Mursamacin: a novel class of antibiotics from soil-dwelling roundworms of Central Kenya that inhibits methicillin-resistant Staphylococcus aureus [version 2; peer review: 2 approved, 1 approved with reservations]

Antibiotic-resistant bacteria, also called “superbugs”, can at worst retrogress modern medicine to an era where even sore throats resulted in death. A solution is the development of novel types of antibiotics from untapped natural sources. Yet, no new class of antibiotic has been developed in clinical medicine in the last 30 years. Here, bacteria from insect-killing Steinernema roundworms found in the soils of Central Kenya were isolated and subjected to specific molecular identification. These were then assayed for production of antibiotic compounds with potential to treat methicillin-resistant Staphylococcus aureus infections. The bacteria were identified as Xenorhabdus griffiniae and produced cell free supernatants that inhibited S. aureus. Fermenting the bacteria for 4 days yielded a heat stable anti-staphylococcal class of compounds that at low concentrations also inhibited methicillin-resistant S. aureus. This class contained two major compounds whose identity remains unknown. Thus X. griffinae isolated from Steinernema roundworms in Kenya have antimicrobial potential and may herald novel and newly sourced potential medicines for treatment of the world’s most prevalent antibiotic resistant bacteria


Introduction
Antibiotic resistant bacteria, otherwise known as "superbugs", are an imminent threat to every existing healthcare system as they could obviate current clinical antibiotics and thereby retrogress humanity to that dark age of lethal sore throats 1 .Of note is methicillin-resistant Staphylococcus aureus (MRSA).In this study, we examine the antimicrobial activity of Xenorhabdus griffiniae fermentation media against MRSA.
MRSA not only causes human diseases such as mastitis, chronic open wound infections and endocarditis, but also livestock diseases such as mastitis in dairy cattle and lameness in poultry and rabbits 2 , that together result in economic losses of billions of dollars to the agricultural sector 3 .In both humans and animals, an MRSA infection can quickly turn lethal.This is because MRSA is resistant to two antibiotic classes, beta lactams and macrolides, and only lipopeptides and glycopetides remain effective 4 ; this development has contributed in large part to the rise of the vancomycin-resistant S. aureus superbugs.A recommended solution 1 to the superbug conundrum is to develop novel classes of antibiotics that can replace those to which disease-causing bacteria have mutated to resist their inhibitory effect.
A potential source of novel anti-MRSA antibiotics are Xenorhabdus bacteria 5 that naturally dwell in the guts of Steinernema roundworms.These 1 mm-long 6 roundworms are found in soils worldwide 7 and live by infecting and killing insects such as moths, caterpillars and weevils.The Xenorhabdus bacteria carried in their gut aid this insect killing lifestyle.Explicitly, Steinernema roundworms enter an insect body and release Xenorhabdus bacteria that secrete insecticidal toxins that quickly kill the insect 8 .To secure this rich food source for the roundworms only, the Xenorhabdus produce an armory of antibiotic compounds that effectively destroy competing soil fungi and microorganisms 9 , a mechanism that has been demonstrated as having medical potential against human diseases 8,[10][11][12][13][14] .Each Xenorhabdus species has been demonstrated to produce its own unique array of antibiotics 11 and this is prompting new studies on the classes of antimicrobial compounds produced.For example, X. cabalinasii JM26 from Jamaica led to the discovery of nemaucin, a novel and highly potent antibiotic compound against methicillin-resistant S. aureus 15 .
Politically, "superbugs" are today's top global health issue exemplified by the 2016 United Nations High Level General Meeting's agenda being antibiotic resistant bacteria: this is only the fourth time in its 80-year history that a health issue has been the reason for this annual meeting.Technically, superbugs have been detected in all 114 countries recently surveyed with the number of pan drug resistant superbugs, those immune to every antibiotic available, on the rise 1 .Accordingly, Kenyan prevalence levels of MRSA have been steadily increasing 16,17 .
Previously 18 we demonstrated that Xenorhabdus bacteria from Kenya can produce antibiotics against MRSA.However, the identity of the species, the antibiotic classes it produces and their inhibitory concentrations remain unknown.Here, we elucidate the specific molecular identity of Kenyan Xenorhabdus isolates and determine the efficacy of produced antimicrobial compounds against MRSA.Our findings highlight a novel antibiotic class designated "mursamacin" 19 obtained from Xenorhabdus bacteria found in Kenyan soils, which is highly active against methicillinresistant S. aureus.

