Agency Additional
Correspondence Letter
GRAS Notice No. GRN 000049

Mr. Fred Degnan
King and Spalding
1730 Pennsylvania Ave., N.W.
Washington, DC 20006-4706

Re: GRAS Notice No. GRN 000049

Dear Mr. Degnan:

The Food and Drug Administration (FDA) is responding to a submission, dated February 23, 2004, that you submitted on behalf of Nestlé USA (Nestlé). The submission is related to GRAS Notice No. GRN 000049, which you previously submitted on behalf of Nestlé in accordance with the agency's proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS)). FDA responded to GRN 000049 with a letter, dated March 19, 2002, stating that the Agency had no questions at the time regarding Nestlé's determination that the subjects of the notice Bifidobacterium lactis strain Bb12 and Streptococcus thermophilus strain Th4 are GRAS for their intended use as ingredients in infant formula. FDA received this submission February 23, 2004, and added it to the administrative record for GRN 000049 as a supplement to the notice.

In the supplement to GRN 000049, Nestlé informs FDA that, in determining the complete DNA sequence of their B. lactis strain and comparing it with sequences in publicly available DNA sequence databases, they found that their B. lactis strain bears a tetracycline resistance gene designated as the tet(W)¹ gene, which encodes the ribosomal protection protein designated TetW. In the supplement, Nestlé presents data and information to support its view that the presence of tet(W) does not affect the safety of this bacterium as an ingredient in infant formula. FDA also reviewed other generally available information relating to this antibiotic resistance gene and the function of its gene product.

Nestlé's supplement includes a summary by a panel of individuals who evaluated data and information relating to tet(W) in B. lactis. Nestlé considers the members of this panel to be qualified by scientific training and experience to evaluate the safety of the presence of the tet(W) gene in B. lactis in the context of its use in infant formula. The summary states that the panel addressed: a) organism-specific findings; b) the distribution of tet(W) in food and microbes; c) the potential for gene transfer via conjugative or mobilized elements; d) antibiotic resistance and susceptibility; e) the contribution of tetracycline use to tetracycline resistance; f) possible clinical consequences and public health issues related to exposure to the tet(W) gene; and g) gaps in current knowledge related to tet(W) prevalence and potential transfer of the gene to other microbes. According to this panel, the presence of tet(W) in B. lactis is neither clinically nor environmentally relevant, has no impact on the safety of B. lactis for its intended use in infant formula, and would not change Nestlé's conclusion that B. lactis is GRAS for its intended use as an ingredient in infant formula.

Questions Regarding Current Knowledge of tet(W) and B. lactis

Nestlé has addressed the following questions relating to tet(W) in their B. lactis strain:

I) Is transfer of tet(W) from B. lactis to other microorganisms likely?

Nestlé states that the B. lactis tet(W) gene is unlikely to be transferred to other microorganisms. Nestlé discussed its current knowledge, based on having determined the DNA sequence of the B. lactis genome. Nestlé reports that the B. lactis tet(W) gene is chromosomally located, and they found a single insertion sequence element located near tet(W), which would not provide a means for interspecies transfer of tet(W). Barriers to transcription or expression of tet(W) or regulatory factors may prevent expression of tet(W) in some bacteria. The most plausible mechanism for transfer of tet(W) from B. lactis: natural transformation of DNA from lysed organisms, would be a remote possibility, because most normal flora of the human intestine are not known to undergo natural transformation⁽²⁾ and the probability of DNA remaining intact in the intestinal tract is low.

Some scientists have inferred from high levels of homology among tet(W) genes from different species that these genes were acquired recently, perhaps by a conjugative transposon-like mechanism. However, in the absence of direct evidence regarding the acquisition of this gene by B. lactis, this hypothesis does not rise above speculation. Overall, Nestlé concluded that the likelihood of transfer of tet(W) from B. lactis to other microorganisms is very low.

II) Would exposure to microorganisms in the food supply and commensal microorganisms bearing tet(W) (or other ribosomal protection protein producing genes) be likely to result in any safety concerns?

