Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas

Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas Jason R. Barash, CLS, MT(ASCP), Jennifer K. Hsia, MPH, and Stephen S. Arnon, MD Objective Because Clostridium botulinum was isolated from powdered infant formula (PIF) fed to an infant in the United Kingdom who subsequently developed infant botulism and from unopened PIF from the same manufacturer, we tested PIF manufactured in the United States for the presence of clostridial spores. Study design Thirty PIF ingested by 19 California infants with botulism within 4 weeks of onset of illness (48% of all patients fed PIF during study) in 2006-2007 were cultured anaerobically to isolate clostridia. All isolated clostridia were identified to the species level and enumerated with standard microbiologic and molecular methods. Results Five of 30 (17%) PIF samples ingested by patients contained clostridial spores. Spores were also found in 7 of 9 (78%) market-purchased PIF samples. Clostridium sporogenes was isolated most frequently, followed by Clostridium butyricum and at least 10 other soil-dwelling clostridial species. No neurotoxigenic clostridia were isolated. The most probable number of clostridial spores in PIF ranged between 1.1 to >23 per 100 g. Conclusions With the notable exception of production of botulinum neurotoxin, C sporogenes is physiologically comparable with proteolytic strains of C botulinum, and both share the same natural reservoir (soils and dust worldwide). The isolation of C sporogenes and potentially pathogenic clostridia from U.S.-manufactured PIF suggests that neurotoxigenic clostridial spores have the potential to be present in these products. (J Pediatr 2010;156:402-8).

B

acteria belonging to the genus Clostridium are characterized as anaerobic, endospore-forming Gram-positive bacilli, whose natural habitat chiefly is soil and marine sediments. The clostridia are also found as normal inhabitants of human and animal intestinal flora and excreta.1-6 Several clostridial species are of medical importance because of the more than 20 protein toxins that they collectively produce.2-6 These toxins include botulinum and tetanus neurotoxins, respectively, the most potent poisons known, as well as the toxins produced by Clostridium perfringens and Clostridium difficile.7 Clostridium botulinum, and rarely neurotoxigenic Clostridium butyricum3,7-9 or neurotoxigenic Clostridium baratii,3,7,10,11 can cause infant botulism (IB), the intestinal toxemia form of botulism and the most common form of human botulism in the United States.12,13 Honey is the most well-documented food source of C botulinum spores for infants and for this reason is universally recognized to be an unsafe food for infants.14-21 However, in the United Kingdom in 2001 a single case of infant botulism occurred and appeared to be linked to powdered infant formula (PIF) that contained C botulinum spores.22 Laboratory investigations of this case reported in 2005 disclosed that the PIF consumed by the patient contained 4 strains of C botulinum type B that could be distinguished by their 4 different amplified fragment length polymorphism (AFLP) patterns and that 2 of these type B strains had colonized the infant.22 Also, 1 of 2 C botulinum type B strains from the patient had a pulsed-field gel electrophoresis (PFGE) pattern that was indistinguishable from the PFGE pattern of 1 of 4 strains isolated from the opened can of formula.23 In addition, an unopened, factory-sealed container from the same lot of formula that was consumed by the patient yielded another C botulinum type B strain with a different PFGE pattern and a fifth AFLP pattern.22,23 In light of the United Kingdom experience, we undertook a 2-year surveillance study of U.S.-manufactured PIF consumed by patients hospitalized with IB for the possible presence of C botulinum and other clostridial species.

AFLP BoNT BSM CMGS EYA FDA IB MPN PFGE rDNA SBA

Amplified fragment length polymorphism Botulinum neurotoxin Botulinum selective medium Chopped meat glucose starch Egg yolk agar Food and Drug Administration Infant botulism Most probable number Pulsed-field gel electrophoresis Ribosomal deoxyribonucleic acid Sheep blood agar

From the Infant Botulism Treatment and Prevention Program, Division of Communicable Disease Control, Center for Infectious Diseases, California Department of Public Health (J.B., J.H., S.A.), Richmond, CA Supported by the California Department of Public Health. The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright Ó 2010 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2009.09.072