Bacterial and nematode strains
MRSA strain 133 cultures were obtained as a gift from Dr. John Ndemi of the Kenya Medical Research Institute, Centre for Microbiology Research, Nairobi, Kenya.Pure nematode cultures of Steinernema roundworm isolates were obtained from the nematode culture collection of Horticulture Research Institute Thika, Kenya.
Using the indirect haemolymph method 20 X. griffiniae strains XN45 and L67 were isolated from Steinernema sp.Scarpo and Steinernema sp.L67 nematodes respectively.The isolates were cultured on Xenorhabdus differential media NBTA, composed of nutrient agar (Himedia) supplemented with 0.0025% (w/v) bromothymol blue (Sigma-Aldrich) and 0.004% (w/v) 2,3,5 triphenyl tetrazolium chloride (Sigma-Aldrich) 20 .Identification of the bacteria as Xenorhabdus was based on the presence of the following characteristics: swarming motility on NBTA of 1% (w/v) agar concentration; swimming motility on NBTA of 0.5% (w/v) agar concentration; and yellow green colony pigmentation on NBTA 5 .Specific identification of the bacteria was done by multi locus sequence typing of the 16s rRNA, serC and recA genes (See Dataset 1).

Molecular methods
Total DNA extraction from the bacterial strains was done using FastDNA®SPIN Kit for Soil (MP Biomedicals, USA).Isolation of a 1397 base pair(bp) 16s rRNA gene fragment was done by Polymerase Chain Reaction (PCR) with primer sequences (Inqaba Biotech) as follows: (27f-AGA GTT TGA TCA TGG CTC AG) and (1391r-ACG GGC GGT GTG TGC) 21 .The genes were amplified in a 25 μl reaction volume containing final concentrations of 0.5 U Q5 DNA polymerase (New England Biolabs, USA), 200μM each dNTP, 2mM MgCl 2, and 0.05μM of each primer.Cycling conditions were set at 98°C for 30 s, 40 cycles of 98°C for 30 s, 42°C for 15 s (first 20 cycles) and 47°C for 15 s (final 20 cycles), 72°C for 1 min and a final extension of 72°C for 2 min (MJ Research PTC-100, USA).Isolations of 670bp serC and 400bp recA gene fragments were done by PCR with primers (recA-FW CCA ATG GGC CGT ATT GTT GA) and (recAREV-TCA TAC GGA TCT GGT TGA TGA A) and (serCF-CCA CCA GCA ACT TTG TCC TTT C) and (serCR-AAA GAA GCA GAA AAA TAT TGC AC) respectively 22 .They were amplified in a 50 μl reaction volume containing final

Amendments from Version 1
In this version, a few typographical errors have been corrected.The contributions of Daniel Masiga, previously only recognised in the Acknowledgments section of version 1, significantly increased.Thus, he is now recognized as a co-author of the research paper.Also, the new Acknowledgments section has been updated to conclusively reflect the role various parties played in bringing this research, over its 8-year journey, to light.

REVISED
concentrations of 2 U MyTaq ® DNA polymerase (Bioline, USA), 200μM of each dNTP, 3mM MgCl 2, and 0.4μM of each primer.For both, cycling conditions were set at 95°C for 1 min, then 40 cycles of 95°C for 15 s, 52°C for 15 s, 72°C for 40 s, with a final extension of 72°C for 5 min (Thermo Scientific Arktik, USA).PCR products were visualized on 1.2% (w/v) agarose gels stained with ethidium bromide at final concentrations of 0.5 μg/ml.Typical electrophoresis conditions were 4V/cm for 72 min.Expected bands were excised and purified with Quick Clean II Gel extraction kits® (Genscript, USA).Products were outsourced for sequencing (Macrogen, Netherlands), and obtained sequences were quality checked, assembled and poor quality base calls trimmed in BioEdit 23 and MEGA6 24 software suites.

