Author's response to reviews Title:Detection of antibiotic resistance in probiotics of dietary supplements Authors: Aloysius T.H. Wong (aloysius.wong@kaust.edu.sa) Davey Y.S. Ngu (daveyngu311@gmail.com) Lydia A. Dan (lad201@mun.ca) Amanda S.L. Ooi (amanda.ooi@kaust.edu.sa) Renee L.H. Lim (reneelim@ucsiuniversity.edu.my) Version:2Date:5 August 2015 Author's response to reviews: see over
Response to reviewer 1 This is a short report from Wong Tze Hern et al. that highlights the discrepancy between the enumerated viable bacteria amounts in probiotics supplements (capsules from different origin) and the claims of the manufacturers. This first finding is not new but corroborates previous studies where mislabeling, overestimation and misidentification of probiotics in various food and health products have also been demonstrated. This highlights the lack of regulation and appropriate labeling on these products, which is of great importance nowadays due to the increasing demand for probiotic-containing foods and supplements. In fact, if the purpose of the probiotics is to regenerate the intestinal microflora, an expected number of live bacteria are needed. We agree with this comment and we have showed in our study that such discrepancy also occurs in dietary supplements. Specifically, we highlighted that 4 out of 5 brands of probiotic supplements have reduced amount of bacteria than that stated on the labels. The second finding of this study shows the presence of antibiotic resistance in lactic bacteria from dietary supplements. The antibiotics tested are suitable as they are against gram positive bacteria. Although not required, the table included as supported data serves as a control to demonstrate that the antibiogram works perfectly. I thank its inclusion as an internal control. However and although this study is a preliminary one, a few experiments are required in order to be ready to be published in the Nutritional Journal. In addition, although the bibliography is adequate and suits with the state of the art and the discussion of the results, newest citations concerning this field should also be included. We thank reviewer 1 for the positive comment. Many probiotic supplements consist of a consortia of bacteria and therefore, requires different recovery methods, for e.g., optimal media and conditions for the recovery of the respective Lactobacilli and Bifidobacterium, as well as careful isolation of the probiotics before identifying the isolated strains by biochemical and 16s rrna sequencing methods. Once this is established, a quantitative MIC tests for antibiotics can be accurately determined. Here, we leverage on the availability of commercial antibiotic discs to perform quick screenings on the antibiogram of the recovered probiotics bacteria to provide a preliminary but important assessment on the antibiotic resistance profile of these increasingly popular supplements. Such information is unfortunately scarce if not absent. Therefore, we believe that it is important to first disseminate these preliminary results in the form of a short report so that consumers and health practitioners are made aware of the potential implications of consuming probiotics health supplements, and can make better informed decisions. Further strains identification and precise quantitative MIC of antibiotics can then follow in a more comprehensive report. We have added more recent references as suggested by reviewer 1, and this has made the manuscript more comprehensive. The references (Sharma P, Tomar SK, Goswami P, Sangwan, Singh R: Antibiotic resistance among commercially available probiotics. Food Res Int 2014, 57:176-195) and (Sharma P, Tomar SK, Sangwan V, Goswami P, Singh R: Antibiotic resistance of Lactobacillus sp. isolated from
commercial probiotic preparations. J Food Safety 2015, doi: 10.1111/jfs.12211) have been added; highlighted in yellow. Major Compulsory Revisions: 1. Lactobacilli are known to be resistant to aminoglycosides such as Streptomycin and Gentamicin. Therefore, higher concentrations should be tested in the antibiogram. The antibiotic discs contain pre-determined amounts of antibiotics (erythromycin 15 mcg; gentamycin 5 mcg). These antibiotic dosage have been determined based on the tolerance of the control organisms in this case, the gram negative E. coli and gram positive S. aureus (see Table S1 in Supporting Data 1), and based on data of antimicrobial susceptibility studies by Korhonen JM et al., 2008, 3, Int. J. Probioctics Prebiotics. These amounts also correspond well with the cut-off values of the European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) 2012 standards (http://www.crl-ar.eu/data/images/faq/eurlrecommended%20cut%20off%20values-24-09-2012.pdf). 2. Results & Discussion section, first paragraph: the authors mention the genes that confer resistance in Lactobacilli, however, they have not demonstrated the presence of such genes in the probiotics from commercial supplements. From my point of view, these results (simple PCRs are only required for it) should be included in the manuscript. We agree that the characterization of resistant genes are required to shed light on how the probiotics confer resistance to antibiotics especially if the identity of the probiotic strains are known. More than 25 genes conferring resistance to various antibiotics have been identified in Lactobacilli (see Gueimonde M et al., 2013, 4:202 Front. Microbiology; http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3714544/). Additionally, resistant to certain antibiotics for e.g. vancomycin, are attributed to the presence of multiple intrinsic vancomycin resistant genes in Lactobacilli such as aac(6 )-aph(2 ), ant(6), aph(3 )-IIIa, and not mobile genetic elements. Linking the observed antibiotic resistance to the gene(s) is therefore complicated by 1) the high number of known (and possibly more unknown) resistant genes in Lactobacilli and 2) the difficulty to distinguish mobile genetic elements from the intrinsic resistant genes; the former deserves more attention since they confer antibiotic resistance through horizontal gene transfer, which is also the focus of this study. Differentiation of mobile genetic elements from intrinsic resistant genes have been the focus of works by Ammor MS et al., 2008, 14:1-3 J. Mol. Microbiol. Biotechnol. and Mayrhofer S et al., 2010, 144:1, Int. J. Food Microbiol.). Here, the isolates have not been characterized with regards to the identity but only with regards to their antibiotic resistant profiles, which is the focus of this short report. We made inference on the type of resistance (intrinsic or acquired) based on the currently known resistant genes in Lactobacilli. We