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Methods Parents of all California infants who have laboratory-confirmed IB are interviewed by our Program to obtain epidemiologic information. Parents of patients who were fed PIF within the 4 weeks preceeding the onset of illness were asked to submit any PIF that remained in the original commercial container from which their child had been fed. Parents were provided with postage-paid shipping supplies and monetary reimbursement for the formula. The study period was January 1, 2005 to December 31, 2006. PIF fed to patients meeting the study inclusion criteria were submitted directly to our laboratory in the original commercial containers. For laboratory coding purposes, each sample was grouped according to formula brand, which was designated by a roman numeral. Brands I and II accounted for most samples received for testing. Brand I formulas accounted for the greatest number of IB case–associated formulas that were submitted. A smaller number of PIF representing 3 other lesser-used brands were submitted and were assigned to groups III, IV, and V. Additionally, containers of Brand II PIF were randomly purchased from area stores so that more samples of Brand II could be tested and compared with Brand I formulas. Contamination control Considerable effort was taken to prevent the possibility of laboratory-introduced spore contamination of the PIF during testing. The work reported was conducted in a closed laboratory room that has extremely low foot-traffic or other movements that would cause any possible dust contaminants to become airborne. Additionally, quality control procedures done monthly to document media sterility determined that no bacterial spore contamination existed within the laboratory. Finally, each time sample testing was undertaken, all work surfaces of the sample testing area, including the edges of overhead shelving, were first decontaminated with a fresh solution of 10% sodium hypochlorite and 70% ethanol. Presampling preparation and sanitization The laboratory bench-top where the samples were processed was lined with absorbent plastic-backed paper and soaked with a fresh solution of 10% sodium hypochlorite. A precleaned balance, 4-position magnetic stir plate and all sterile glassware and sterile consumable items were placed on this paper in preparation for sampling. Finally, the formula containers, which were received in clean, zip-sealed, plastic bags, were placed on the decontaminated bench-top. A fresh solution of 10% sodium hypochlorite was used to sanitize all external surfaces of the zip-sealed plastic bags before they were opened to remove the commercial PIF containers. Once removed from the plastic bag, all external formula container surfaces were sanitized with 10% sodium hypochlorite. Extra care was given to sanitizing the plastic cap of previously opened containers or the perforated pop-top seam of factory-sealed containers before these containers were entered for sampling.

Sampling For factory-sealed containers or for open containers with greater than 100 g of powder, 100.0 g of powder were sampled in 4 portions of 25.0 g each with a sterile, single-use sampling spoon. Each sample portion was transferred to individual, preweighed sterile beakers to which 10 volumes of Butterfield’s diluent24 prewarmed to 50 C was added. Sterile stir bars were added, and the solution was stirred on a magnetic stir-plate until the powder dissolved. The dissolved powder was transferred to individual 500-mL sterile conical centrifuge bottles and spun in a centrifuge at 3450  g at 4 C for 60 minutes. The supernatant was vacuum-aspirated with sterile glass Pasteur pipettes and discarded; the pellets from the 4 centrifuge bottles were combined and then resuspended in 10 mL of Butterfield’s diluent. For previously opened containers with less than 100 g of PIF, all available formula was tested. Culture and botulinum toxin culture screen The entire volume of the resuspended pellets (when 100 g was sampled) was equally distributed into a set of 10 culture tubes each containing 20 mL of prereduced chopped meat glucose starch (CMGS) broth. Doing so enabled the calculation of a single 10-tube most probable number (MPN) of spore-forming bacteria contained in the 100.0 g of PIF.25 When less than 100 g of PIF was sampled, up to 5 tubes of CMGS broth were inoculated. For all samples the inoculated, CMGS broth was heat-shocked in a 70 C water bath for 15 minutes to select for spore-forming bacteria, immediately cooled, and then incubated at 35 C for up to 2 weeks. CMGS cultures of PIF visually confirmed to have growth at 1 and 2 weeks of incubation were screened for botulinum neurotoxin (BoNT) and also subcultured for isolation and identification of Clostridium species. To accomplish these tasks, broth culture was removed from the bottom of each tube and diluted 1:4 in gelatin phosphate diluent. The diluted broth culture was spun in a centrifuge at 3450 g at 4 C for 15 minutes. The supernatant was filter sterilized through a 0.45-mm syringe filter and retained for BoNT screening, which was performed with the standard mouse bioassay.26 The pellet was subcultured onto 2 plates, 4% egg yolk agar (EYA) and botulinum selective medium (BSM),27 both made with a base of brain-heart infusion. EYA and BSM cultures were incubated either in anaerobe jars or an anaerobe chamber (Sheldon Mfg., Cornelius, Oregon) and incubated for 48 to 72 hours at 35 C. All diluents and media were prepared in-house. Organism identification EYA and BSM cultures were examined for colonies exhibiting typical characteristics of the genus Clostridium.2-4,28-30 Suspicious colonies were subcultured to 2 plates of brain-heart infusion agar containing 5% sheep blood (SBA) and to 1 tube of CMGS and then incubated as above. One SBA plate was incubated aerobically to test for the aerotolerant clostridia.4,28-30 All spore-forming, Gram-positive bacilli that grew anaerobically, or those that were aerotolerant and 403