Phylogenetic analysis
Phylogenetic reconstruction was performed using a multi locus concatenate of 16s rRNA, serC and recA genes sequences, that jointly constituted 2076 positions.A dataset of n=13 (1= from this study and 12= public databases) was used that contained the 11 strains of Xenorhabdus and a Photorhabdus luminiscens (the out-group sequence) that had public database sequences of all three genes (see data files).Database sequences were checked for quality and ambiguous nucleotides resolved in the MEGA6 software suite 24 .Multiple sequence alignments were performed in the same suite using the MUSCLE algorithm 25 .The evolutionary history was inferred by the ML method based on the Generated Time Reversible (GTR) Model (500 bootstraps).Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.The analysis involved 13 nucleotide sequences.All positions containing gaps and missing data were eliminated resulting in a total of 2076 positions used in the reconstruction.Xenorhabdus DNA sequences generated in this study were deposited in the DNA databank of Japan with the partial 16s rRNA gene sequences of X. griffiniae L671, L672, L673, L675, XN45 assigned the following accession numbers respectively; AB987698.1,AB987700.1,AB987701.1,AB987699.1,AB987697.1.The partial recA, serC gene sequences of X. griffiniae L67 and XN45 were assigned LC096094, LC096092 and LC096093, LC096091 respectively.Percentage sequence similarities between gene sequences from this study and other 16s rRNA, recA and serC gene database sequences was determined using blast searches 26 .

Bacterial fermentation
Fermentation was done using X. griffiniae XN45 bacterial cultures.Multiple colonies (2-3) of an individual isolate were selected, inoculated into 5 ml of Luria Bertani (LB) media containing 1% tryptone, 0.5% yeast extract and 1% NaCl (w/v) and incubated on a rotatory shaker at 150 rpm at 33°C for 24 h.These served as 1% (v/v) starter inocula.Sterile LB media (500 ml) was dispensed into sterile 1-L Erlenmeyer flasks, with starter cultures (5 ml) thereafter inoculated and LB media incubated at 150 rpm at 33°C for 355 h, 180 h and 108.5 h.LB with no inoculum was also incubated to serve as a control for sterility.After fermentation, cells were removed by centrifugation of broths at 20,000 g for 25 min at 4°C (Beckman Avanti J-25, USA) followed by decanting cell free supernatants (cfs).These were heat-treated by autoclaving at 121°C and 15 p.s.i for 20 min to yield a sterile heat stable fraction of the whole broth extract (antibiotic) that was designated "mursamacin".These were stored at 4°C until use.

Broth macro dilution inhibition assay against MRSA
The broth macro dilution assay was prepared as previously described 11 with modifications, using antibiotics obtained from each of the fermentation durations (180.5 h and 355 h) as the antibiotic and MRSA as the test bacterium.MRSA overnight cultures were inoculated into each dilution, at a final concentration of 2.3×10 4 cfu/ml, and then incubated for 18 h at 37°C without agitation.The following controls were included in every replicate: negative control of 2× LB media inoculated with bacteria without antibiotic; sterility control of 2× LB media with no inoculated bacteria; and sterility control of undiluted antibiotic with no inoculated bacteria.
After incubation, turbidity of each dilution was measured by determining the A600 nm (See dataset files).This was used in the following formula, modified from Houard, Aumelas 13 that included a correction factor for inhibition by the broth media to calculate the percentage growth inhibition of bacterial cultures by an antimicrobial.
Where g = A600 nm of bacteria in broth culture without antibiotic, and g x = A600 nm of bacteria in broth culture with antibiotic.

