Pitting Microbes against One AnotherTo quell infectious disease, investigators are testing whether other microbes canhelp to hold pathogens in check

Shannon Weiman

To quell a variety of infectious diseases, scientistsare engaging nonpathogenic bacteria and virusesto counteract the culprit microbial pathogensthat so far resist more conventional control ap-proaches. This strategy is being pursued against arange of pathogens, including those responsiblefor causing dengue fever in humans, Americanfoulbrood in bees, and bleaching in coral, accord-ing to several investigators who described theirrespective approaches during the 2012 ASMGeneral Meeting, held in San Francisco, Calif.,last June.

Although microorganisms traditionally wereviewed as the problem, now they are being eyedat least sometimes as potential allies in the faceof the complex challenges that infectious diseasescan pose. Not so long ago, that challenge wasframed almost exclusively in bellicose meta-phors. More recently, that tone is softening asnew alliances take shape, and investigators ex-plore amore subtle set of possibilities for treatingmedical, agricultural, and ecological problemswith infectious components.

Bacteria Burden Insects ThatCarry the Dengue Virus

The virus responsible for causing dengue fever inhumans infects as many as 100 million individu-als per year, according to Scott O’Neill ofMonashUniversity in Australia, who spoke during theopening session symposium, “Harnessing thePower of Microbes for Biomedicine.” The num-bers of people who develop dengue fever exceedthose who become infected with other arthro-pod-borne viruses, called arboviruses, includingthe West Nile and yellow fever viruses. Denguefever thus is the leading cause of illnesses anddeath among arbovirus-caused infections, and itsimpact is second only to that of malaria amongtropical diseases.

Although endemic to the tropics, the denguevirus recently beganmoving intomore temperateareas, carried along with its main vector host,the Aedes aegypti mosquito. “Dengue hemor-rhagic fever has expanded from Southeast Asiato 28 countries in theWesternHemisphere,” saysO’Neill. “The severity and frequency of dengueoutbreaks is rapidly increasing across the world.”Indeed, incidence of dengue has risen 30-foldin the past 50 years, despite various attempts toslow the spread of disease. Vaccine development,antiviral drugs, and strategies aimed at reducingmosquito populations all failed to prevent, treat,or control dengue thus far, leaving 40% of theworld’s population at risk.

O’Neill is taking a novel approach, harnessingbacteria to thwart the dengue virus. Specifıcally,he and his collaborators infect the mosquitoesthat harbor this virus with Wolbachia pipientis,reasoning that this new biological burden on thevector mosquito may reduce its capacity to sup-port viral replication and transmission to addi-tional humans.

Wolbachia species are found in 65% of all in-sect species, including 28% of mosquito speciesthatwere surveyed. In general, these intracellular,maternally inherited bacteria form symbiotic re-lationships with their hosts, in many cases pro-

SUMMARY

➤ To quell a variety of infectious diseases, scientists are engaging nonpatho-genic bacteria and viruses to counteract the culprit microbial pathogensthat so far resist more conventional control approaches.

➤ One approach to controlling the dengue virus involves infecting mosqui-toes that serve as its vector with bacteria that impair both the insect andthe virus.

➤ Bacteriophage may help to control the bacteria responsible for causingAmerican foulbrood in honeybees, a devastating disease that can contrib-ute to colony collapse.

➤ Phage may also help to control bacterially caused bleaching of coral reefs.

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tecting their hosts against pathogens and para-sites, including nematodes and viruses as well asthose that cause malaria. “An important featureofWolbachia is its ability to induce resistance toa variety of pathogens, including dengue virus,in its insect hosts,” says Zhiyong Xi of MichiganState University in East Lansing, who collabo-rates with O’Neill.

Although the mechanism remains to be deter-mined,Wolbachia can inhibit dengue replicationin mosquito cell lines and in live insects, accord-ing to O’Neill, Xi, and their collaborators as wellas other researchers. For example, Wolbachiamay boost the insect immune system, priming itto attack the dengue virus. Host immune genesare up-regulated after being infectedwithWolba-chia, O’Neill reports. Meanwhile, Xi and his col-laborators fınd reactive-oxygen-species (ROS)-dependent activation of the Toll pathway andantimicrobial peptide defensin DEFD expres-sion. Another possibility is that the Wolbachiabacteria may outcompete the dengue virus forinsect host cell resources. Dengue replication is

inversely correlated withWolbachia density, andthe virus is excluded from cells and tissues withvery high densities of those bacteria, according toboth Xi and O’Neill.