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formed colonies with typical clostridial appearance, were identified. Biochemical characterization was performed with API 20a (Biomerieux, Hazelwood, Missouri) and Rapid Ana II (Remel, Lenexa, Kansas) anaerobe identification kits. Kit-based identifications were determined with biochemical profiles and other tests ancillary to the kits that were specified by the manufacturer in their identification code books. Gram stain appearance, phase-contrast microscopy (for spore location) and colony and other cultural characteristics were used to verify species-level identification.29,30 When indicated, pure cultures were screened for BoNT as above. The identity of most isolates was either confirmed or established using 16S rDNA sequencing,31 which was necessary because of the inherent limitations of using biochemical identification kits for identifying anaerobic bacteria.32

Results A total of 39 powdered infant formulas were submitted and tested. Of these, 30 PIFs were associated with 19 laboratory-confirmed cases of IB, and 9 formulas were marketpurchased. Brand I accounted for 20 case-associated samples, and Brand II accounted for 5 case-associated samples. The 5 other case-associated formula samples were from 3 additional brands identified as Brands III, IV, and V. The 9 additional cans of PIF (‘‘surveillance samples’’) purchased from local stores consisted of 8 cans of Brand II (to balance the number of samples tested for the 2 major brands) and a single can of Brand III (to match the single patient-associated sample of Brand III). Thirty-one percent (12/39) of the formula samples were found to harbor clostridial spores. Clostridium species were isolated from 3 different brands of PIF associated with 5 infant botulism patients; these were 2 samples of Brand I, 2 samples of Brand II, and 1 sample of Brand III. Additionally, 6 of 8 Brand II surveillance samples and the single brand III surveillance sample contained spores of several different clostridial species. Two of the Brand II surveillance samples contained spores from at least 4 different clostridial species. No spores of Clostridium botulinum, neurotoxigenic C baratii or neurotoxigenic C butyricum were detected in any of the PIF tested. However, at least 12 other different Clostridium species were isolated and identified. Notable among the clostridial species found were C perfringens, C septicum, C novyi/haemolyticum and C sporogenes. Additionally, the MPN of Clostridium spores/100 g of PIF was determined to range between 1.1 and >23 organisms (Table).25 Clostridium sporogenes was the most frequently isolated Clostridium species; 8 of the 12 formula samples (67%) that contained clostridial spores harbored this organism. All C sporogenes isolates were identified by cultural and biochemical characteristics, and all isolates tested by 16S rDNA sequence identification of a 1312 base-pair segment were further confirmed to be C sporogenes (Figure). Most (75%) C sporogenes isolates had a 100% rDNA sequence identity to C sporogenes ATCC 3584, the type strain of this species that 404

Vol. 156, No. 3 was originally isolated from soil. Two of the C sporogenes isolates were atypical, in that minimal to no lipase activity was demonstrated on EYA cultures, and therefore these colonies were initially thought not to belong to this species. However, 16S rDNA sequencing matched these strains to the Genbank listing for Clostridium sporogenes subspecies tusciae with a 100% sequence identity. This subspecies was renamed as C sporogenes biovar pennavorans before publication of the work that described this strain (personal communication with F. Canganella, April 28, 2008).33 All C sporogenes isolates were confirmed by the mouse bioassay to be negative for BoNT production (i.e., none were C botulinum); assaying for possible production of BoNT is always necessary to differentiate definitively between these 2 closely-related species.2,3,26 In addition to C sporogenes and C sporogenes biovar pennavorans, the PIF were found to contain 10 other clostridial species (Table). Almost all the PIF harbored other spore-forming bacteria. These spore-formers were presumptively identified as belonging to the genus Bacillus on the basis of gram stain appearance, colony structure, and strong aerobic growth on SBA. Some PIF contained multiple species of Bacillus spores. Characterization of these aerobic organisms beyond presumptive identification as Bacillus was not performed.

