Team:Caltech/Biosafety

General Concerns

There are risks in probiotic engineering that must be addressed before our system can be implemented in humans. One common risk is increasing the chance of bacterial sepsis for those who are immune deficient. Other risk factors include the impact on premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea¹. It is also unclear how probiotics affect host metabolic activities. However, the unmodified Nissle 1917 strain has been shown to be safe for long term use².

The engineered probiotic will persist in the gut longer if antibiotics are used to suppress other gut flora³. However, such resistance may pass over into pathogenic strains and cause future problems for the host. The concern of introducing foreign genes into the gut is described below for each individual project.

Subproject Concerns

Oxidative Burst

• Short Term: There are no safety concerns beyond that of typical E. coli lab strains. Considering that 260 mM H₂O₂ is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H₂O₂ produced by the engineered strain should not be a health concern to anyone working in the lab.
• Long Term: Production of hydrogen peroxide is not a normal occurrence in the large intestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine are listed below:
  • bowel irritation⁴.
  • killing of the native gut flora.
  • damage to gut epithelial cells.
  • transmission of the ability to produce peroxide to other gut flora.

Phage Pathogen Defense

• Phage therapy is generally not harmful to the host due to phage specificity to bacterial hosts. However, separate complications associated with phage therapy exist:
  • Often times, there are pathogenic and benign strains of the same species of bacteria, and when targeting a species of pathogen, the associated benign strains could be destroyed as well.
  • Bacterial lysis leads to a release of endotoxins within the host, this could lead to a variety of side effects including fever and toxic shock[http://en.wikipedia.org/wiki/Herxheimer_reaction]
  • Phage lysogeny can often induce pathogenesis in otherwise harmless species by carrying virulence genes in the phage genome (Vibrio cholerae is a classic example). We can avoid this problem by switching to phage variants which are only lysogenic in our engineered host.

Lactose Intolerance

• Synthetic LacY/Z Plasmid: There are no known safety concerns regarding this plasmid. The only foreign enzyme not present in humans that is expressed is β-Galactosidase, which simply acts as a lactase. Several lactase preparations are formally 'Generally Recognized as Safe' by the FDA[http://www.cfsan.fda.gov/~rdb/opa-g132.html] and we expect no difficulties in finding a suitable lactase.
• Lysis Cassette Plasmid: We expect little effect of this plasmid, as there would be a strong selective pressure against lateral transmission. However, bacterial lysis can lead to undesirable side effects as described above.

Vitamin Production

• Overexpression of Folate in the Gut: While an excess of folate (Vitamin B9) in the colon would need further testing, the risk of toxicity from overdose is very low, since folate is water-soluble[http://en.wikipedia.org/wiki/Folate]. The tolerable upper intake level for folate is around 1 mg, and many cereals contain 100% of the recommended daily dose[http://www.cdc.gov/ncbddd/folicacid/cereals.htm].
• Folate biosynthesis plasmids: While these plasmids are constitutively high copy, these plasmids produce intermediates for folate, which again, has a very low risk of toxicity.
• pABA biosynthesis plasmids para-aminobenzoic acid (pABA) is also an intermediate for folate that is commonly used in sunscreen to absorb UV radiation. There are no known serious toxic effects of pABA overdose, though there has also not been extensive testing[http://www.sciencelab.com/xMSDS-4_Aminobenzoic_acid-9922876].

Population Variation

• The current constructs are regulatory in nature and present few safety concerns aside from those involved in the treatment subprojects. Minor concerns are discussed below:
  • In the simple FimE constructs, when the promoter starts in the configuration pointing upstream, DNA upstream of the constructs may be transcribed by the cell.
  • In the final design, the terminator that sits in the population variation generator may not be 100% efficient. Thus, the efficiency of the terminator should be tested before genes that are hazardous when co-expressed are placed into the system.
  • The engineered FimE system may interfere with natural fimbriae expression in Nissle 1917 or other gut flora. Since fimbriae are important for intestinal colonization, this could affect the intestinal composition⁵.

References

1. Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.
2. Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84.
3. Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A 2005 Aug 23; 102(34) 11993-8.
4. Ackermann M, Stecher B, Freed NE, Songhet P, Hardt WD, and Doebeli M. Self-destructive cooperation mediated by phenotypic noise. Nature 2008 Aug 21; 454(7207) 987-90.
5. Stentebjerg-Olesen B, Chakraborty T, and Klemm P. Type 1 fimbriation and phase switching in a natural Escherichia coli fimB null strain, Nissle 1917. J Bacteriol 1999 Dec; 181(24) 7470-8.