Fractionation of lyophilized powders of mursamacin
To extract an organic heat stable mursamacin class of antibiotics, cfs from a 108.5 h fermentation reaction were lyophilized to yield a yellow powder, whose measured amounts were dissolved in known volumes of methanol, and vortexed for 2 min to yield a dark orange methanol extract.These were centrifuged at 20,000 g for 13 min at room temperature to separate liquid methanol extracts from insoluble residues.Methanol extracts were pipetted into sterile 1.5 ml tubes while pelleted solids were aseptically air dried and then incubated at 37°C overnight to evaporate residual methanol.To provide a negative control, the same procedure was repeated for lyophilized powders of the fermentation media.
The difference in weight of powder before and after methanol extraction was then measured to determine the concentration of total dissolved compounds in the methanol extracts.
Broth microdilution assay of organic heat stable mursamacin antibiotics against MRSA This was modified from a previously described method 27 .To determine the minimum inhibitory concentrations (MICs) against MRSA, 100 μl of known concentrations of organic heat-stable fractions of mursamacin antibiotics were dispensed into sterile 96-well micro-titre plate starting wells.These were left in a biological safety cabinet to evaporate methanol resulting in visible orange solid residues; each was then re-dissolved in 200 μl RPMI media supplemented with 5% (v/v) LB 28 .Every other well along each row was filled with 100 μl RPMI media supplemented with 5% (v/v) LB; 100 μl of starting well mixture was then dispensed to the subsequent well resulting halving the antibiotic concentration.This was repeated for all wells along the rows resulting in a 2-fold dilution series.MRSA inocula (10 μl) were used that had been previously prepared from plate cultures dissolved in physiological saline to a turbidity of 0.5 Mcfarland standard (ca.concentration= 2.6×10 6 cfu/ml), then subjected to 10 -3 dilution in RPMI media supplemented with 5% (v/v) LB.
A positive control row of Daptomycin and negative control of methanol extract of LB media only was incorporated in every replicate.Plates were incubated at 37°C for 21 h without agitation.Experiment was performed in 10 replicates in three reproductions.

High performance liquid chromatographic analysis
To identify the compounds contained in the organic heat-stable fraction of mursamacin antibiotics, analytical reverse phase chromatography of the methanol extract was performed as previously described 13 and modified to use of a c18 column (Aglient Zorbax Eclipse Plus C18; 3.5um, 4.6 ×100 nm) under isocratic conditions of 60% acetonitrile and uv detection of 224 nm.

Results and discussion
Xenorhabdus griffiniae strains are found in Kenya The Xenorhabdus isolates identified were most closely related to Xenorhabdus griffiniae (Figure 1).The apt thresholds for Xenorhabdus species identification based on sequence similarities are currently considered to be 98.65% and 97% for 16s-rRNA and for both recA and serC gene fragments respectively 29,30 .In this study, sequences similarities to X. griffiniae strains, including type strains, were 99.52%, 98.57% and 97.68% for 16s rRNA, recA and SerC respectively (See data files) Xenorhabdus griffiniae has only previously been isolated from Indonesia 31 , Malaysia 32 and South Africa 33 ; therefore our data strongly suggest a new strain of X. griffiniae originating from Central Kenya.