Discussion It has been determined that PIF is not a sterile product and that making it sterile is not feasible.34 In 2001 powdered infant formula contaminated with Cronobacter (formerly Enterobacter) sakazakii caused several infants to become ill, and 1 died.35 Accordingly, the U.S. Food and Drug Administration (FDA) concluded that a required microbiologic standard should be set specifically for possible C sakazakii and Salmonella species contamination of PIF.36 The FDA also concluded that a required microbiologic standard for organisms other than C sakazakii and Salmonella species in PIF was not needed.36 In reaching these conclusions, the FDA cited the 2006 United Nations Joint Food and Agriculture Organization and World Health Organization meeting, which concluded that only C sakazakii and Salmonella species have been clearly linked to illness in infants fed PIF.36,37 The identification of C botulinum type B spores in a PIF in the United Kingdom with an evident link to a 2001 case of laboratoryconfirmed type B infant botulism suggests that PIF may serve as a vehicle of clostridial spores that in certain circumstances may become pathogenic to the infant.22,23 The PIF fed to the 5-month-old patient with IB in 2001 in the United Kingdom contained 0.38 (95% CI, 0.09-1.51) C botulinum spores per 100 g.22 Assuming an even distribution of C botulinum spores in the PIF can, the U.K. patient ingested in total fewer than 3.42 spores (95% CI, 0.8113.59), the number that would have been in the entire 900g powder-containing can. Our study design tested up to 100 g PIF from the commercial PIF containers in order to have the remaining PIF available for repeat testing if any Barash, Hsia, and Arnon

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Table. Clostridium species isolated from U.S.-made PIF Positive sample

Brand*

Sample origin

Clostridium species identified

MPN/100 g (95% CI)

1 2 3 4 5 6

I I II II II II

P P P P S S

7

II

S

8

II

S

9

II

S

10

II

S

11

III

S

12 N/A N/A

III IV V

P P P

C bifermentans Clostridium species† C sporogenesz C sporogenesz C beijerinckii C butyricum C sporogenes bv. pennavoransz,x C butyricum C cochleariumz C septicumz C sporogenesz C butyricum C novyi/haemolyticumz C sporogenesz C lundensez C sporogenes bv. pennavoransz,x C butyricum C innocuum C tyrobutyricumz C subterminalez C sporogenesz C perfringens C sporogenesz No Clostridium species Isolated No Clostridium species Isolated

1.1 (.05-5.9) >23 (12-N) 5.1 (1.6-13) 1.1 (.05-5.9) 1.1 (.05-5.9) 1.1 (.05-5.9) 3.6 (0.91-9.7) 1.1 (.05-5.9) 1.1 (.05-5.9) 1.1 (.05-5.9) 3.6 (0.91-9.7) 2.2 (0.37-8.1) 2.2 (0.37-8.1) 2.2 (0.37-8.1) 1.1 (.05-5.9) 3.6 (0.91-9.7) 1.1 (.05-5.9) 1.1 (.05-5.9) 1.1 (.05-5.9) 1.1 (.05-5.9) 2.2 (0.37-8.1) 1.1 (.05-5.9) 6.9 (2.5-15) — —

P, Patient; S, surveillance. *Brand I accounted for 20 case-associated samples, Brand II accounted for 5 case-associated samples, and Brands III, IV and V accounted for 5 case-associated samples. Eight surveillance samples for Brand II and 1 surveillance sample for Brand III were purchased (see Methods). †Organism identifiable to genus level only. zIdentification confirmed by 16S rDNA sequence analysis xListed in Genbank as C sporogenes ssp. tusciae; current nomenclature for this species is C sporogenes biovar pennavorans.33

