Thursday, June 5, 2008

Suggestion to reduce antibiotic resistant skin infections

in Hospitals, Nursing Homes and Hospices using topical ammonia oxidizing bacteria

Skin and wound infections involving antibiotic resistant organisms are serious problems in medical care. Hospitals and nursing homes present difficult infection control challenges because diverse patients carry different organisms and presenting different immune system status. The large influx of patients, staff, visitors and supplies, with diverse resident microorganisms ensures that a hospital is an open system in terms of microbiology. It is suggested that introducing and maintaining a dominant population of autotrophic ammonia oxidizing bacteria in the hospital and patient environment may significantly reduce hospital transmitted infections while also reducing selection for antibiotic resistance.

The conventional approach to infection control is the liberal use of soap and detergents to remove soil, use of chemical disinfectants [1] including oxidants such as bleach, iodine and hydrogen peroxide; cell membrane disruptants such as alcohols, cationic surfactants and phenolics and the rare and judicious use of antibiotics. Antibiotics are agents which interfere with a microorganism's internal metabolism in a fairly specific way (as opposed to non-specific oxidants and membrane disruptants). Some antimicrobial agents are being used indiscriminately such as triclosan, which inhibits lipid synthesis in target organisms.[2] Use of triclosan containing cleaning products does lead to increased resistance of exposed microorganisms to triclosan and also to other antibiotics [3] (the therapeutic and statistical significance of this is complex, a single resistant clone may be medically significant even if most clones stay susceptible). Use of antibacterial household products does not reduce the incidence of disease symptoms. [4]

Antibiotics should be used sparingly because use eventually leads to antibiotic resistance. Every time an antibiotic is used, organisms are exposed to different levels as the administered dose is absorbed, transported throughout the body, and finally metabolized or excreted. If the exposure level is less than the toxic dose, the organism will survive and if resistant will pass that resistance down to its descendents. Non-target non-pathogenic organisms can develop resistance, and this can be transferred to pathogens via plasmids. Even non-antibiotic disinfectants, such as pine oil can cause broader resistance to antibiotics through upregulation of multi-drug resistance pathways. [5] These are ATP powered transporters which excrete target compounds including antibiotics. Upregulation of these efflux pathways can cause broad spectrum resistance to many antibiotics and chemical disinfectants. [6]

In addition to the problem of resistance, killing all microorganisms including non-pathogens leaves those niches completely open for the next organism that appears. Re-inoculation of surfaces cannot be avoided because microorganisms are ubiquitous in the environment and on patients. An adult human contains more bacterial cells than human cells. [7] Mostly in the gut, but every surface exposed to the external environment including the skin, mouth, gut all have characteristic resident microorganisms. The resident commensal microbiota is a normal part of what protects against infection by pathogenic organisms. When that normal flora is disrupted, as with systemic antibiotics, the now empty niche can be colonized by pathogens, as for example a yeast infection following antibiotics.

Many pathogenic organisms can survive long term on dry inert surfaces, days, weeks and even months. [8]

I am working with organisms previously unrecognized as commensal Autotrophic Ammonia Oxidizing Bacteria (AOB). AOB are widely known in the environment, where they perform the first step in the process of nitrification, the oxidation of ammonia to nitrite. They are found in virtually all soils and all sources of water including ground, surface and sea water. I have found that AOB live on the external surfaces of many eukaryotes, where they provide a first line of defense in keeping those organisms from acquiring surface infections.

AOB are obligate autotrophs. They derive ATP only from oxidation of ammonia into nitrite and are incapable of deriving ATP from oxidation of organic compounds. There has been no report of an infection with any of these organisms and it is likely that an infection is not possible. They produce no toxins, and have no transporters to excrete them. They are slow growing with optimum doubling times of ~10 hours. They are incapable of growth on any media used to isolate pathogens.

The major product of commensal AOB is nitrite, produced from ammonia in sweat or bodily secretions. Acidified nitrite is a very potent anti-microbial and has been shown to kill antibiotic resistant organisms such as MRSA and VRE. [9] The normal pH of the skin is about 4, sufficiently acid that nitrite disproportionates to NO and NO2 and sufficiently acid that nitrite has potent antimicrobial effects. AOB are virtually completely resistant to nitrite, acidified nitrite, NO, even NO2 at levels that would be acutely toxic to other organisms (e.g. 600 ppm NO, 100 ppm NO2) [10]. Acidified nitrite is synergistically (~100x) toxic to some organisms in the presence of H2O2 and lactate. [11] Lactic acid bacteria are a large class of commensal organisms, particularly on the skin and vagina where some of the most persistent produce H2O2 [12], and because they lack hemes are fairly resistant to NOx toxicity. It is our hypothesis that AOB are natural human commensal organisms, and in conjunction with Lactic acid bacteria provide a first line of defense against skin pathogens for humans and for many eukaryotes.