The overreplicating strain of Wolbachia,calledwMelPop, reduces the lifespan of its nativehost Drosophila melanogaster by 50%. This fea-ture led O’Neill to think that this strain wouldmake a good candidate for controlling dengue,which is transmitted by mosquitoes only towardthe end of their 30-day lifespan. If the wMelPopstrain could kill or otherwise hampermosquitoesduring the 7–14 days in which dengue virusesreplicate within this host insect, it could drasti-cally reduce dengue virus transmission, he says.

O’Neill adapted wMelPop to infect Aedesaegypti, the primary mosquito vector for thedengue virus, but a species that does not naturallyharbor Wolbachia. Once adapted to the mos-quito, wMelPop retains its life-shortening pheno-type vis a vis its new mosquito host, and alsoinhibits dengue replication within that host.However, wMelPop does not sustainably in-

FIGURE 1

Larvae of Aedes aegyptimosquitoes, the main vector for the viruses that cause dengue fever. Researchers are usingthe bacterium Wohlbachia pipientis to inhibit dengue virus replication within the vector in attempts to controlspread of the disease. (Photo © Andy Crump, TDR, World Health Organization/Science Source.)

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fect mosquito populations, according to O’Neill.“Wolbachia strains that provide greater disrup-tion to dengue transmission also confer greaterfıtness costs to the mosquito host, and successfulinvasion for dengue control therefore requires aWolbachia strain that balances these two effects,”he says.

O’Neill and his collaborators redirected theirefforts to anotherWolbachia strain,wMel, whichlacks the overreplication phenotype and exacts alesser toll on host fıtness. Because wMel infectsmosquitoes raised in the lab and inhibits denguevirus under those protected conditions, O’Neillnext sought to test this dengue-control scheme infıeld trials.

Following regulatory approval from Austra-lian government offıcials and after receivingcommunity support, O’Neill released approxi-mately 300,000wMel-infectedmosquitoes at twosites in north Queensland, Australia. The trialsproved successful, with Wolbachia invading na-tive populations to 80–100% penetrance withina few months and stabilizing thereafter. Individ-ual mosquitoes from these communities exhibitantidengue effects when recaptured and tested inthe laboratory. However, the impact on denguewithin human populations remains to be deter-mined.

O’Neill plans to continue monitoring theseparameters in Australia, and hopes to expandfıeld testing to sites in Vietnam, Thailand, andother countries where dengue is prevalent. “Wehave demonstrated that it is possible to produceWolbachia-infected mosquito populations thatcan act as ‘nursery’ areas for future human-assisted collection and further dispersal ofWolbachia-infected mosquitoes, without theneed to rear additional mosquitoes in an insec-tary,” he says. “This should provide a strategyfor sustainable dengue control at low cost, with arelatively simple deployment system suitable forimplementation in developing countries.”

Wolbachia may also be useful in curbing ma-laria, according to ThomasWalker of theUniver-sity of Queensland, Australia, who collaborateswith O’Neill. The wMelPop strains ofWolbachiacan inhibit replication of various Plasmodiumspecies—the parasites that cause malaria—within their mosquito hosts, which do not natu-rally harbor that bacterial species. Immune acti-vation and competition for resources may con-tribute to this effect. “Plasmodium is dependenton host lipids, suggesting cholesterol could be a

critical nutrient required by bothWolbachia andthe mosquito-borne pathogen,” Walker says.

However, application of this approach re-quires stable transfection of theAnopheles speciesof mosquitoes, which are responsible for har-boring and transmitting Plasmodium parasites.While Wolbachia strains can be maintained inAnophelesmosquito cell lines, all attempts to sta-bly transfect these species have failed. Ultimately,the successful use of Wolbachia against malariaand other vector-borne diseases will rest on theability to transfect the bacteria intomosquito spe-cies that serve as vectors for pathogens and para-sites, and the subsequent integration of those bac-teria into wild mosquito populations.