C botulinum-containing samples were found. If C botulinum were present in commercial U.S. PIF at the same MPN concentration as it was in the positive U.K. PIF, our sampling size may have been too small to detect it. The ID50 of C botulinum spores for patients with IB is not known but was estimated 30 years ago from studies of spore-containing honeys to be approximately 10 to 100 organisms per patient.21 In PIF manufactured in the United States, we found the concentration of C sporogenes spores to be 2.2 to 6.9 per 100 g (95% CI, 0.37-15.0). The median age and the median body mass for 264 California infant botulism patients 2000-2008 were 3.67 months and 5.9 kg, respectively. For such a child, the AAP Pediatric Nutrition Handbook38 recommends an energy intake of 86 kcal/kg/d (see chart p. 244), which can be provided by 760 mL of 0.67 kcal/mL (20 kcal/ oz) formula that is reconstituted from 101.5 g of formula powder. Hence, a typical California patient with IB fed the PIF containing the C sporogenes reported herein would ingest approximately 2.2 to 7.0 (95% CI, 0.38-15.2) C sporogenes spores every 24 hours. C sporogenes is closely related to C botulinum and may serve as a potential indicator organism for C botulinum. Clostridium sporogenes was the most frequently isolated Clostridium species encountered in this study and was found in PIF produced by 2 manufacturers, 1 major (Brand II), and 1 minor (Brand III). With the notable exception of the ability to produce botulinum neurotoxin, C sporogenes is physiologically and genetically similar to the C botulinum strains

(Group I, proteolytic) that produce botulinum toxin types A, B and F (Figure).2,3,6,28-30 Hence, C sporogenes may be thought of as an atoxigenic variant of proteolytic C botulinum. Although C sporogenes and C botulinum also inhabit and share the same natural environment of soils and dust worldwide, the relative ratio of these 2 organisms in their natural habitat has apparently not been determined. Studies of soil samples in our laboratory have identified the simultaneous presence of both species in the same soil samples (unpublished data). For these reasons, C sporogenes may serve as a potential indicator organism in powdered infant formulas for the possible presence of soil contamination and for the possible presence of C botulinum. In addition to C sporogenes, 11 other clostridial species were identified in PIF (Table). One of these species was C sporogenes biovar pennavorans, which was isolated recently in Italy from sulfur-containing thermal mud and which has a unique ability to produce a feather-degrading keratinase.33 Of the other clostridial species found in PIF, C bifermentans, C novyi/haemolyticum, C perfringens, C septicum, and, to a lesser extent, C cochlearium, C innocuum, and C subterminale are of known medical importance as potential pathogens.4-6,28 Also, several species of solventogenic clostridia isolated from PIF are of industrial importance. For example, atoxigenic strains of C butyricum produce butyric acid, which has a broad array of industrial uses,39 and C beijerinckii is used for butanol fuel production.40,41 Clostridium tyrobutyricum, commonly found in the dairy environment, may

Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas

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Figure. Dendrogram of 16s rDNA genes of C sporogenes and C botulinum Group I (type A and proteolytic types B and F). A neighbor-joining 16s rDNA dendrogram that was created to show the genetic relatedness of 28 Clostridium species31 was expanded to include 5 C sporogenes strains isolated from powdered infant formula (Table). These 5 strains (highlighted in blue) included 3 C sporogenes strains (07R-0043, 07R-0075, 07R-0082, Genbank accession numbers GU139702, GU139703, and GU139704, respectively) and the 2 C sporogenes ssp. tusciae strains (07R-0035, 07R-0044, Genbank accession numbers GU139705 and GU139706, respectively). The portion of the expanded dendrogram that shows the close relationship of the C sporogenes and C botulinum Group I strains is displayed here. The 16s rDNA gene sequences (1312 nucleotides) of the 3 displayed C sporogenes strains differ slightly from the two C sporogenes ssp. tusciae strains. (C sporogenes ssp. tusciae was recently renamed C sporogenes bv. pennavorans33). The distance scale represents 3 mutations per 100 nucleotides. 1, The toxin-type for this strain of C botulinum was not available.