AOB are associated with corrosion of natural stone, particularly calcareous stone. [13] The mechanism seems to be production of low pH via oxidation of ammonia to nitrite, the low pH then traps more ammonia from the atmosphere and the low pH causes loss of NO/NOx. Too low a pH reduces NH3 availability to the bacteria, too high a pH causes loss to the atmosphere. The growth rate is higher on calcareous rock likely because as the rock dissolves it releases trace minerals needed for growth. The only substrates needed are ammonia, CO2, O2 and minerals.

While NO/NOx species are broadly antimicrobial, the main mechanism for preventing surface infection may not one of organism death (which organisms can evolve resistance to) but rather disruption of quorum sensing (evolved resistance to disruption of quorum sensing is much more difficult and may necessitate loss of pathogenicity). .

Many microorganisms communicate by what is called quorum sensing. [14] Organisms producing a specific chemical which diffuses away, but when a sufficient number of clones of the organism are present the local concentration builds to a level that triggers a change to a different phenotype. Usually this transition is associated with the expression of virulence factors, adherence, toxins, exporters, proteases, biofilm formation. If these virulence factors are not expressed, the organism remains non-virulent even if it is a virulent strain. Virulence factors can only cause disease if they are expressed. Even transient interference with quorum sensing blocks formation of abscesses by Staphylococcus aureus. [15]

Gram-negative bacteria often use acyl homoserine lactones as quorum sensing compounds. Some eukaryotes interfere with these signals as part of their defense against infection. These interference mechanisms include oxidation [16] by hypochlorite, superoxide and NOx, and displacement by halogenated furanones. [17]

Nitric oxide is a signaling molecule used by some biofilm formers to transition from a biofilm phenotype to a planktonic type. That is, low NO triggers the transition to form a biofilm and high NO inhibits it. Bacteria in biofilms are much more resistant to antimicrobial agents both antibiotics and antiseptics. The inhibitory concentration in a biofilm is higher sometimes by more than a factor of 500. [18] Many infections particularly persistent infections are characterized by the formation of biofilms [19] which is not surprising because therapeutic doses cannot be arbitrarily increased to counter the increased resistance of biofilms. Nitrite inhibited the formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis, and caused dissociation of biofilms already formed. [20] When formation of quorums sensing compounds is blocked, even agents that are virulent pathogens exhibit reduced dissemination and reduced mortality. [21] The main virulence factor of Staphylococcus epidermidis is formation of a biofilm and this is triggered through quorum sensing. [22]

An advantage of the AOB as commensal organism suppressing heterotrophic pathogenic organisms is that AOB have very simple genomes [23] and an almost complete inability to metabolize organic compounds. It is quite likely that they would be extremely slow to evolve resistance to antibiotic compounds (if at all) because they lack precursor metabolic pathways that can be adapted to metabolizing them. If they are unable to evolve resistance, they would be unable to transmit that resistance via a plasmid to a pathogen. Because they grow ~30 times slower (10 hours vs. 20 minutes), if they could evolve resistance they would do so much slower (if at all).

It is expected that AOB are safer than the lactobacilli which are commonly consumed in large quantities as yogurt and which are specifically used as probiotics. [24] Very rarely liver abscesses and endocarditis has been attributed to lactobacilli strains which have been indistinguishable from those used by the patients as food. [25] These associations are thought to be opportunistic infections rather than primary infections. [26] Introduction of some Lactic acid bacteria into nude athymic mice does not cause illness. In any case AOB don't excrete any proteases to degrade structural proteins and are unable to metabolize animal products if they did (unlike Lactic acid bacteria). AOB are obligate aerobes and so can't colonize the gut. The only place they can live is on the external skin, where they can live long term (years) subsisting only on natural secretions (unpublished data). During long term growth on human skin AOB do suppress other bacteria including bacteria that cause body odor (unpublished data).