X. griffiniae produces heat stable anti-staphylococcal compounds
The growth of methicillin-resistant S. aureus was inhibited when cultured in X. griffiniae cell-free supernatants (cfs) that we termed 'mursamacin' (Figure 2).Furthermore, cfs were obtained by various X. griffiniae fermentation durations and had the following percentage of growth inhibition against methicillin-resistant S. aureus at neat concentrations: 81% (cfs from 180 h ferment), 94% (cfs from 355 h ferment) (Figure 3).In both instances, cfs were heat-sterilized by autoclaving and pH adjusted to that of the control (6.6-7.0),indicating that inhibition was due to heat stable compounds contained therein.
Previous studies 9, [11][12][13]34,35 have demonstrated that Xenorhabdus bacteria are prolific antimicrobial producers with each species producing its unique array of antibiotics that often contained novel compounds. Consitent with these reports, our data demonstrate unprecedented evidence of heat-stable X. griffiniae antimicrobials and further suggests that their production is affected by how long X. griffiniae is cultured in fermentation media.An organic heat-stable fraction of mursamacin antibiotics is highly effective against methicillin-resistant S. aureus An organic heat-stable fraction of mursamacin antibiotics inhibited methicillin-resistant Staphylococcus aureus at a standardized concentration of 8.25 μg/ml while its negative control-organic extract of autoclaved fermentation media only-displayed growth at all concentrations confirming this inhibition as due to antibiotic compounds only (Table 1).However, this concentration was 17-fold higher than the positive control Daptomycin that gave 0.5 μg/ml.
Currently, there are two major clinical antibiotics with inhibitory concentrations against methicillin-resistant S. aureus that are considered effective: Daptomycin (0.5 μg/ml) and Vancomycin (2 μg/ml) 4 .On the other hand, the larger majority today's clinical drugs have inhibitory concentrations against methicillin-resistant S. aureus considered that are considered ineffective; Azithromycin (128μg/ml), Amoxicillin/Clavulanic acid (64 μg/ml), Cefriaxone (64 μg/ml) and Erythromycin (32 μg/ml) and Imipenem (16 μg/ml) 4 .Yet our data demonstrate a heat stable class of compounds with an inhibitory concentration of 8.25 μg/ml.This strongly suggests that antimicrobials contained in this class are even more potent against methicillin-resistant S. aureus, as pure compounds.
Further high performance liquid chromatographic analysis revealed that this fraction contained two major compounds, eluted at 1.862 min and 2.775 min and absorbed at 224 nm when dissolved in methanol (Figure 4).Previous studies have characterized classes of organic Xenorhabdus antibiotics that   were highly effective against S. aureus 11,36 and had peaks absorption ranges from 200-230 nm 15 .Of note, the PAX lipopeptides 10 isolated X. cabanillasii and X. nematophila were highly effective against MRSA at concentrations of 0.5 μg/ml.Yet in contrast to our results, no class has been characterized as heat stable and isolated from X. griffiniae.
In conclusion, we demonstrate that X. griffiniae antibiotic compounds termed "mursamacin" contained an organic heat stable fraction are highly effective against methicillin-resistant S. aureus and antimicrobial activity seems to be attributed to two dominant uncharacterized compounds.This may offer a founding stone for further development of clinical drugs from X. griffiniae, giving hope to thousands of patients affected by methicillin-resistant S. aureus infections.Last Para, Line last 3 rd "designated "mursamacin"19 obtained from Xenorhabdus bacteria" Name the spp. of Xenorhabdus bacteria used.

Methods
Page 3, Phylogenetic analysis, line 12, (Nei and Kumar 2000), according to reference style name should not be included despite use number and this reference is cited in text but not mentioned in Reference part of the manuscript.

1.
Page 3, Bacterial fermentation, line 1, only one bacterial strain was used for fermentation what about the second strain mentioned in the manuscript? 2.
Page 3, Bacterial fermentation, line last 4 th , "These were heat-treated by autoclaving……" If these were further autoclaved, even after inoculation, the antibiotics produced might get degraded.Explain.

3.
Page 3, Bacterial fermentation, line 5 th , The ideal temperature for growth of Xenorhabdus spp. is 28 o C, then why 33 o C had been used for bacterial fermentation? 4.
Page 4, High performance liquid chromatographic analysis, line 1, "To identify the compounds….." How can compounds be identified using HPLC only?This part is not explained well as per scientific standard.

6.
Instead of nm it should be mm and C-18 should be written like this not as author wrote.
If my questions would be answered with full justification, I would give my full approval for this manuscript.
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.If autoclaved supernatant will be used, why centrifugation and filtration methods were used to remove the cells?

3.
Not only autoclaved but also non-autoclaved cell-free supernatants should have been tested against S. aureus bacteria as well.

4.
Tube-A in Figure-2 looks like there are several tubes in a beaker!This photo needs to be renewed.

5.
Competing Interests: No competing interests were disclosed.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.I accept it but some little missing information should be added to get my approval (see details).The subject of paper is extremely important, since the "super bugs", that is the dramatically increasing numbers of multiresistant pathogens is a real danger for mankind and pose new challenge to the antibiotics researcher, and the research concept outline in this publication is excellent.

Reviewer
As for the title: it is appropriate for the content of the article.
The abstract represents a suitable summary of the work.As for the article content: the design, methods and analysis of the results from the study has been properly explained and they are appropriate for the topic being studied.
The information for the conclusions: the conclusions are sensible, well-balanced and justified on the basis of the results of the study.Enough information has been provided to be able to replicate the experiment.The data are presented in a usable format and all the data we need to understand has been provided.