anticipate that further characterization of the probiotic bacteria isolated from these supplements coupled with increasing genetic information will in future, reveal the type of resistant genes present in the isolates of these probiotic supplements. 3. Results & Discussion section: line 120, page 7, the authors state that the overestimation of probiotic amounts in four of the five products tested suggest If I am not wrong, in Bi, Bg, Cn and L products was observed an underestimation of live bacteria with respect to the data sheet Therefore, the overestimation cited by the authors refers to the data sheet of the products, is this right? Please, specify in order to avoid errors when reading this part of the manuscript. Yes, this is correct. We apologize for not making it clear or causing confusion to the readers. The overestimation refers to that of the data sheet or labels. We have now rephrased..the manufacturers have deliberately overestimated the probiotic content of the products in an attempt to compete for consumers [16]. This finding seems to agree with other reports concerning mislabeling, overestimation and misidentification of probiotics in various food and health products.... This change is highlighted in yellow in the text. Minor Essential Revisions: 1. Methods section, first paragraph: MRS media is not only selective for Lactobacilli but also for every lactic acid bacteria that includes Bifidobacterium, Streptococcus thermophilus etc, also present in the products tested in this study. This highlights that the antibiotic resistance results presented in this paper are not only concerning the Lactobacilli but also to the other species included in the capsules. MRS media only specifically selects for Lactobacilli. In order to grow Bifidobacteria, the MRS media must be supplemented with 0.05% cysteine and must be grown under anaerobic conditions. Here, we grow under aerobic conditions and did not add cysteine into the growth media. As for S. thermophilus cultivation, milk-based media is required in addition to other conditions such as ph and temperature. Please see Ashraf and Shah, 2011, 149:3, Int. J. Food Microbiol. for the media and conditions optimized for different probiotic strains. As such, we believe that the antibiotic resistant results are unique to the isolates from Lactobacilli only. 2. Although the use of English is in general fine, there are some sentences that should be reworded such as: Abstract, Background: This can risk clinical complications Introduction, first paragraph: delete prevention after cancer Introduction, last paragraph: rephrase the last two sentences in order to make them clearer. We have made the suggested changes. Highlighted in yellow in the text.
Response to reviewer 2 Major Compulsory Revisions 1. Enumeration of probiotic in supplements: The conclusion reached by the author on the fact that the content in probiotic is largely under the claimed content for probiotic Bi, Bg and L is certainly biased. The method used for enumeration particularly the fact that the sample were dissolved in water is not compliant with the ISO 15214:1998 standard method on enumeration of lactic acid bacteria which recommends tryptone salt as a reconstitution buffer and which is used usually by manufacturer for the release of their products. The fact that water was used can be detrimental on the survival of the probiotic because of the osmotic stress. We dissolved the powdered sample in water and immediately plate on the de Man, Rogosa and Sharpe (MRS) media, and do the same for each runs, mainly to dilute off the preservatives and additives present in the capsule. About 1 g of the powdered sample is dissolved in 1 ml water, and at this ratio, there are residual salts and preservatives to buffer the osmotic shock caused by water. Because we immediately plate the re-suspended sample, probiotic bacteria is only exposed to water for about 1 min. Therefore, majority of the probiotic bacteria in the water re-suspended sample should be viable and can be recovered on MRS media. Water or water-based methods for dissolving powdered samples have also been used in other reports (see Sharma et al., 2015, J Food Safety, doi: 10.1111/jfs.12211. Furthermore as claimed by the manufacturer, other genera are present in the mix (e.g. bifidobacteria & streptococci). Bifidobacteria & streptococci do usually not grow on MRS which could explain part of the difference. I would suggest that the author repeats the enumeration to include all genera contained in the products and according to internationally recognized methodologies. We have explicitly mention in the methods (page 5, line 94 highlighted in yellow) and in supporting data that we have excluded contributions of other probiotic strains from the bacteria count. For example, brand Bg has 2,000 x 10 6 CFU/capsule and 3 different but equal (unless specifically stated) amounts of probiotic bacteria according to the datasheet. Since, two of them are Lactobacilli, we have adjusted the expected amount to 1,333 x 10 6 CFU/capsule, and compared out enumeration to this expected amount (see Figure 1). The discrepancy observed here is therefore a true reflection of live bacteria (Lactobacilli) overestimation by the manufacturers. 2. Antibiotic resistance profiling: I am missing several aspects to accept the conclusions reached by the author on the fact that the probiotic mixes tested contains acquired mobile genetic elements for antibiotic resistance. To me, the method used for determining the antibiotic resistance has several defaults: 1) It is only semi quantitative 2) Has been performed with a mix of strain.
