The Use of Boosted Regression Trees to Predict the Occurrence and Quantity of Staphylococcus aureus in Recreational Marine Waterways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

This study explains the use of a predictive boosted regression trees (BRT) model to forecast the abundance of Staphylococcus aureus in the Tampa Bay estuary and uncover environmental variables linked to the occurrence of this microbial pathogen. Samples have been collected from 7 recreational sites. The paper has been convincingly written to present the data with in the limits of objectives taken in this study. There are some concerns/suggestions that may be addressed. 

1. S. aureus has been identified on the basis of PCR amplification of nuc element, which is common for quick detection of S. aureus. However, this method is not 100% specific and sensitive. Though authors have mentioned that PCR non-responsive isolates were checked by 16S amplification. But again, it has not been given if these were S.aureus specific 16 S primers, or 16s sequence homology has been used instead.

2. Section 2.1.1, the coordinates of sampling sites may be included. Its not given in figure 1 as well.

3. For the statement in line 48: give reference.

4. Under section 3, is there any need to write the list of heads first, followed by their separate descriptions? is this list needed?

5. Some part of the sampling has been done during the time of COVID-19. This has been mentioned too in line 318, where variations has been explained due to covid. It would be better if the lockdown / restrictions' month are discussed, instead of whole year.  As I know it was from march till Sept 21. was there any sampling done in 21 after Sept? or before march? were 2019 data also in accordance to 2022?

6. Its unclear if the S. aureus isolates were of clinical significance? Or were MRSA or multi drug resistant. No such effort had been considered in research design. In that case, how does the background of research as explained in Introduction section, is justified.

 

 

 

Author Response

Reviewer 1

This study explains the use of a predictive boosted regression trees (BRT) model to forecast the abundance of Staphylococcus aureus in the Tampa Bay estuary and uncover environmental variables linked to the occurrence of this microbial pathogen. Samples have been collected from 7 recreational sites. The paper has been convincingly written to present the data with in the limits of objectives taken in this study. There are some concerns/suggestions that may be addressed. 

1. aureus has been identified on the basis of PCR amplification of nuc element, which is common for quick detection of S. aureus. However, this method is not 100% specific and sensitive. Though authors have mentioned that PCR non-responsive isolates were checked by 16S amplification. But again, it has not been given if these were S.aureus specific 16 S primers, or 16s sequence homology has been used instead.

Thank you for this feedback. In order to address the quick detection method of PCR we did some additional sequence confirmation of nuclease amplicons as well as 16S amplicons from nuclease negative PCR isolates. The following text was inserted at lines 145-151 and lines 163-167 to address reviewer #1 comment 1.

 

Inserted at lines 145-150

 

“Specifically, nuclease primers used were designed from a 966 bp sequence of the nuclease A gene from the S. aureus Foggi strain to amplify a 279 bp sequence [33], [34]. The 279 bp amplicon reports 100% identity to NCBI Accession #CP150135.1. A random sub-sampling of S. aureus nuclease PCR amplicons were sequenced to confirm positive identity of isolates as well as control S. aureus N315 strain amplicons (NCB1 Accession # NC_002745.2).”

 

Inserted at lines 162-166

“Specifically, the 16S primers target a region of the 16S sequence used for phylogenetic analysis of a wide range of bacteria [35], amplifying a 1465 bp sequence. Further, internal 16S sequencing primers are being used for microbial identification and current data indicate when Staphylococcus genus are detected they are not aureus species specific (unpublished data).”

 

2. Section 2.1.1, the coordinates of sampling sites may be included. Its not given in figure 1 as well.

 

We agree with the reviewer and add the coordinates for each sampling site in lines 111 – 116 and add graticule lines to Figure 1 lines 226-229.

 

Inserted at lines 111-116

 

“Water samples for microbial analysis were systematically collected from the following 7 recreational sites in Tampa Bay; Gandy Beach (GB) (-82.59566, 27.87459), Ben T. Davis (BD) (-82.57884, 27.97045), Cypress Pt. Park (CP) (-82.54697, 27.95016), Picnic Island (PI) (-82.55415, 27.85183), Davis Island (DI) (-82.44721, 27.902972), Bahia Beach (BB) (-82.47657, 27.72894), and E. G. Simmons Park Beach (EGS) (-82.47278, 27.74772) (Figure 1).”

 

3. For the statement in line 48: give reference.

We agree with reviewer 1 and added the below references to text in line 48.

2. K. D. Goodwin, M. McNay, Y. Cao, D. Ebentier, M. Madison, and J. F. Griffith, ‘A multi-beach study of Staphylococcus aureus, MRSA, and enterococci in seawater and beach sand’, Water Research, vol. 46, no. 13, pp. 4195–4207, Sep. 2012, doi: 10.1016/j.watres.2012.04.001.

 

3. F. Hassard et al., ‘Abundance and Distribution of Enteric Bacteria and Viruses in Coastal and Estuarine Sediments—a Review’, Front Microbiol, vol. 7, p. 1692, Nov. 2016, doi: 10.3389/fmicb.2016.01692.

 

8. B. F. Froeschke, A. Williams, and R. Seferian, ‘Spatial distribution and antibiotic susceptibility of Staphylococcus aureus from discharge into the Hillsborough River, Tampa, FL’, Florida Scientist, vol. 82, no. 1, pp. 1–8, 2019.

