole                   -         -          -            -
production (SIM)

Motility (SIM)           -         -          -            -

Mannitol                 -         -          -            +
fermentation

Sucrose                  -         -          -            +
fermentation

Glucose                  -         -          -            +
fermentation

MR/VP                   -/-       -/-        -/-          +/-

Nitrate reduction        +         +          +            +

                                  Colorado
                      Colorado   whole body   Colorado
                      surface    homogenate    surface

Shape                  Cocci       Cocci        Cocci

Gram                     +           +            +

KOH *                    -           -            -

Capsule                  -           -            -

Endospore                -           -            -

Aerotolerance           FAN         FAN          OA

Catalase                 +           +            +

Oxidase                  -           -            -

Gelatinase               -           +            -

Coagulase                -           -            -

Urease                   +           -            -

Amylase                  -           -            -

Mannitol salts agar
(growth/mannitol
fermentation)           +/-         +/-        -/NA *

Eosin methylene         -/NA        -/NA        -/NA
blue agar

Blood agar               GH          GH          GH

Citrate                  -           -            +

Lactose                  -           -            -
fermentation (TSI)

Sucrose                  +           -            +
fermentation (TSI)

Sulfur                   -           -            +
reduction (TSI)

Sulfur                   -           -            +
reduction (SIM)

Indole                   -           -            -
production (SIM)

Motility (SIM)           -           -            -

Mannitol                 +           -            -
fermentation

Sucrose                  +           +            -
fermentation

Glucose                  +           -        No growth
fermentation

MR/VP                   -/-         +/+          -/-

Nitrate reduction        +           -            -

Table 3. Identity of unknown bacterial isolates.

Isolate                          Genus and species

Harlan surface (1)               Staphylococcus arlettae
Harlan surface (2)               Staphylococcus arlettae
Harlan whole body homogenate     Small, unidentified colony
Harlan whole body homogenate     Staphylococcus epidermidis
Maryland whole body homogenate   Not identified
Colorado whole body homogenate   Micrococcus spp.
Colorado surface                 Kocuria kristinae
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Author:Cockburn, Chelsea; Amoroso, Matt; Carpenter, Marc; Johnson, Brittany; McNeive, Rachel; Milleri, Adam
Publication:Entomologica Americana
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2013
Words:4469
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