Many pathogens express urease which hydrolyzes urea into ammonia raising the pH of infected skin and tissues. If this ammonia were oxidized into nitrite by AOB, they would lower the pH. If the skin did become infected, any infectious organisms would metabolize proteins into amino acids and then deaminate them releasing ammonia. An AOB biofilm would oxidize that ammonia into nitrite and NO, killing or inhibiting the organism. The pH of the site where the infection is would be high due to the ammonia released. The pH dependence of surface tension (lower at high pH) would wick those fluids to regions of low pH, where the AOB are turning the cation (ammonium) into an anion (nitrite). This is thought to be a major factor on the skin where natural pH gradients distribute sweat to regions of highest AOB activity. The lowest pH that AOB can attain is limited by the availability of ammonia (ionized ammonium is not metabolized) and the decomposition of nitrous acid. A pH below 4 cannot be generated by AOB.

I have been growing these bacteria in an organic free media simulating human sweat, distilled water plus minerals and ammonia. The only source of organic is what the AOB fix from CO2 utilizing the ATP they derive by oxidizing the ammonia to nitrite. The growth media ends up being about 40 mM in nitrite, perhaps 20 times levels observed in vivo in human saliva (2 mM) [27]. Human saliva naturally contains nitrite derived from nitrate concentrated 10x over plasma levels in saliva then reduced to nitrite by tongue commensal bacteria. This nitrite is a normal part of the antimicrobial system of the mouth and gut. [28] Mice and rats have survived for years on drinking water containing 5,000 ppm sodium nitrite (72 mM) [29].

In a hospital or nursing home setting there are many surfaces which cannot be sterilized. The surface in closest proximity to a patient's wound is the patient him/herself. Patients cannot be sprayed with disinfectant for a variety of reasons, and no disinfectant is self-renewing the way a natural biofilm is. An agent that was completely natural, sufficiently mild, odorless, which actively suppressed bacteria via mechanisms which did not lead to resistance and was self-renewing could be a useful addition to normal infection control procedures. One method of use might be to spray the patient down after bathing. Another use might be to apply to surfaces after cleaning. Perhaps applying to surfaces not cleaned as regularly, the undersides of beds, walls, floors. Perhaps applying to bedding and garments.

A major problem is incontinence and skin injury due to ammonia from hydrolysis of urea in urine. AOB oxidize ammonia to nitrite, suppressing the heterotrophic bacteria that hydrolyze urea to ammonia. Formation of nitrite lowers pH, turning toxic ammonia into non-toxic ammonium. The pH can't get below about 5-6 because the availability of ammonia goes down (they don't utilize ammonium). NO and nitrite are vasodilators and would be expected to increase circulation in affected regions. The NO/NOx level can't get to locally toxic levels because hyperemia occurs first which then carries the NO/NOx away.

This is an approach that may have some advantages for in patient long term care.

[1] McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999 Jan;12(1):147-79. Review. Erratum in: Clin Microbiol Rev 2001 Jan;14(1):227.

[2] McMurry LM, Oethinger M, Levy SB. Triclosan targets lipid synthesis. Nature. 1998 Aug 6;394(6693):531-2.

[3] Aiello AE, Marshall B, Levy SB, Della-Latta P, Larson E. Relationship between triclosan and susceptibilities of bacteria isolated from hands in the community. Antimicrob Agents Chemother. 2004 Aug;48(8):2973-9.

[4] Larson EL, Lin SX, Gomez-Pichardo C, Della-Latta P. Effect of antibacterial home cleaning and handwashing products on infectious disease symptoms: a randomized, double-blind trial. Ann Intern Med. 2004 Mar 2;140(5):321-9.

[5] Moken MC, McMurry LM, Levy SB. Selection of multiple-antibiotic-resistant (mar) mutants of Escherichia coli by using the disinfectant pine oil: roles of the mar and acrAB loci. Antimicrob Agents Chemother. 1997 Dec;41(12):2770-2.

[6] Poole K. Efflux-mediated antimicrobial resistance J. Antimicrob. Chemother., July 1, 2005; 56(1): 20 - 51.

[7] Xu J, Gordon JI. Inaugural Article: Honor thy symbionts. Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10452-9.

[8] Kramer A, Schwebke I, Kampf G. How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect Dis. 2006 Aug 16;6:130.

[9] Rao A, Jump RL, Pultz NJ, Pultz MJ, Donskey CJ. In vitro killing of nosocomial pathogens by acid and acidified nitrite. Antimicrob Agents Chemother. 2006 Nov;50(11):3901-4.