Viruses Help to Control Bacterial AmericanFoulbrood Disease in Honeybees

American foulbrood disease (AFD) is anotherinstance inwhichmicroorganisms are being eval-uated as a means for treating an infectious dis-ease. However, in this case, viruses are beingused to combat bacterial infections, according toPenny Amy and Diane Yost of the University ofNevada, Las Vegas, and their collaborators. Theiraim is to identify and then harness viruses—bac-teriophages—to rid honeybees of the bacterialinfection that leads to AFD, which may also con-tribute to colony collapse disorder. While phagetreatment of honeybee larvae is yet to be fullytested, this approach appears promising in theongoing battle against AFD,which has continuedfor nearly a century. Amy and Yost presentedtheir fındings during the poster session, “Mor-phogenesis, Evolution andEcology,” convened aspart of the ASM General Meeting in San Fran-cisco last June.

American foulbrood disease is caused by thegram-positive bacterium Paenibacillus larvae.These highly contagious bacteria infect and killhoneybee larvae, leading ultimately to collapse ofentire hives. “Honeybee health is of great concerndue to the importance of honeybees as pollinatorsin the agriculture of the United States and there-fore the current and future food supply,” Yostsays. Moreover, despite its name, AFD is wide-spread and a highly destructive brood diseaseaffecting honeybees worldwide, and second onlyto the parasiticmiteVarroa destructor as themosteconomically important disease of honeybeesglobally, according to bee expert Elke Genersch

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of the Institute for Bee Research in Neuendorf,Germany.

Conventional antibiotics pose numerousproblems when used in treating American foul-brood, according to Genersch. They disrupt thevitality and longevity of bees, partly by disruptingbenefıcial gut microbiota, and compromise thequality and safety of honey that the bees produce.More importantly, antibiotics are not particularlyeffective in controlling the disease, particularlyas P. larvae continue to develop resistance tosuch treatments. “Antibiotics only suppress clin-ical symptoms and mask the disease, but cannotcure it,” she says.

Incineration is the most effective treatmentagainstAFDbecause it kills growingP. larvae andspores. Although local authorities may insist thatbee keepers burn infected hives to prevent thespread of AFD, this approach is extremely costlyand threatens agricultural productivity in generalbecause honeybees pollinate crops. Indeed, fruits,nuts, and vegetables are among the crops thathoneybees pollinate, accounting for about one-

third of the U.S. food supply. “Honeybees are themost economically valuable pollinators of cropand fruit monocultures worldwide,” Generschsays.Within the United States, honeybee pollina-tion was valued at $19 billion per year in 2010,according to a study conducted by agronomists atCornell University in Ithaca, N.Y.

In their search for an alternative and moresustainable way of treating AFD, Yost and hercollaborators identifıed dozens of bacteriophagesthat infect and kill P. larvae bacteria. They iso-lated these viruses from environmental sources,including desert and garden soil, hive wax,honey, flowers, plants, compost, dead bees, andcosmetics containing beeswax. These phages arelytic to various strains of P. larvae, but not otherbacteria such as Escherichia coli. Of particularimportance for the bees, the phages apparently donot affect the microbiota of the honeybee gut orother bacteria that are found in hives. Yost andher collaborators plan to characterize the phagefurther, including through DNA profıling andelectron microscopy.

FIGURE 2

Honeybees in a hive. American foulbrood disease is an economically devastating disease caused by the bacteriumPaenibacillus larvae. Researchers are working on treatments with phage that could help protect bee colonies fromthe disease. (Photo © fatchoi/iStockphoto.)

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“Phage therapy is a safer solution than antibi-otics because phages are already naturally presentin the environment and not harmful to humansor bees,” Yost says. “We are not introducing for-eign or genetically modifıed organisms into theenvironment.” Treating AFD with phage carriesadvantages over antibiotics. P. larvae are lesslikely to develop resistance against phages, partic-ularly if several phage types are applied simulta-neously. Also, unlike antibiotics, phages will notinfect or affect nearby animals or plants.