Some detail:
The paper reports the discovery of a new, heat stable antimicrobial (probably peptide) complex effective against the "superbug" MRSA in both in vitro tests and in larger scale.It is a very important discovery, even if we are still rather far to get a commercial product.The conception and the applied methods are suitable, and the discovery and proper molecular identification of the antibiotic producing Xenorhabdus griffiniae from Kenya is very fortunate and great.
○ I think that the quality of the research and the article is scientifically sound.
○ I have only one important question: Table 1: The reader need the definition how the "Standardized MIC" values were calculated from the "Average MICs".It seems to be a result of a statistical calculation, but I cannot be seen the source as mentioned.the related text in "Methods" and "Results and discussions" chapters: Some details related to "high performance liquid chromatographic analysis" is badly missing, such as the specific name of the method, the data of column, the eluting media, and at least an approximated molecular size of the two active fraction 1,862 and 2775.

○
If my question got an answer and my remark were taken into consideration, I would give my full Approval.
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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Figure 1 .
Figure 1.Phylogenetic tree of Xenorhabdus species based on the maximum likelihood method.The tree was based on a concatenate of ss-rRNA, recA, and serC gene sequences and was reconstructed with a general time reversal model and test of phylogeny of 500 bootstrap replicates.X. griffiniae isolated from this study (red triangle) clustered in the X. griffiniae clade (red), and was most closely related to X. griffiniae from Malaysia.

Figure 2 .
Figure 2. Inhibition of methicillin-resistant S. aureus by mursamacin antibiotics.MRSA cultures were incubated with (tube a) and without (tube b) mursamacin antibiotics respectively.A clear tube denotes no bacterial growth while turbid tube denotes bacterial growth.

Figure 3 .
Figure 3. Growth inhibition of methicillin-resistant S. aureus by heat stable fractions of mursamacin antibiotics produced by varying fermentation durations.The longer fermentation duration (355 h) produced antibiotics that were generally more inhibitory to methicillinresistant S. aureus.Dotted line graphs represent linear equations derived from the raw inhibition values.The high R-squared values demonstrate that the concentration of the antibiotic was predominantly responsible (97-98%) for the level of percentage growth inhibition.

MICS of a,b,c are averages of 3 , 4 , 3
replicates respectively (See data files).*These are projected MICs values should tests be done under standard assay conditions.

Figure 4 .
Figure 4. HPLC Chromatographs of organic mursamacin antibiotics.Top chromatograph represents the solvent only while bottom chromatograph is of solvent containing organic mursamacin antibiotics.Two dominant compounds, indicated by the red arrows, were detected in this fraction.

Dataset 1 .
Raw data of antibiotics from soil dwelling roundworms of Central Kenya inhibiting methicillin resistant Staphylococcus aureus http://dx.doi.org/10.5256/f1000research.9652.d136966All raw data supporting the study are provided.Introduction 3 rd Para, Line 9 "The Xenorhabdus produce an armory of antibiotic compounds…."Can author define some names of antibiotic compounds produced by Xenorhabdus spp.1.

Reviewer
Report 03 November 2016 https://doi.org/10.5256/f1000research.10401.r17200© 2016 Hazir S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Selcuk Hazir Department of Biology, Faculty of Arts and Science, Adnan Menderes University, Aydin, Turkey Mueller-Hinton media should have been used in antimicrobial tests instead of Luria Bertoni (LB).

1 .
The optimum growth temperature of Xenorhabdus spp. is 28 o C. Why authors used 33 o C for bacterial fermentation?2.
Report 31 October 2016 https://doi.org/10.5256/f1000research.10401.r16762© 2016 Fodor H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Habil Andras Fodor Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA I consider the publication of Awori RM, Ng'ang'a PN, Nyongesa LN and Amugune NO, entitled as " Mursamacin: a novel class of antibiotics from soil-dwelling roundworms of Central Kenya that inhibits methicillin-resistant Staphylococcus aureus" is as follows as a well conducted research.I have read this submission.

○
Fig 4 and the related text in "Methods" and "Results and discussions" chapters: Some details related to "high performance liquid chromatographic analysis" is badly missing, such as the specific name of the method, the data of column, the eluting media, and at least an approximated molecular size of the two active fraction 1,862 and 2775.