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Vol. 156, No. 3 contaminate pasteurized or unpasteurized milk intended for use in cheese manufacturing, and is the main cause of a metabolic, gas-induced, ‘‘late-blowing’’ defect in cheese manufacturing.40,42 Also isolated from PIF was C lundense, another newly-described clostridial species that was originally isolated from bovine rumen.43 Three of the clostridial species found in PIF, namely, C butyricum, C perfringens and C innocuum, were also found among 12 clostridial species identified (as spores) in a year-long study of the fecal flora of 10 healthy Australian infants.44 The common feature shared by the clostridial species found in these PIF is that they are all soil-dwelling organisms and, to a lesser extent, also inhabitants of mammalian (eg, dairy cattle) intestinal tracts. The wide array of different clostridia identified in the PIF is also noteworthy. Brand II formula appeared to contain more clostridia than the other brands tested, especially in its unopened cans, and was the only brand found to contain 3 or more different clostridial species in a single unopened formula sample. However, we cannot exclude the possibility that clostridial spores entered the case-associated cans of PIF after they were opened in the patients’ homes. Nonetheless, we consider this possibility unlikely because the market-purchased surveillance cans of PIF with intact factory seals were found to contain the greatest numbers of different clostridial species (Table). Our findings raise the public health concern that naturally occurring C botulinum spores may potentially exploit the same access pathways used by C sporogenes and the other clostridia found in these PIF and may place infants at risk of contracting infant botulism. The presence of C sporogenes and other clostridial spores in PIF does not prove or disprove that C botulinum coexists in PIF; instead, it indicates that such a circumstance could occur. In this regard, the fact that no C botulinum was isolated from the formula samples tested and that there has been no recent rise in the number of hospitalized cases of infant botulism in the United States likely indicates that our finding of soil-dwelling clostridial spores in commercial PIF does not constitute a public health threat. However, the scope of our study was limited by the relatively small number of PIF that could be obtained for evaluation. A more extensive study might provide additional perspective on whether there exists a possible public health hazard from the potentially pathogenic clostridial spores that may be present in commercial PIFs in the United States. n We thank Karen Hill of Los Alamos National Laboratories and William Probert, Jim Ware, Silvia Moler, Janet Ely, and Kimmi Schraeder of the Microbial Diseases Laboratory, California Department of Public Health, for their contributions of the 16S rDNA identifications. Submitted for publication Jun 2, 2009; last revision received Aug 25, 2009; accepted Sep 30, 2009. Reprint requests: Stephen S. Arnon, MD, Infant Botulism Treatment and Prevention Program, Division of Communicable Disease Control, Center for Infectious Diseases, California Department of Public Health, 850 Marina Bay Parkway, Room E-361, Richmond, CA 94804. E-mail: stephen.arnon@cdph. ca.gov.

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23. Johnson EA, Tepp WH, Bradshaw M, Gilbert RJ, Cook PE, McIntosh EDG. Characterization of Clostridium botulinum strains associated with an infant botulism case in the United Kingdom. J Clin Microbiol 2005;43:2602-7. 24. Murano EA, Hudnall J. Media, reagents and stains. In: Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods. 4th ed. Washington, DC: American Public Health Association; 2001. p. 601-48. 25. US Food and Drug Administration, Center for Food Safety and Applied Nutrition. Bacteriological Analytical Manual Online. Appendix 2: Most probable numbers from serial dilutions. 2006. Available online at: http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ BacteriologicalAnalyticalManualBAM/ucm109656.htm. Accessed May 12, 2009. 26. Centers for Disease Control and Prevention. Botulism in the United States (1899–1996). Handbook for Epidemiologists, Clinicians, and Laboratory Workers. 1998. U.S. Department of Health, Education and Welfare, CDC, Atlanta, Georgia. 27. Mills DC, Midura TF, Arnon SS. Improved selective medium for the isolation of lipase-positive Clostridium botulinum from feces of human infants. J Clin Microbiol 1985;21:947-50. 28. Johnson EA, Summanen P, Finegold SM. Clostridium. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of clinical microbiology. 9th ed. Washington, DC: ASM Press; 2007. p. 889-910. 29. Holdeman LV, Moore WEC. Anaerobe laboratory manual. Blacksburg, Virginia: Virginia Polytechnic Institute Anaerobe Laboratory; 1972. 30. Jousimies-Somer HR, Summanen P, Baron EJ, Citron DM, Wexler HM, Finegold SM. Wadsworth-KTL Anaerobic bacteriology manual. 6th ed. Belmont, CA: Star Publishing; 2002. 31. Hill KK, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, et al. Genetic diversity among botulinum neurotoxin-producing clostridial strains. J Bacteriol 2007;189:818-32. 32. Summanen P, Jousimies-Somer H. Comparative evaluation of RapID ANA and API 20A for identification of anaerobic bacteria. Eur J Clin Microbiol Infect Dis 1988;7:771-5. 33. Ionata E, Canganella F, Bianconi G, Benno Y, Sakamoto M, Capasso A, et al. A novel keratinase from Clostridium sporogenes bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds. Microbiol Res 2008;163:105-12. 34. United States Food and Drug Administration, Center for Food Safety and Applied Nutrition. Contaminants and natural toxicants subcommittee of the food advisory committee: Volume II. 2003. Available online at: http://www.fda.gov/ohrms/dockets/ac/03/transcripts/3939t2.doc. Accessed May 12, 2009. 35. Centers for Disease Control and Prevention. Enterobacter sakazakii infections associated with the use of powdered infant formula-Tennessee, 2001. Available online at, http://www.ncbi.nlm.nih.gov/pubmed/ 12002167. Accessed May 12, 2009. 36. United States Food and Drug Administration. 21 CFR Parts 106 and 107: Proposed Rule. Available online at, http://edocket.access.gpo.gov/2006/ pdf/E6-12268.pdf. Accessed May 12, 2009. 37. Joint Food and Agriculture Organization/World Health Organization meeting. Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting Report. FAO Headquarters, Rome, Italy, 16-20 January, 2006. Available online at: ftp://ftp.fao.org/ag/agn/jemra/e_sakakazii_ salmonella.pdf. Accessed May 12, 2009. 38. American Academy of Pediatrics. Pediatric Nutrition Handbook. 5th ed. Elk Grove Village, IL: American Academy of Pediatrics; 2004. 39. Guo-qing H, Kong Q, Chen Q, Ruan H. Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB. Zhejiang Univ Sci B 2005;6:1076-80. 40. Le Bourhis AG, Dore J, Carlier JP, Chamba JF, Popoff MR, Tholozan JL. Contribution of C. beijerinckii and C. sporogenes in association with C. tyrobutyricum to the butyric fermentation in Emmental type cheese. Int J Food Microbiol 2007;113:154-63. 41. Manish P, Blaschek HP. Butanol production by hypersolventproducing mutant Clostridium beijerinckii BA101 in corn steep

