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Abstract
Bed bugs (Cimex lectularius L.) have resurged in the United States and globally. Bed bugs are hematophagous ectoparasites of humans and other animals, including domestic pets, chickens, and bats, and their blood feeding habits contribute to their potential as disease vectors. Several species of Bartonella are re-emergent bacterial pathogens that also affect humans, domestic pets, bats and a number of other wildlife species. Because reports of both bed bugs and Bartonella have been increasing in the U.S., and because their host ranges can overlap, we investigated whether the resurgences of these medically important pathogens and their potential vector might be linked, by screening for Bartonella spp. in bed bugs collected from geographic areas where these pathogens are prevalent and from bed bugs that have been in culture in the laboratory for several years. We screened a total of 331 bed bugs: 316 bed bugs from 36 unique collections in 29 geographic locations in 13 states, 10 bed bugs from two colonies maintained in the laboratory for 3 yr, and 5 bed bugs from a colony that has been in culture since before the recent resurgence of bed bugs. Bartonella spp. DNA was screened using a polymerase chain reaction assay targeting the 16S–23S rRNA intergenic transcribed spacer region. Bartonella DNA was not amplified from any bed bug, but five bed bugs from four different apartments of an elderly housing building in North Carolina contained DNA sequences that corresponded to Burkholderia multivorans, an important pathogen in nosocomial infections that was not previously linked to an arthropod vector.
Citation: Saenz VL, Maggi RG, Breitschwerdt EB, Kim J, Vargo EL, Schal C (2013) Survey of Bartonella spp. in U.S. Bed Bugs Detects Burkholderia multivorans but Not Bartonella. PLoS ONE 8(9): e73661. https://doi.org/10.1371/journal.pone.0073661
Editor: Subba Reddy Palli, U. Kentucky, United States of America
Received: April 26, 2013; Accepted: July 19, 2013; Published: September 9, 2013
Copyright: © 2013 Saenz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2008-35302-04565 from the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (to ELV and CS), a grant from the Pest Management Foundation (to ELV and CS), the Blanton J. Whitmire endowment at North Carolina State University, and scholarships to VLS from the North Carolina Pest Management Association and the Structural Pest Management Training and Research Facility at North Carolina State University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Bed bugs (Cimex lectularius L.) are an important resurging pest in urban centers globally, including the U.S. [1]. These hematophagous ectoparasites feed mainly on humans but they also occasionally feed on other animals including bats, chickens, and domestic pets such as cats [2], [3]. Because of their blood-feeding and commensal association with their hosts, there is great concern about the potential of bed bugs to vector disease organisms. More than 45 potential human pathogens have been isolated in association with bed bugs, but none has been shown to be transmitted from bed bugs to humans (review: [4]). Furthermore, experimental infection of bed bugs with pathogens in the laboratory showed that hepatitis B virus could persist in the insects and their feces for up to 5 wk [5], and the spotted fever group rickettsia, Rickettsia parkeri, could last up to 2 wk in the insects [6].
Bartonella is a genus of emerging and re-emerging facultative intracellular bacterial pathogens found throughout much of the industrialized world [7]. Many new Bartonella species have been described in recent years in conjunction with an expanding host range, and evidence supports transmission by a wide range of blood-sucking arthropod vectors that include ticks, sand flies, biting flies, fleas, and lice [8]. These bacteria are the causative agents of several diseases, including Cat Scratch disease (CSD) (Bartonella henselae), Carrion’s disease (Bartonella bacilliformis) and Trench fever (Bartonella quintana). In the U.S., cats are the main reservoir of B. henselae, humans and dogs are incidental hosts, and CSD is common with the highest B. henselae seroprevalence found in cats in warm and humid areas of the Southeast, Hawaii, coastal California, and the south central plains regions [9]. The cat flea (Ctenocephalides felis (Bouchè)) is the primary vector of B. henselae, but the pathogen can also be transmitted by cat bites and/or scratches contaminated with flea feces. Trench fever is also prevalent in the U.S. in homeless shelters; it is transmitted by the body louse (Pediculus humanus humanus L.), and humans are considered the main reservoir [10]. Based upon these patterns, detection of either B. henselae or B. quintana in bed bugs from the U.S. would seem more probable than other less prevalent Bartonella species that occasionally infect humans.
