Exploring the Diversity and Antibiogram of the Soil around a Tertiary Care Hospital and a University Precinct in Southern India: A Pilot Study
, Cassandra R. Stanton 1, Jayson Rose 1, Colleen J. Thomas 1,3,4, Ashley E. Franks 1,5, Sumana M. Neelambike 2, Sumana Kumar 6, Steve Petrovski 1 and Anya E. Shindler 1,*
Round 1
Reviewer 1 Report
The article is written well but there are still minor corrections need to be corrected. Specially figures and tables are corrected. In the tables still bacterial names are written some place italic and some places non-italic. Bacterial species (Spp.) should be non-italic etc.
Author Response
We thank Reviewer 1 for their thoughtful comments and suggestions. Please find our responses below:
- “The article is written well but there are still minor corrections need to be corrected. Specifically, figures and tables are corrected. In the tables still bacterial names are written some places italic and some places non-italic. Bacterial species (spp.) should be non-italic etc”.
Thank you for identifying this inconsistency. In the revised manuscript, no bacterial species (spp) are in italicized text (main text, Table 4, and Appendices (A1, A2, A3 and A4). The following figures have been modified to improve the clarity of the image, and were further arranged in the increasing order.
Figure 5 was divided into three separate images – Figure 5, 6 and 7 to improve the clarity of the images. Figure 8 was modified to arrange the horizontal scale naming each sample in the increasing order.
Reviewer 2 Report
This is an interesting work, although there are certain aspects that the authors could develop.
There is a lack of aspects related to the soil, such as properties that indicate the similarities or differences between the soils analyzed (basically pH, texture, organic matter content). Also, how the samples were taken: depth, mode of collection, criteria, where the samples were taken, whether in gardens or on roads. How many subsamples were taken to form the established point sample (Lines 88-100).
In Line 296 it is indicated that the organisms isolates were subjected to Gram staining, why if the MacConkey medium is specific to selectively isolate Gram-negative?
In the discussion section, information already presented in the results section is repeated to a large extent, so I suggest that these repetitions should be avoided.
The authors state that they know of no similar studies, and yet they cite 2 related studies (Line 398, and citation 58 refers to a similar study).
No explanations are mentioned for the results obtained, specifically the fact that the microbial diversity in both locations is the same (Lines 400-402) and that it is the effect of anthropogenic activities in those locations (Lines 402-403), however they do not cite what those anthropogenic activities are.
The Discussion section is more a description of the situation than a discussion of the results obtained.
The work, in its present presentation, I do not suggest its publication. The authors should explain and emphasize the aspects indicated.
Author Response
We thank Reviewer 2 for their thoughtful comments and suggestions. Please find our responses below:
- “There is lack of aspects related to the soil, such as soil properties that indicate the similarities or differences between the soils analyzed (basically pH, texture, organic matter content). Also how the samples were taken: depth, mode of collection, criteria, where the samples were taken, whether in gardens or on roads. How many subsamples were taken to form the established point samples (Line 88-100).”
To the first point, the physiochemical characterization of soil samples collected from the two precincts was not within the scope of the present study but would be useful to consider in future work. Thank you for this suggestion. Our limited resources were used to address the key aim of identifying different bacteria present in the soil, including any antibiotic-resistant bacteria which could further contribute to the dissemination of antibiotic resistance genes. We note that others have published work on soil bacterial diversity without reporting on soil aspects such as pH (eg., Mandal et al ; Environmental Monitoring and Assessment, 2019, 191, 778-813).
To the second point, we have added more detail to the methods section. New content is shown in red colour text below (revised manuscript page 3, lines 89-103).
