Are Phages the An­swer?

by S. Mar­vin Fried­man

The emer­gence of mul­ti­ple drug-re­sis­tant bac­te­r­ial strains, the preva­lence of re­cal­ci­trant biofilm con­fig­u­ra­tions, and the re­luc­tance of the phar­ma­ceu­ti­cal in­dus­try to ini­ti­ate new an­tibi­otic dis­cov­ery pro­grams have led to the de­vel­op­ment of a for­mi­da­ble pop­u­la­tion of bac­te­r­ial pathogens that is in­creas­ingly dif­fi­cult to con­trol. Af­ter a long but suc­cess­ful era of re­search that had all but elim­i­nated se­ri­ous threats from bac­te­r­ial in­fec­tions, we are now fac­ing this dire prob­lem once again. In re­sponse, re­searchers have re­cently been ex­plor­ing al­ter­na­tive ap­proaches to an­tibi­otic ther­apy in­clud­ing iden­ti­fy­ing chem­i­cal agents that an­tag­o­nize quo­rum sens­ing and thus pre­vent pop­u­la­tion-wide ex­pres­sion of vir­u­lence genes, as well as em­ploy­ing ei­ther in­tact bac­te­rio­phages or their iso­lated lysins to di­rectly kill their path­o­genic bac­te­r­ial hosts. Lysins kill Gram-pos­i­tive bac­te­ria by hy­drolyz­ing the pep­ti­do­gly­can in the cell wall, thereby caus­ing cell ly­sis. Gram-neg­a­tive bac­te­ria are im­mune to their ac­tion be­cause their outer mem­brane does not al­low the lysins ac­cess to their pep­ti­do­gly­can. I will now sum­ma­rize two re­cent pa­pers that use in­tact phages to com­bat two im­por­tant bac­te­r­ial pathogens, both in vitro and in vivo.

Pseudomonas. aerug­i­nosa colonies show­ing the cha­racteristic green color. Source: Glo­ria Delisle, Mi­crobe Li­brary, ASM.

One of the im­por­tant ap­pli­ca­tions for phage ther­apy is for treat­ing cys­tic fi­bro­sis (CF). CF is an in­her­ited ge­netic dis­or­der where a de­fec­tive en­zyme re­sults in the pro­duc­tion of un­usu­ally vis­cous, sticky mu­cus and chlo­ride-con­tain­ing se­cre­tions in ducts and body cav­i­ties. The lungs, in par­tic­u­lar, are se­ri­ously com­pro­mised and are read­ily in­fected, typ­i­cally by Pseudomonas aerug­i­nosa. Ini­tial col­o­niza­tion usu­ally oc­curs dur­ing early child­hood. The en­su­ing chronic in­fec­tion even­tu­ally causes death due to res­pi­ra­tory fail­ure in 80–95% of CF pa­tients. Treat­ment of these pa­tients is im­peded by the mul­ti­ple mech­a­nisms of an­tibi­otic re­sis­tance har­bored by these strains of P. aerug­i­nosa and by their abil­ity to form biofilms in the lung.

(A) Growth of lux-tagged Pseudomonas biofilms on the sur­face of a CF-bronchial ep­ithe­lia cell mono­layer. Light was mea­sured 1, 5, and 24 h. (B) Read­ings from 6 wells. Source

A re­search team has re­cently in­ves­ti­gated the ef­fi­cacy of phage ther­apy in the treat­ment of CF. They iso­lated two phages (φMR299‑2 and φNH‑4) from sewage and showed that both were vir­u­lent for P. aerug­i­nosa. The viri­ons of one have the iso­met­ric cap­sids and short tails char­ac­ter­is­tic of mem­bers of the fam­ily Podoviri­dae, whereas the other pos­sesses an iso­met­ric cap­sid and long con­trac­tile tail and there­fore is clas­si­fied in the My­oviri­dae. The au­thors used equal num­bers of the two phages in all ex­per­i­ments.