Post-resurgence efforts to screen bed bugs for human pathogens, including Bartonella, are few and relatively recent. Lowe and Romney [11] detected antibiotic resistant bacteria in five bed bugs collected from patients that were infected with the bacteria in Vancouver, British Columbia. They concluded that bed bugs can be vectors of methicillin-resistant Staphylococcus aureus and vancomycin resistant Enterococcus faecium. Richard et al. [12] screened 18 individual bed bugs from French warships for Rickettsia spp., Bartonella spp., and Anaplasma spp. These authors did not detect Rickettsia or Bartonella in the screened insects, but they found a single sample containing an Anaplasma-like bacterium “Candidatus Midichloria mitochondrii,” an endosymbiont of ticks [13]. To date, few studies have examined the vectorial capacity of bed bugs by screening wild populations for disease agents. Even fewer vector competency studies have been performed to implicate bed bugs as disease vectors.
Bartonella spp. have been detected in eastern bat bugs (Cimex adjunctus Barber) collected in two bat caves from the southeastern U.S. [14]. Cimex adjunctus is found in bat roosts, occasionally invading buildings in bat roosting sites [2]. It is a close relative of C. lectularius and co-infestation of human-built structures by both species can sometimes occur [2]. Because bats are relatively new hosts of Bartonella [15], and C. lectularius can occasionally feed on bats [2], [16], it is plausible that C. lectularius could also harbor bat-adapted Bartonella species. Given that both C. lectularius and several Bartonella species are resurgent in the U.S. in association with humans, bats, domestic pets, and wildlife, our objective was to investigate if the resurgence of bed bugs could represent a potential source of Bartonella transmission. We screened for Bartonella spp. in bed bug populations from geographic areas where B. henselae bacteremia is prevalent in feral and pet cat populations.
Results and Discussion
We did not detect any positive PCR products for Bartonella spp. in any of the 316 bed bugs freshly collected in the field between 2005 and 2010. Furthermore, we did not detect Bartonella spp. DNA in any of the bed bugs maintained in our cultures, including 10 bed bugs from two colonies maintained in the laboratory for 3 yr and five bed bugs from the Fort Dix (Harold Harlan) colony, which was originally collected in 1973, well before the recent resurgence. The endosymbiont Wolbachia is highly prevalent in bed bug populations [17], and we successfully amplified bacterial DNA from all 10 bed bugs screened with 16S rDNA universal primers. These results confirm that our negative PCR results for Bartonella are due to absence of Bartonella DNA and not PCR interference. Our findings suggest that bed bugs are an unlikely vector of Bartonella spp. However, as Bartonella are emerging pathogens, adaptation to new hosts in conjunction with an expanding range of vectors suggests that vector biologists should remain vigilant to the possibility of Bartonella occurring in bed bugs in the future [7]. The ecological interactions between Bartonella and bed bugs may be dynamic and could have changed since our collections were completed. We targeted our collections to regions of the United States where B. henselae seroprevalence is high in cats due to frequent flea exposure (Fig. 1). It is possible that other geographic regions, other Bartonella species, and other sites within the regions in which we collected might show different results. Vector competency studies, in which bed bugs are infected with the bacteria to determine the fate of the bacteria and the ability of bed bugs to infect a host, should be investigated to completely rule out bed bugs as potential vectors of Bartonella [18].
Bed bug collection sites (circles, N = 29) superimposed on a distribution map showing the percentage of pet cats with B. henselae antibodies from 33 geographic regions throughout the U.S. (adapted from Jameson et al. 1995 with permission from Oxford University Press). The highest seroprevalence of B. henselae is in warm, humid areas, especially in the southeastern U.S.
We screened 99 bed bugs from 10 apartments of an elderly housing building in Raleigh, North Carolina (Table 1). Five bed bugs from four different apartments yielded PCR products of the expected size for Bartonella spp. (400-600 bp), close to the Bartonella henselae control (604 bp). When these amplicons were sequenced, the alignments of the five sequences (Genebank accession numbers: KF286544-KF286548) with BlastX were 98% similar to a sequence of Burkholderia multivorans, accession number YP001949472.1. As these amplicons were generated using Bartonella 16S-23S intergenic spacer primers, it is possible that more specifically designed primers for B. multivorans may detect higher prevalence of this pathogen in bed bugs. B. multivorans is recognized as an important pathogen in nosocomial infections of patients with cystic fibrosis [19], although it has not been linked to an arthropod vector. Amplification and sequencing of B. multivorans DNA from multiple bed bugs in multiple but nearby apartments suggests that this association should be investigated further to determine whether bed bugs are competent to vector B. multivorans between humans.