“Figure 1 illustrates the eight sampling sites used in the study to assess the microbial diversity of the soil surrounding JSS Hospital versus JSS University, which were ~6 km apart. The soil samples were collected from the top 10 cm of the soil with a sterile spatula and glove (separate ones for every sampling site). The samples were collected from the gardens around both the precincts and the sampling locations (n=4 from each precinct; hospital and university) were chosen with the following criteria in mind: building entrance and exit points, places with most human interactions and, for the hospital precinct - location of the nearby sewage treatment plant. In brief, hospital soil samples were collected from the gardens near the entrance (H1), exit (H2), lawns near the east gate (H5; closer to the parking area), and west gate (H6). The university soil sampling sites also focused on the gardens adjacent to a university building. They were collected from the east front garden (U3), west side garden (U4), north garden on the right of the entrance (U7) and north garden on the left of the entrance (U8). Four sub-samples were obtained from each sampling spot. Briefly, a gram (1g) of soil was collected along each of the 4 cardinal directions (north, south, west and east) from each sampling spot and placed into a sterile Nasco soil sampling bag. Next, the samples were pooled to become the representative soil sample from that site. Thus, a total of 4 g of soil was obtained from each sampling site, to establish each site. point along the four directions were. obtain samples sampling site. A total of 4g of soil was obtained from each sampling point. The same procedure was repeated for all soil sampling sites.”
- “In Line 296 it is indicated that the organisms isolated were subjected to gram staining, why if the Mac Conkey medium is specific to selectively isolate gram-negative?”
Although MacConkey medium is selective to Gram-negative bacteria, Gram-positive cocci such as Staphylococci and Enterococci are also known to grow in Mac Conkey medium. Hence, we performed Gram staining to exclude any Gram-positive cocci. We have added further text in the revised manuscript for clarity on this point (page 13, lines 315-317.)
“As certain Gram-positive cocci like Staphylococcus and Enterococcus have the ability to grow on Mac Conkey medium, Gram staining was performed to select the Gram-negative organisms and exclude Gram-positive cocci.”
- In the discussion section, information already presented in the results section is repeated to a large extent so I suggest that these repetitions should be avoided.
We have avoided repetition of the results in the Discussion section of the revised manuscript. The text indicated below (from original manuscript, Pages 15-17 has been removed).
“Among the eight soil samples collected, H1, H2, H5 and H6 were soil sampling sites from the hospital and U3, U4, U7 and U8 were soil sampling sites from the university. With respect to the locations evaluated in our study, the tertiary care hospital had 3 entrance gates and one rear exit. The university sampling sites included the gardens adjacent to one university building.”
“A high value of Shannon diversity indicates a greater diversity. Among all the samples. The U3 site was the most diverse with Shannon diversity 4.68, followed by H2 site ((Shannon diversity, 4.54)”
“Additionally, this study identified 17 statistically diverse phyla and found the predominant phyla, in order of prevalence, to be Proteobacteria, Acidobacteriota, Actinobacteriota, Firmicutes, Cyanobacteria, Bacteroidota, Chloroflexi, Entotheonellaeota, Myxococcota, Gemmatimonadata, Planctomycetota, Armatimonadata, Nitrospirota, Verrucomicrobiota Bdellovibrionota, Patescibacteria and Elusimicrobiota. The study identified 27 classes of statistically diverse bacteria and the top 10 classes of bacteria in the order of abundance were Gammaproteobacteria, Alphaproteobacteria, Bacilli, Cyanobacteria, Actinobacteria, Thermoanaerobaculia, Entotheonellia, Bacteroidia, Blastocatellia and Myxococcia.”
“Our study identified the bacterial diversity around a hospital and university precinct and observed that the university and hospital precinct was similarly diverse. This could possibly be an effect of anthropogenic activities around the hospital settings.”
- The authors state that they know of no similar studies, and yet they cite 2 related studies (Line 398, and citation 58 refers to a similar study).
We apologize for not being clear about this point. Additional text is included in the revised manuscript Discussion section to clarify why our study findings are unique (red-coloured text, below. Page 17, Lines 431-435)
“Although these studies were performed in the soil, different environments were compared. For example, the soil microbiota and its resistome between a conventional and organic farming system (29), the waste soil and sediment microbiome from three rivers and one dumping site polluted by solid and liquid wastes from domestic, hospital and municipal premises (30), etc. However, to our knowledge, this is the first study exploring the microbial diversity in the soil around a hospital setting.”