The re­searchers em­ployed bi­o­lu­mi­nes­cence imag­ing to as­sess the abil­ity of this phage mix to kill Pseudomonas cells in biofilms on the sur­face of a mono­layer of a cys­tic fi­bro­sis bronchial ep­ithe­lial cell line. They in­oc­u­lated con­flu­ent mono­lay­ers with two lux-tagged Pseudomonas strains, one mu­coid, the other non-mu­coid. They also pro­vided argi­nine, which is re­quired for good biofilm for­ma­tion. Af­ter 24 hours, bi­o­lu­mi­nes­cence in­dica­tive of the grow­ing bac­te­r­ial biofilm in­creased one hun­dred-fold. Most of the lux-tagged Pseudomonas cells re­sisted wash­ing, in­di­cat­ing that they were an­chored to the ep­ithe­lial mono­layer. Nu­mer­ous Pseudomonas cells were packed within the biofilm ma­trix and at­tached to the ep­ithe­lial cells, as shown by stain­ing with Cal­co­fluor white, a flu­o­res­cence dye that binds to poly­sac­cha­rides in biofilm ma­tri­ces.

Is the phage mix­ture ef­fec­tive against the Pseudomonas biofilms? When the Pseudomonas cells were cul­tured for 24 hours in the pres­ence of the phages, Cal­co­fluor white stain­ing showed only weak and open ma­tri­ces, in­di­cat­ing con­sid­er­able dis­rup­tion of the biofilm ar­chi­tec­ture. Phage titers in­creased about one hun­dred-fold dur­ing the 24 hour pe­riod, con­firm­ing that sub­stan­tial phage repli­ca­tion had oc­curred. In ad­di­tion, di­rect plat­ing con­firmed that this re­duced lu­mi­nes­cence re­sulted from the killing of Pseudomonas cells by the phages. The con­clu­sion is that the phage mix­ture was ef­fec­tive in killing Pseudomonas cells em­bed­ded in biofilms grow­ing on a bronchial ep­ithe­lial cell line.

Flu­o­res­cent im­age of 24-h-old cul­ture of P. aerug­i­nosa cells grown on a CF-ep­ithe­lial cell mono­layer af­ter Cal­co­fluor white stain­ing. Stain­ing con­firms that P. aerug­i­nosa strain NH57388A (A) and strain MR299 (B) are em­bed­ded in an exopoly­saccharide struc­ture prior to phage ex­po­sure. Af­ter 24‑h in­cu­ba­tion with mixed phages, ma­tri­ces be­come open and, with re­duced num­bers of cells for both NH57388A (C) and MR299 (D). (E) The phage titers over the 24‑h in­cu­ba­tion. Source

To test ef­fec­tive­ness of the phages in vivo, they in­fected 8 week-old fe­male BALB/c mice in­tranasally with lux-tagged Pseudomonas and con­firmed the pres­ence of the la­beled bac­te­ri­ain the lungs 2 hours later. No­tice the sim­ple el­e­gance of this model. Not only do the Pseudomonas cells lo­cal­ize al­most eclu­sively in the res­pi­ra­tory tract, but their where­abouts can be seen by sim­ply tak­ing the mice to a dark room. At the 2 hour point, the re­searchers di­vided the mice into con­trol and test groups and in­oc­u­lated the test group in­tranasally with the phage mix­ture. At 6 hours, the lu­mi­nes­cence recorded in lungs of the con­trol group had reached its max­i­mum level, a three-fold in­crease. The lu­mi­nes­cence recorded from the phage-treated mice de­creased sig­nif­i­cantly dur­ing the same pe­riod. It sounds promis­ing.

The story of phages as ther­a­peu­tic tools goes back to their ear­li­est his­tory. In 1919, D'Herelle, one of their co-dis­cov­er­ers, suc­cess­fully treated a pa­tient with dysen­tery. Over the course of time, phage ther­apy has had its ups and downs. The al­lure of us­ing phages when other mea­sures fail has con­tin­ued and cur­rent day re­search may well re­store them to ade­served place in the an­timi­cro­bial ar­ma­men­tar­ium.