Materials and Methods
Sample Collection
Adult bed bugs were sampled from 36 unique collections in 29 different geographic locations spanning 13 states (Table 1, Fig. 1). A total of 331 bed bugs were screened individually for Bartonella spp. DNA, 2–10 bed bugs per collection (Table 1). Bed bugs were collected by pest control companies, collaborators, or by us and in all cases the resident or owner of the property gave permission to collect bed bugs from the site. In most locations specimens collected from a single room within an apartment or building were placed in a single collection vial, but in some samples bed bugs from multiple rooms were combined by the collector in a single vial. Most of the screened bed bugs (N = 316 bed bugs) were freshly field-collected and immediately stored in ethanol, but two collections were reared in the lab for approximately 3 yr before they were screened (N = 10 bed bugs). Additionally, we screened five bed bugs from the Fort Dix (Harold Harlan) colony which had been in culture since 1973, well before the bed bug resurgence in the late 1990s (Table 1).
DNA Extraction and PCR Amplification
Bed bugs were surface sterilized by rinsing them with sterile water and 95% ethanol, and they were processed individually throughout the entire screening process. Total genomic DNA was extracted from individual adult bed bugs using the phenol-chloroform methodology described by Taggart et al. [20]. DNA concentration of samples was standardized to 20 ng/µL. Bartonella genus screening was performed using a polymerase chain reaction (PCR) assay targeting the 16S–23S ribosomal RNA intergenic transcribed spacer (ITS). The 16S–23S rRNA ITS region has been successfully used for the molecular diagnostic of Bartonella spp. from blood-sucking arthropods, including ticks and lice [21], [22]. For our screening, we used forward primer 438s (5′- GGTTTTCCGGTTTATCCCGGAGGGC-3′) and reverse primer 1100as (5′-GAACCGACGACCCCCTGCTTGCAAAGCA-3′) [23]. The detection limit observed in 100% of 10 replicate PCR reactions was 2.5 DNA copies of B. henselae. Bed bug DNA was spiked with 2.5 copies of B. henselae DNA to determine if PCR inhibitors would interfere with successful amplification. No PCR inhibitors were detected in bed bug DNA. Additionally, to ensure that we could amplify bacterial DNA from bed bug DNA samples, we randomly chose 10 bed bug DNA samples from 10 distinct geographic locations and amplified bacterial DNA using bacteria specific 16S rDNA universal primers: 27F (5′- AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [24]. Bacterial DNA was amplified from all 10 samples, but amplicons were not sequenced.
Bartonella spp. PCR amplification was performed in a 25 µL final volume reaction containing 12.5 µL of Premix Ex Taq™ (Takara Bio Inc.) and 7.5 µL of molecular grade water (Epicentre, Madison, WI). The reaction mixture was completed by adding 0.2 µL of forward and reverse primers (each at 30 µM) and 5 µL of either bed bug DNA template, positive control (with 2.5 copies of Bartonella DNA) or water (as PCR negative control). Amplification of the rRNA ITS region was performed under the following conditions: a hot start cycle of 94°C for 2 min, followed by 55 cycles of denaturing at 94°C for 15 s, annealing at 68°C for 15 s, and extension at 72°C for 18 s. Amplification was completed by an additional cycle of 72°C for 1 min to allow complete extension of PCR products. All amplification products were separated by electrophoresis in a 2% agarose gel and visualized under ultraviolet light with ethidium bromide. Positive and negative controls were used in all reactions and consisted of genomic DNA extracts of B. henselae, and molecular-grade sterile water, respectively. If a sample was found to be positive, the PCR reaction was purified using the QIAquick® PCR purification kit (QIAGEN, Valencia, CA) and sent to an external laboratory for sequencing. Alignment of sequences was performed with the program BlastX in order to identify bacteria at the genus and species levels.
Acknowledgments
We are grateful to Elsa Youngsteadt, J. Swobada, Rafael Valle, Ray Quesenberry, Robert Nagy, Josh Campbell, Shelly Morton, Trevor Wallace, Jim Nakanye, Lon Okamoto, Mauk Duvee, Ernii Spinella, Bob Cook, Michael Hinrichs (Orkin Pest Control), Frank Fowler (McNeely Pest Control), John Dunbar (Terminix), Jeff White (Cooper Pest Solutions) and Rose Pest Solutions for generously providing field-collected bed bugs, and Harold Harlan for providing the Ft. Dix colony. Pedro Diniz, Loganathan Ponnusamy, Richard Santangelo, and Ayako Wada-Katsumata provided valuable technical assistance.
Author Contributions
Conceived and designed the experiments: VLS RGM EBB ELV CS. Performed the experiments: VLS RGM. Analyzed the data: VLS RGM ELV CS. Contributed reagents/materials/analysis tools: VLS RGM EBB JK ELV CS. Wrote the paper: VLS RGM EBB JK ELV CS.
References
- 1.
Potter M, Haynes KF, Rosenberg B, Henriksen M (2011) Bugs without borders: Defining the bed bug resurgence. PestWorld. November/December: 4–15.
- 2.