Additionally, we have replaced citation 30 (Tanentzap et al) for a more suitable publication that supports the context (De Mandal et al). Finally, we have withdrawn citation 58 (Horga) since the work is only a proceedings communication and we can find no evidence the study has been peer-reviewed and published.
“A preliminary study performed in the United States, to compare the percentage of antibiotic resistant soil bacteria between two different soil environments (school vs. hospital), revealed a higher percentage of antibiotic resistant bacteria closer to hospitals”
- No explanations are mentioned for the results obtained, specifically the fact that the microbial diversity in both the locations is the same (Lines 400-402) and that it is the effect of anthropogenic activities in those locations (Line 402-403), however they do not cite what those anthropogenic activities are.
The Discussion section is more a description of the situation than a discussion of the results obtained.
Thank you for highlighting the opportunity to further expand the discussion of our findings. We have now included additional text in the revised manuscript red text as below. Page: 17, Lines 436-446.
“The broad similarity of bacterial communities detected in soil at both precincts could be explained by the fact that the hospital and university were situated only 6km from each other and experienced the same climatic conditions. However, the differences in the relative abundances of different bacteria between the sampling sites may be attributable to different anthropogenic activities in the study environments. For example, there may have been an influence of the sewage treatment plant and laboratories at the hospital precinct (H2, H5). One of the sampling sites was also near a canteen in the hospital grounds (H5). The hospital entrance site (H1) experiences large numbers of people entering and exiting the hospital daily; many human-human as well as human-microbe interactions would be expected to occur at this site. One sampling site (H6) was close to the emergency department where many patients and visitors are either waiting or relaxing outside in the gardens.”
Additionally, we have revised the Discussion text on pages 16-17. New content is shown (red-coloured text) below.
Page number: 16, Lines- 383-388
“Among all the samples, U3 was the most diverse (Shannon diversity, 4.68). This university sampling site was in a garden bed, where the soil would have experienced little disturbance. H2 had the second highest diversity in the hospital and was the sampling site closest to the sewage treatment plant. Additionally, it was a little-disturbed area of the hospital premises (having less movement of people), laboratories were close by, as was a sewage treatment plant (⁓77 m away).”
Page number: 16-17 Lines - 390-401
“A total of 32 phyla identified among all the samples collected, of which 17 were statistically diverse. Among the 32 phyla identified, 17 were statistically diverse and Proteobacteria was the most abundant with the highest abundance in hospital H6 collected from the west gate. This site also contained a high proportion of unidentified bacteria. This is possibly because the west gate was close to the emergency department of the hospital and had heavy patient and visitor traffic, providing increased opportunity for human interactions. Prolonged time spent by patients or visitors in the gardens could also be contributing to the exchange of bacteria between humans and soil. Here, people consume food, with likely variable hand hygiene practices and this could contribute to the exchange of healthy and unhealthy bacteria between humans and soil. This can result in unhealthy or antibiotic resistant bacteria ending up in the soil and further possibilities of tracking these into the hospitals or taken up by other immunocompromised people. Gammaproteobacteria was the most abundant class in the study with Pseudomonas being the most abundant organism identified and present in all the samples.”
Page number: 16-17 Lines - 401-408
“Considering the relative abundance of different phyla and classes, it was observed that Sample H5 was different from the other samples. This could be due to different factors like the water availability, organic manure, differences in the amount of certain nutrients like nitrogen and phosphorus present in the area or due to an effect of pollutants present in the soil. Clinically relevant bacteria belonging to Enterobacteriaceae family were present in the soil from H1, H2 and H6 (hospital) and U3 (university). Their presence in the hospital precinct raises concern as these bacteria could have moved from the hospital to the soil via humans or could pose a threat of moving from the soil to hospitals to infect humans.”