Cur­rently, phage ther­apy is suc­cess­fully prac­ticed in sev­eral Eu­ro­pean coun­tries such as Geor­gia, Poland, and Rus­sia. In the United States, the Food and Drug Ad­min­is­tra­tion has re­cently ap­proved the use of phage on food to pre­vent con­t­a­m­i­na­tion of meat and poul­try. Re­luc­tance to ex­tend the use of phage ther­apy to con­trol bac­te­r­ial in­fec­tions in hu­mans stems from con­cerns over im­muno­genic re­ac­tions due to the pres­ence of large num­bers of phage in the cir­cu­la­tion. In ad­di­tion, the re­lease of en­do­tox­ins from Gram-neg­a­tive bac­te­ria at­tacked poses an­other po­ten­tial prob­lem. Lastly, de­fined dosages can­not be for­mu­lated since suc­cess­ful treat­ment re­leases more prog­eny phage, thus po­ten­tially in­creas­ing the "dosage" avail­able to re­in­fect host cells.

Mice were in­fected with non­mu­coid P. aerug­i­nosa MR299 (A) and mu­coid NH57388A (B). Test mice (+) were treated with the phage mix. Phage was given 2 h af­ter in­fec­tion with Pseudomonas. Con­trol mice (−) did not re­ceive the phage mix. Source

Paul et al. have over­come these ob­jec­tions by en­gi­neer­ing a lysin-de­fi­cient phage. For this pur­pose, they mod­i­fied a tem­per­ate phage (P954) that in­fects Staphy­lo­coc­cus au­reus. Be­cause me­thi­cillin-re­sis­tant strains of S. au­reus (MRSA) in­fect about 94,000 peo­ple in the U.S. each year, re­sult­ing in al­most 19,000 deaths, new ther­a­pies are ur­gently needed. The re­searchers dis­rupted the na­tive lysin gene car­ried on the prophage by in­tro­duc­ing the chlo­ram­pheni­col acetyl trans­ferase (cat) gene via ho­mol­o­gous re­com­bi­na­tion us­ing a plas­mid con­struct. A lysin-de­fi­cient phage does not de­grade the bac­te­r­ial cell wall and thus can­not lyse the cell, but the phage still pro­duces a holin pro­tein that cre­ates gap­ing holes in the in­ner mem­brane at the close of the phage repli­ca­tion cy­cle. As a re­sult, the cell can no longer carry out res­pi­ra­tion and death rapidly fol­lows. To test the ef­fi­cacy of lysin-de­fi­cient phage P954, they in­jected im­muno­com­pro­mised mice IP with the MRSA iso­late B911 at a dose that causes 80% mor­tal­ity. IP ad­min­is­tra­tion of lysin-de­fi­cient phage P954 im­me­di­ately and 2 hours af­ter chal­lenge fully pro­tected the mice from the lethal ef­fect of MRSA. Im­por­tantly, the lysiin-de­fi­cient phage alone was nei­ther toxic nor lethal for the mice. Thus, lysin-de­fi­cient phage P954 ap­pears to be an ex­cel­lent can­di­date for treat­ing dif­fi­cult MRSA in­fec­tions in hu­mans.

Phage ther­apy has had a check­ered and in­tense his­tory. The fi­nal word may not be in, but stud­ies such as these make it ap­pear that it has a sub­stan­tive fu­ture. That would be a good thing, not only for cys­tic fi­bro­sis pa­tients, but per­haps for all who suf­fer from re­cal­ci­trant in­fec­tions.

Ref­er­ences

Ale­mayehu D, Casey PG, McAu­li­ffe O, Guinane CM, Mar­tin JG, Shana­han F, Cof­fey A, Ross RP, Hill C (2012). Bac­te­rio­phages φMR299‑2 and φNH‑4 can elim­i­nate Pseudomonas aerug­i­nosa in the murine lung and on cys­tic fi­bro­sis lung air­way cells. mBio, 3 (2). PMID 22396480

Paul VD, Sun­dar­ra­jan S, Ra­jagopalan SS, Har­i­ha­ran S, Kem­pashana­iah N, Pad­man­ab­han S, Sri­ram B, Ra­machan­dran J (2011). Ly­sis-de­fi­cient phages as novel ther­a­peu­tic agents for con­trol­ling bac­te­r­ial in­fec­tion. BMC mi­cro­bi­ol­ogy, 11. PMID 21880144

Mar­vin is Pro­fes­sor Emer­i­tus in the De­part­ment of Bi­o­log­i­cal Sci­ences at Hunter Col­lege of CUNY in New York City, and an As­so­ciate Blog­ger for Small Things Con­sid­ered.