Usinger R (1966) Monograph of Cimicidae. College Park: Entomological Society of America 585 p.
- 3. Clark S, Gilleard JS, McGoldrick J (2002) Human bedbug infestation of a domestic cat. Vet Rec 151: 336.
- 4. Delaunay P, Blanc V, Del Giudice P, Levy-Bencheton A, Chosidow O, et al. (2011) Bedbugs and infectious diseases. Clin Infect Dis 52: 200–210.
- 5. Blow JA, Turell MJ, Silverman AL, Walker ED (2001) Stercorarial shedding and transtadial transmission of hepatitis B virus by common bed bugs (Hemiptera:Cimicidae). J Med Entomol 38: 694–700.
- 6. Goddard J, Varela-Stokes A, Smith W, Edwards KT (2012) Artificial infection of the bed bug with Rickettsia parkeri. J Med Entomol 49: 922–926.
- 7. Cutler SJ, Fooks AR, Van der Poel WHM (2010) Public health threat of new, reemerging, and neglected zoonoses in the industrialized world. Emerg Infect Dis 16: 1–7.
- 8. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR (2010) Bartonellosis: an emerging infectious disease of zoonotic importance to animals and human beings. J Vet Emerg Crit Car 20: 8–30.
- 9. Jameson P, Greene C, Regnery R, Dryden M, Marks A, et al. (1995) Prevalence of Bartonella henselae antibodies in pet cats throughout regions of North America. J Infect Dis 172: 1145–1149.
- 10. Brouqui P (2011) Arthropod-borne diseases associated with political and social disorder. Annu Rev Entomol 56: 357–374.
- 11. Lowe CF, Romney MG (2011) Bedbugs as vectors for drug-resistant bacteria. Emerg Infect Dis 17: 1132–1134.
- 12. Richard S, Seng P, Parola P, Raoult D, Daboust B, et al. (2009) Detection of a new bacterium related to ‘Candidatus Midichloria mitochondrii’ in bed bugs. Clin Microbiol Infect 15: 84–85.
- 13. Sassera D, Beninati T, Bandi C, Bouman EAP, Sacchi L, et al. (2006) ‘Candidatus Midichloria mitochondrii’, an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle. Int J Sys Evol Microbiol 56: 2535–2540.
- 14. Reeves WK, Loftis AD, Gore JA, Dasch GA (2005) Molecular evidence for novel Bartonella species in Trichobius major (Diptera: Streblidae) and Cimex adjunctus (Hemiptera: Cimicidae) from two southeastern bat caves, USA. J Vector Ecol 30: 339–341.
- 15. Reeves WK, Rogers TE, Durden LA, Dasch GA (2007) Association of Bartonella with the fleas (Siphonaptera) of rodents and bats using molecular techniques. J Vector Ecol 32: 118–122.
- 16. Balvin O, Munclinger P, Kratochvil L, Vilimova J (2012) Mitochondrial DNA and morphology show independent evolutionary histories of bedbug Cimex lectularius (Heteroptera: Cimicidae) on bats and humans. Parasitol Res 111: 457–469.
- 17. Sakamoto JM, Rasgon JL (2006) Geographic distribution of Wolbachia infections in Cimex lectularius (Heteroptera: Cimicidae). J Med Entomol 43: 696–700.
- 18. Klempner MS, Unnasch TR, Hu LT (2007) Taking a bite out of vector-transmitted infectious diseases. N Engl J Med 356: 2567–2569.
- 19. Coenye T, Mahenthiralingam E, Henry D, LiPuma JJ, Laevens S, et al. (2001) Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol 51: 1481–1490.
- 20. Taggart JB, Hynes RA, Prodöhl PA, Ferguson A (1992) A simplified protocol for routine total DNA isolation from salmonid fishes. J Fish Biol 40: 963–965.
- 21. Billeter SA, Miller MK, Breitschwerdt EB, Levy MG (2008) Detection of two Bartonella tamiae-like sequences in Amblyomma americanum (Acari: Ixodidae) using 16S–23S intergenic spacer region-specific primers. J Med Entomol 45: 176–179.
- 22. Sasaki T, Tomita T, Sawabe K, Kobayashi M, Seki N, et al. (2006) First molecular evidence of Bartonella quintana in Pediculus humanus capitis (Phthiraptera:Pediculidae), collected from Nepalese children. J Med Entomol 43: 110–112.
- 23. Beard AW, Maggi RG, Kennedy-Stoskopf S, Cherry NA, Sandfoss MR, et al. (2011) Bartonella spp. in feral pigs, Southeastern United States. Emerg Infect Dis 17: 893–895.
- 24.
Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. New York: John Wiley and Sons. 115–175.