Presence of Soil-Dwelling Clostridia in Commercial Powdered Infant Formulas

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50 Years Ago in THE JOURNAL OF PEDIATRICS Current Immunization Problems Gaisford W, Feldman GV, Perkins FT. J Pediatr 1960;56:319-30

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accinologists from Manchester and London provide primary data and much insight surrounding problems of providing protection for infants against major pathogens of 1960. They ask the same questions of each vaccine—questions that we ask today. Is an effective vaccine antigen available? Is protection important? Can the vaccine be given at the age when protection is most needed? Is it safe? With these questions in mind, the authors focus on 4 vaccines—Bacillus-Calmette Guerin (BCG), diphtheria and tetanus toxoids, and whole-cell pertussis vaccines—as well as possibilities and overarching problems of vaccinating in the neonatal period. The full article is worth reading. A few facts that stand out to this reader follow. Efficacy of BCG against severe pulmonary or central nervous system tuberculosis is not questioned, because ‘‘most paediatricians. . . have watched infants grow up in homes where there are contact patients, and have compared the mortality and morbidity from tuberculosis in vaccinated and unvaccinated infants and children.’’ Currently we recognize remarkable variability in effectiveness of BCG vaccines across countries (from 2% to >80%). But we forget that primary outcomes for efficacy of BCG were prevention of morbid and fatal tuberculosis, not M. tuberculosis infection. Combining these toxoids in balanced proportions enhances their immunizing potential, especially for the diphtheria component. The major advance in preventing neonatal tetanus, however, followed immunization of mothers whose infants are protected by transplacental antibody. Active infant vaccination can be deferred until 1 to 2 months of age. To enhance antigenicity of the pertussis component of DPT, an adjuvant such as aluminum hydroxide or phosphate is necessary. In 1960, in Great Britain, the concern of adjuvant-associated ‘‘provoked paralysis’’ following natural acquisition of poliomyelitis was relevant, leading to the recommendation of maternal immunization with inactivated poliovirus vaccine during pregnancy to protect the infant against this potential collateral effect of DPT. Although some 1960 risks for neonates (diphtheria, tetanus, tuberculosis, poliomyelitis) have been eliminated by public health measures and immunization in many countries, the problem of neonatal pertussis persists. The problems and possibilities of neonatal protection in 1960 live on—with the cocooning strategy of administration of Tdap vaccine after delivery to all contacts of neonates (so-called cocoon strategy) difficult to implement, and immunization of mothers during pregnancy to afford passive protection currently under study for its potential blunting effect on active immunization of the infant. Sarah S. Long, MD Section of Infectious Diseases St. Christopher’s Hospital for Children Philadelphia, Pennsylvania 10.1016/j.jpeds.2009.09.074

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