The P. larvae-infecting phage remain viablefor at least three months, based on experimentsdesigned to mimic conditions in the hive andlarval gut, according to Yost. “A hive could besprayed with phage whether it is known to beinfected or not, and once the bee larvae ingest thephage, they would be protected against infectionfor a few months,” she says. The presence ofphage in and around the hive would also targetbacteria not yet ingested by bee larvae, whetherfrom cross contamination with other hives orfrom germinating spores.

Phage Eyed to Combat Coral Bleaching

Eugene Rosenberg of Tel Aviv University in Is-rael is also harnessing bacteriophage, but in thiscase as a potentialmeans for defending coral reefsagainst bacterial infections. “Over the past 30years, there has been an approximately 30%worldwide decline in the coral population, largelydue to emerging diseases,” he says. “At the globalscale, coral bleaching is the most serious diseasethreatening coral reefs.” Rosenberg presentedsome of his recent results on this research duringthe poster session “Regulation, Replication, Re-combination, and Biotechnology,” during the2012 ASM General Meeting in San Francisco.

Bacterial pathogens cause coral bleaching, ac-cording to Rosenberg. He and his colleagues haveidentifıed several species that cause bleachingin specifıc corals of the Mediterranean, IndianOcean, and Red Sea. The bacteria produce a toxinthat inhibits photosynthesis by endosymbioticalgae, which provide coral with oxygen and car-bon-containing nutrients. Without algae to pro-

FIGURE 3

Bleached coral, a widespread problem in warm ocean environments. Researchers are looking at phages to fight oneof the bacterial pathogens that cause coral bleaching, Thalossomonas loyana. (Photo © Lee Chin Yong/iStockphoto.)

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vide these essential nutrients, the growth rate ofthe coral is impaired, as is its reproductive capac-ity, making it susceptible to secondary diseases.“If the process is not reversed within a few weeksor months, depending upon the specifıc coralspecies and conditions, the coral dies, since amajor portion of a coral’s nutrition comes fromthe photosynthetic products of algae,” Rosenbergsays.

Treating infected corals is challenging. “Im-munization and antibiotic treatment, two widelyusedmethods for controlling humandiseases, arenot applicable to coral disease because coralshave no adaptive immune system, and introduc-tion of antibiotics in an open system such as acoral reef is not practical,” Rosenberg says. Bac-teriophages overcome at least some of those dis-advantages.

For example, the phage called BA3 infects andkills one of the bacterial pathogens, Thalossomo-nas loyana, that infect corals—in this case, theone responsible for white plague-like (WPL) dis-ease in Favia favus coral in the Red Sea. Whenadded to diseased coral, this phage stops diseaseprogression and prevents its transmission toother corals. Phage-treated corals suffered only5% tissue death, compared to 65% in untreatedcontrols, according to Rosenberg. In these fıeldexperiments, phage-treated diseased corals trans-mitted the pathogen to only 1 of 19 surrounding

healthy corals. In comparison, untreated-dis-eased corals transmitted infection to 11 out of 18surrounding healthy corals.

However, timing is crucial; phage must be ap-plied within 24 hours after T. loyana infects thecoral to prevent damage, Rosenberg continues.Hence, he adds, “Phage therapy may be morevaluable in preventing the spread of diseaserather than curing an already infected coral.” Inlaboratory experiments, phage treatments effec-tively inhibit transmission of the bacterial patho-gens independent ofwhen the phages are applied.Another concern is that the target pathogen, T.loyana, is likely to develop resistance to phage.“One possibility for overcoming this potentialproblem is to carry out phage therapy in the fıeldwith more than one T. loyana-specifıc lyticphage,” he says.

All in all, Rosenberg is optimistic about usingphage to treat coral disease, emphasizing howenvironmentally safe it is. For instance, BA3treatment does not harm other species of nearbycoral, and is also safe for food and aquacultureapplications. Another advantage is that phagereplicate to replenish the supply, he adds. “Thephage multiplies rapidly at the expense of its hostbacterium, increasing the phage titer for moreeffective control of the pathogen.” Moreover, thephage is highly specifıc, sparing benefıcial micro-organisms within the coral community.

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