Page number: 17, Lines – 412-414
“The identified isolates are clinically relevant bacteria capable of causing infection in humans and hence the low-level resistance developed by soil bacteria could be alarming. “
Page number: 19, Lines – 510-518
“Yet another study compared the antibiotic resistance genes in the soil between different land-use types in Great Britain. Significantly higher levels of antibiotic resistant bacteria across agricultural land, further highlighting how geographical location and management practices followed in a particular area also influenced the presence of resistant bacteria (57). Relative bacterial compositions and antibiotic resistance in soil and water studied at different sampling environments such as dairy water canal, residential garden soil and lake by hospitals displayed resistance (58). Similarly, the influence of human beings in a hospital area witnessing constant use of antibiotics needs to be monitored. Strict management practices need to be in place to avoid improper waste disposal or leaks in the sewage system.”
Reviewer 3 Report
1) Experimental : It is not clear how the samples were numbered. It would be of better understanding if the numbers were H1, H2, H3 and H4 / U1, U2, U3 and U4. Otherwise, what about H3 and H4, U1 and U2? explain why their were disgarded
2) Figure 5 can be improved : the quality is low, graphs do not have the same size and legenders are not readible.
on the horizontal scales samples are not cited in an "increasing" manner (H1 H2 U3 U4 H5 H6...). Why? if no specific reason, please change it
Considering the Relative abundance of different phyla (5A), and classes (5B) sample H5 seems to be different than other samples. could you explain or discuss?
3) in fig 6 : on the horizontal scales samples are not cited in an "increasing" manner (H1 H2 U3 U4 H5 H6...). Why? if no specific reason, please change it
Author Response
We thank Reviewer 3 for their thoughtful comments and suggestions. Please find our responses below:
- Comments: “Experimental: It is not clear how the samples were numbered….Explain why they were disgarded”.
We provided unique numbers (1-8) to every sampling site to avoid repetition of the same numbers in both the settings as this could lead to mistakes during analysis. Hence, H3, H4 and U1, U2 were disgarded. To be clear, irrespective of the sampling precinct, the samples were numbered consecutively based on the order of collection. The first two samples were collected from the hospital and numbered 1,2 while the next two samples were collected from the university and numbered 3, 4. Similarly, the next two, 5 and 6 were collected from the hospital while 7 and 8 from the university. After sample collection, the abbreviations of the precinct (Hospital (H) and University (U)) were placed preceding the sampling spot. Hence the names H1, H2, U3, U4, H5, H6, U7 and U8. This was done to avoid repeating the same numbers during analysis.
- ) Comments: “Fig 5 can be improved: the quality is low, graphs do not have the same size and legenders are not readable”.
The content presented in Figure 5 (original manuscript) is now presented in three different images to improve the quality (Figure 5, Figure 6 and Figure 7, revised manuscript).
Page: 10, Lines: 268-272,
Page: 11, Lines: 283-288, 290-295
B.) Fig 5, on the horizontal scales samples are not cited in an increasing manner (H1, H2, U3, U4, H5, H6, U7, U8). Why? If no specific reason, please change it.
Figure 5 (Figure 7, revised manuscript) has been modified to arrange the horizontal scales in the increasing order. Page 11, Line: 290-295
C.) Comments: “Considering the relative abundance of different phyla (5A), and classes (5B), sample H5 seems to be different than the other samples, could you explain or discuss?”
The following has been inserted in the Discussion text, Page: 16-17, Lines 402-405
“Considering the relative abundance of different phyla and classes, it was observed that Sample H5 was different from the other samples. This could be due to a different factors like the water availability, differences in the amount of certain nutrients like nitrogen and phosphorus present in the area or due an effect of pollutants present in the soil.”
- Comments: “In Fig 6: on the horizontal scales samples are not cited in an increasing manner (H1, H2, U3, U4, H5, H6..). Why? If no specific reason please change it.”
The heatmap image has been modified (sample numbers in increasing manner) in the revised manuscript.
Page: 12, Lines: 303-307
Round 2
Reviewer 2 Report
The authors have carefully considered the suggestions provided. While the focus of this study is not on soil analysis, providing information about the soil characteristics is important in order to potentially explain the results. As soil is an ecosystem, its properties can influence the presence or absence of particular bacterial communities. Therefore, the inclusion of this information can aid in understanding the findings.