[10] Schmidt I, Hermelink C, van de Pas-Schoonen K, Strous M, op den Camp HJ, Kuenen JG, Jetten MS. Anaerobic ammonia oxidation in the presence of nitrogen oxides (NO(x)) by two different lithotrophs. Appl Environ Microbiol. 2002 Nov;68(11):5351-7.

[11] Kono Y, Shibata H, Adachi K, Tanaka K. Lactate-dependent killing of Escherichia coli by nitrite plus hydrogen peroxide: a possible role of nitrogen dioxide. Arch Biochem Biophys. 1994 May 15;311(1):153-9.

[12] Vallor AC, Antonio MA, Hawes SE, Hillier SL. Factors associated with acquisition of, or persistent colonization by, vaginal lactobacilli: role of hydrogen peroxide production. J Infect Dis. 2001 Dec 1;184(11):1431-6.

[13] Mansch R, Bock E. Biodeterioration of natural stone with special reference to nitrifying bacteria. Biodegradation. 1998;9(1):47-64.

[14] Schauder S, Bassler BL. The languages of bacteria. Genes Dev. 2001 Jun 15;15(12):1468-80. Review.

[15] Wright JS 3rd, Jin R, Novick RP. Transient interference with staphylococcal quorum sensing blocks abscess formation. Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1691-6.

[16] Rothfork JM, Timmins GS, Harris MN, Chen X, Lusis AJ, Otto M, Cheung AL, Gresham HD. Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the NADPH oxidase in host defense. Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13867-72.

[17] Hentzer M, Riedel K, Rasmussen TB, Heydorn A, Andersen JB, Parsek MR, Rice SA, Eberl L, Molin S, Høiby N, Kjelleberg S, Givskov M. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology. 2002 Jan;148(Pt 1):87-102.

[18] Olson ME, Ceri H, Morck DW, Buret AG, Read RR. Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can J Vet Res. 2002 Apr;66(2):86-92.

[19] Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999 May 21;284(5418):1318-22. Review.

[20] Schlag S, Nerz C, Birkenstock TA, Altenberend F, Götz F. Inhibition of staphylococcal biofilm formation by nitrite. J Bacteriol. 2007 Nov;189(21):7911-9.

[21] Lesic B, Lépine F, Déziel E, Zhang J, Zhang Q, Padfield K, Castonguay MH, Milot S, Stachel S, Tzika AA, Tompkins RG, Rahme LG. Inhibitors of pathogen intercellular signals as selective anti-infective compounds. PLoS Pathog. 2007 Sep 14;3(9):1229-39.

[22] Xu L, Li H, Vuong C, Vadyvaloo V, Wang J, Yao Y, Otto M, Gao Q. Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis. Infect Immun. 2006 Jan;74(1):488-96.

[23] Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M, Hauser L, Hooper A, Klotz M, Norton J, Sayavedra-Soto L, Arciero D, Hommes N, Whittaker M, Arp D. Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J Bacteriol. 2003 May;185(9):2759-73.

[24] Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr. 2006 Jun;83(6):1256-64; quiz 1446-7.

[25] Sipsas N, Zonios D, Kordossis T. Safety of Lactobacillus strains used as probiotic agents. Clin Infect Dis 2002; 34:1283–4 .

[26] Ishibashi N, Yamazaki S. Probiotics and safety. Am J Clin Nutr. 2001 Feb;73(2 Suppl):465S-470S.

[27] Pannala AS, Mani AR, Spencer JP, Skinner V, Bruckdorfer KR, Moore KP, Rice-Evans CA. The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic Biol Med. 2003 Mar 1;34(5):576-84.

[28] Dykhuizen RS, Frazer R, Duncan C, Smith CC, Golden M, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense. Antimicrob Agents Chemother. 1996 Jun;40(6):1422-5.

[29] National Toxicology Program. Toxicology and carcinogenesis studies of sodium nitrite (CAS NO. 7632-00-0) in F344/N rats and B6C3F1 mice (drinking water studies). Natl Toxicol Program Tech Rep Ser. 2001 May;495:7-273.


Tsu Dho Nimh said...

Yes, truly strange indeed. Quorum sensing explains the "healthy carrier" very nicely.

Makes me feel strange knowing I have voting by chemical going on in my guts, but I'll get over it.

daedalus2u said...

It isn't just your gut, it is everywhere you have bacteria.

SweetMarie said...
This comment has been removed by the author.