1) We agree that the method is semi-quantitative mainly because we conducted antibiotic resistance screening using commercial antibiotic discs and not with broth dilution method. Antibiotic discs only allow qualitative and semi-quantitative assessments of the anitbiogram of the isolated probiotic bacteria. We selected this method because it is a rapid and well-established assessment (i.e. frequently used in QC of food manufacturing companies) for antibiotic resistant screenings of food products (Liu C et al., 22:5, Biomed. Environ. Sci.) and plant extracts (Ahmad and Beg, 2001, 74:2, J. Ethnopharmacology). In addition, we conducted the screens according to the instructions provided by the disc manufacturers and accompanied by the control microorganisms (E. coli and S. aureus; see Supporting Data 1) which validates the effectiveness of the antibiotics used. 2) The antibiotic resistant screenings were performed on single colony isolates of the respective dietary supplements, thus the tests represent resistance conferred by pure bacteria isolates and not a mixture of strains. Specifically, we first recover the bacteria on MRS plates and then select single colonies for antibiotic resistant screenings. The European Food Safety Authorities have edited clear guidelines on how to perform such antibiotic resistance profiling. It can be found at this address: http://www.efsa.europa.eu/en/efsajournal/doc/2740.pdf. The method described follows the ISO 10932:2010 methodologies, using micro dilutions to calculate a minimum inhibitory concentration (MIC). They have also defined clear MIC for each group/species. As mentioned, we did not conduct the broth microdilution method but the disc diffusion method, which gives qualitative (or semi-quantitative at best) assessment of the probiotic isolates. This is sufficient to highlight the presence of antibiotic resistance in isolates of dietary supplements. Further works to determine the MIC using more sensitive methods such as broth microdilution can be done once the resistant isolates have been characterized with regards to their identities. As one can see in this document, the MIC obtained from the streptomycin and gentamycin are relatively high for most of the species claimed to be present in the mixes, indicating an intrinsic resistance of several of the genera which contradicts the conclusions reached by the author. As the author has tested the mixes using a mix of strain with a semi-quantitative methodology it is impossible to compare it with the extensive work performed by EFSA. I recommend that the author repeats the testing with internationally recognized and quantitative method and with isolated strains. In this way it will be possible to compare the values obtained with existing data. Only after this, if the MIC is above the one of EFSA, it will certainly indicate an acquired resistance and recommendation for sequencing of this isolates to check genetic determinants could be made. We agree that the observed resistance to aminoglycosides streptomycin and gentamycin is likely due to intrinsic resistance rather than a transfer event. We have made it clearer in the text (page 8, line 146 149; highlighted in yellow).
We thank reviewer 2 for the suggestions. We note that both the antibiotic diffusion disc and broth microdilution methods are recognized internationally and widely used (see review: Mayrhofer et al., 2008, 74:12, Appl. Environ. Microbiol. and Jorgensen and Ferraro, 2009, 19:11, Clin. Infect. Dis). The primary aim of this short communication is to highlight the presence of antibiotic resistance in isolates of dietary supplements and to do this, the antibiotic discs method is suitable in our opinion because it is rapid, convenient and allow for screening against multiple antibiotics at once. In order to compare with standard values of EFSA, the identity of the isolates must first be determined before a narrower in-depth study of antibiotic resistance is performed with the broth microdilution method. The genetic determinants can also be identified once the strain ID is confirmed. It is therefore common for broad screenings to be first made with using diffusion disc especially if information (antibiogram) of isolates from dietary supplements is scarce if not absent. Minor Essential Revisions 1. Enumeration of probiotic in supplements: Growth conditions (time, temperature & aerobic/anaerobic conditions) are not mentioned in the text. As to my remark above this is important to know what these conditions were since they can also affect the final enumeration result. We have now specified the growth conditions in the detailed methods in Supporting Data (highlighted in yellow). Discretionary Revisions 1. The difference of amount in bacteria is expressed in %. This kind of difference can also be expressed in log which makes more sense from a microbiology point of view. Furthermore as the methodology used does not seem to be an internationally recognized method, what is the reproducibility of this method from one lab to one other and are the values within this reproducibility? We expressed in % to enable convenient comparison between the experimentally determined amounts with the claims of the manufacturers, which is the message we wanted to put across to readers. We also mentioned that (page 6, line 119) despite the discrepancy, the enumerated amounts are all above the threshold of 10 6 CFU/capsule. In the enumeration, we used the drop plate method which is recognized internationally and is considered superior to the regular spread plate method (see review: Herigstad B et al., 2001, 44:2, J. Microbiol. Meth.), and as you can see from Figure 1A, our representative images of the MRS plates show good bacterial growth. As for the antibiotic resistant screenings, we used commercial antibiotic discs and run the tests in parallel with the control bacteria (E. coli and S. aureus) (see supporting data). The antibiotic discs method is a qualitative (and semiquantitative) method that is internationally recognized to determine if the pure bacterial isolate or microorganism mixtures in plant extracts or biological sample is resistant to the tested antibiotics.