Nestlé summarized published data indicating that tetracycline resistance is common among lactic acid bacteria used in food and states that genes "annotating as tetracycline resistance are now being found commonly in a large number of lactic acid bacteria." Recent publications reported that the tet(W) gene is common among human commensal gut bacteria. It was the second most frequently detected tetracycline resistance gene among tetracycline resistant bacteria cultivated from the human oral cavity, and in one study, was detected in children who lacked previous exposure to tetracycline. Consequently, the consumption of organisms bearing this gene would not represent new exposure of this gene to humans ingesting food containing tet(W)-bearing strains. A recent publication reported that tetracycline resistance, including that encoded by tet(W), is widespread, particularly in farm environments.

The organism for use in the proposed infant formula B. lactis (also known as Bb12) is commonly used in a number of food products in the U.S. and other developed countries. These products have been marketed for several years; they include yogurts and dairy-based drinks, among which is yogurt marketed in the U.S. specifically for infants, and infant formulas containing this ingredient have been consumed for several years overseas. There are no known adverse events attributed to consumption of tetracycline resistant microbes used in food processing.

III) Would the presence of a tetracycline resistant organism in infant formula affect therapeutic use of tetracycline in this population?

Nestlé states that distribution of a tetracycline resistant B. lactis to the population of intended users (children under one year old) would not compromise clinical use of tetracycline for the following reasons: Tetracycline would not ordinarily be administered to the infant population. The Committee on Infectious Diseases of the American Academy of Pediatrics does not recommend the use of tetracycline in children younger than eight years old. In most cases, alternative antibiotics would be used. However, for those instances where physicians would desire using tetracyclines, including nonbacterial uses such as protozoan infections, the TetW protein or other ribosomal protection proteins in ingested microorganisms would not affect the efficacy of tetracyclines in targeted pathogens.

Conclusions

Nestlé has presented data and information to show that the presence of a tetracycline resistance element in B. lactis does not present a safety problem and does not change their conclusion that the organism is GRAS for use as an ingredient in infant formula. Nestlé supports this conclusion with the following information: (1) current knowledge provides no likely mechanism for transfer of tet(W) from B.lactis to pathogens; (2) B. lactis has been used extensively in food products; there are no reports of problems with antibiotic use in consumers of these products; (3) tetracycline resistance is pervasive in the microbial world, even in the microflora of subjects who have never been exposed to tetracycline; thus, the widespread presence of bacteria with tet(W) indicates that exposure to B. lactis containing tet(W) would not likely introduce a tetracycline resistance element new to most subjects; and (4) tetracycline is not normally administered to infants in the United States.

Based on the information provided by Nestlé, as well as other information available to FDA, the agency has no questions at this time regarding Nestlé's conclusions that the presence of the tet(W) gene in their B. lactis strain does not affect the safety of the intended use of B. lactis as an ingredient in infant formula and that the discovery of the tet(W) gene in B. lactis does not change their previous conclusion that B. lactis is GRAS for its intended use as an ingredient in infant formula. The agency has not, however, made its own determinations regarding the safety of the tet(W) gene in B. lactis or the GRAS status of the subject use of B. lactis as an ingredient in infant formula. As always, it is the continuing responsibility of Nestlé to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

In accordance with proposed 21 CFR 170.36(f)(2)(iii), a copy of the text of this letter is available for public review and copying on the homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

Sincerely,

Laura M. Tarantino, Ph.D.
Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition

Reference

Levy, S.B., L.M. McMurry, V. Burdett, P. Courvalin, W. Hillen, M.C. Roberts, and D.E. Taylor. 1989. Nomenclature for Tetracycline Resistance Determinants. Antimicrob. Agents Chemother. 33:1373-1374.

---

⁽¹⁾Conventions set by Levy, et al. (Ref. 1) use parentheses to distinguish different tetracycline resistance elements from sets of structural genes and regulatory elements that may be present within a single resistance element and are designated without parentheses (e.g., tetA and tetR).

⁽²⁾ Helicobacter pylori, resident in the stomach and the agent attributed to causing stomach ulcers and other stomach conditions can be naturally competent; however, current evidence is that only DNA sequences with Helicobacter-specific recognition sequences are transformed.