 

14. M. Tosic, J. D. Restrepo, A. Izquierdo, S. Lonin, F. Martins, and R. Escobar, ‘An integrated approach for the assessment of land-based pollution loads in the coastal zone’, Estuarine, Coastal and Shelf Science, vol. 211, pp. 217–226, Oct. 2018, doi: 10.1016/j.ecss.2017.08.035.

 

4. Under section 3, is there any need to write the list of heads first, followed by their separate descriptions? is this list needed?

Thank you for noticing this editorial error. The list of heads should be removed as indicted by strike through on document lines 206-213.

 

5. Some part of the sampling has been done during the time of COVID-19. This has been mentioned too in line 318, where variations has been explained due to covid. It would be better if the lockdown / restrictions' month are discussed, instead of whole year.  As I know it was from march till Sept 21. was there any sampling done in 21 after Sept? or before march? were 2019 data also in accordance to 2022?

Thank you for the feedback. We added the below text to specify the sampling dates centered around COVID lockdown in Florida in lines 330 – 335.

Lines 330 - 335

“It is possible that the long term temporal patterns seen are due to the increase of recreational uses during the COVID pandemic. Specifically, a rapid increase in the occurance of S. aureus in Febuary 2020 (Figure 5, month 2) followed by sustained high levels of S. aureus in March and April 2020 (Figure 5, months 3 and 4). Additionally, a decrease in S. aureus follows in May 2020, coinciding with reopening of Florida. (Figure 5, month 5).”

 

6. Its unclear if the S. aureus isolates were of clinical significance? Or were MRSA or multi drug resistant. No such effort had been considered in research design. In that case, how does the background of research as explained in Introduction section, is justified.

Thank you for your insightful comment. While MRSA was not the primary focus of our study, it's important to note that the Staphylococcus aureus isolates we collected were indeed of clinical significance. S. aureus is notorious for causing staph infections, which can range from mild skin infections to more severe conditions such as pneumonia and bloodstream infections. Additionally, these bacteria can be transmitted through various environmental sources, including water bodies. Although our research did not specifically target MRSA or multi-drug resistant strains, the presence of clinically significant S. aureus isolates underscores the relevance of our study. By focusing on developing a predictive model based on environmental parameters, we aimed to shed light on the potential pathways and factors influencing the presence and spread of pathogenic bacteria in aquatic environments. This broader perspective allows us to explore the complexities of microbial ecology and contribute to the development of strategies for mitigating public health risks associated with waterborne pathogens.

Reviewer 2 Report

Comments and Suggestions for Authors

After reviewing the manuscript the authors submitted, I found your manuscript to be an interesting results.  However, I would like you to present the specificity and copy number of the gene the authors selected for quantitative analysis via the NCBI blastn module, though you cited information a reference journal.

Quantitative analysis of target bacteria In order to perform PCR, it is necessary to first confirm whether the gene used is specifically present in the bacteria. Then, it is pointed out that it is meaningless to proceed without clear data on that point. So, first of all, I suggest that authors use the NCBI BLASTn module to confirm the specificity of the gene.

 

Author Response

Reviewer 2

After reviewing the manuscript the authors submitted, I found your manuscript to be an interesting results.  However, I would like you to present the specificity and copy number of the gene the authors selected for quantitative analysis via the NCBI blastn module, though you cited information a reference journal.

We appreciate review 2’s suggestion and agree that the NCBI accession numbers improve the methods section. To address this comment, we inserted the following at lines 144-150.

 

“Specifically, nuclease primers used were designed from a 966bp sequence of the nuclease A gene from the S. aureus Foggi strain to amplify a 279 bp sequence (Shortle 1983, Brakstad 199). The 279bp amplicon shown 100% identity to NCBI Accession #CP150135.1. A random sub-sampling of S. aureus nuclease positive PCR products were sequenced to confirm positive identity of isolates as well as control S. aureus N315 strain amplicons (NCB1 Accession # NC_002745.2).”

 

Quantitative analysis of target bacteria In order to perform PCR, it is necessary to first confirm whether the gene used is specifically present in the bacteria. Then, it is pointed out that it is meaningless to proceed without clear data on that point. So, first of all, I suggest that authors use the NCBI BLASTn module to confirm the specificity of the gene.

We agree with reviewer 2 that it is necessary to use a gene that is specific to the bacterial species being investigated. The following two sections of text have been added to the methods section to include NCBI accession information and two additional references to address this comment.

Inserted at lines 145-150

 

“Specifically, nuclease primers used were designed from a 966 bp sequence of the nuclease A gene from the S. aureus Foggi strain to amplify a 279 bp sequence [33], [34]. The 279 bp amplicon reports 100% identity to NCBI Accession #CP150135.1. A random sub-sampling of S. aureus nuclease PCR amplicons were sequenced to confirm positive identity of isolates as well as control S. aureus N315 strain amplicons (NCB1 Accession # NC_002745.2).”

 

Inserted at lines 162-166

“Specifically, the 16S primers target a region of the 16S sequence used for phylogenetic analysis of a wide range of bacteria [35], amplifying a 1465 bp sequence. Further, internal 16S sequencing primers are being used for microbial identification and current data indicate when Staphylococcus genus are detected they are not aureus species specific (unpublished data).”