Vibrio parahaemolyticus from Migratory Birds in China Carries an Extra Copy of tRNA-Gly and Plasmid-Mediated Quinolone Resistance Gene qnrD

ABSTRACT Vibrio parahaemolyticus is a marine bacterium coming from estuarine environments, where the migratory birds can easily be colonized by V. parahaemolyticus. Migratory birds may be important reservoirs of V. parahaemolyticus by growth and re-entry into the environment. To further explore the spreading mechanism of V. parahaemolyticus among marine life, human beings, and migratory birds, we aimed to investigate the characteristics of the genetic diversity, antimicrobial resistance, virulence genes, and a potentially informative gene marker of V. parahaemolyticus isolated from migratory birds in China. This study recovered 124 (14.55%) V. parahaemolyticus isolates from 852 fecal and environmental (water) samples. All of the 124 strains were classified into 85 known sequence types (STs), of which ST-2738 was most frequently identified. Analysis of the population structure using whole-genome variation of the 124 isolates illustrated that they grouped into 27 different clonal groups (CGs) belonging to the previously defined geographical populations VppX and VppAsia. Even though these genomes have high diversity, an extra copy of tRNA-Gly was presented in all migratory bird-carried V. parahaemolyticus isolates, which could be used as a potentially informative marker of the V. parahaemolyticus strains derived from birds. Antibiotic sensitivity experiments revealed that 47 (37.10%) isolates were resistant to ampicillin. Five isolates harbored the plasmid-mediated quinolone resistance (PMQR) gene qnrD, which has not previously been identified in this species. The investigation of antibiotic resistance provides the basic knowledge to further evaluate the risk of enrichment and reintroduction of pathogenic V. parahaemolyticus strains in migratory birds. IMPORTANCE The presence of V. parahaemolyticus in migratory birds' fecal samples implies that the human pathogenic V. parahaemolyticus strains may also potentially infect birds and thus pose a risk for zoonotic infection and food safety associated with re-entry into the environment. Our study firstly highlights the extra copy of tRNA as a potentially informative marker for identifying the bird-carried V. parahaemolyticus strains. Also, we firstly identify the plasmid-mediated quinolone resistance (PMQR) gene qnrD in V. parahaemolyticus. To further evaluate the risk of enrichment and reintroduction of pathogenic strains carried by migratory birds, we suggest conducting estuarine environmental surveillance to monitor the antibiotic resistance and virulence factors of bird-carried V. parahaemolyticus isolates.

spread of the bacterium between aquatic environments and other sources, including fish, though they also suggest that the bird V. parahaemolyticus may constitute a bird-adapted variant. You have stated the purpose of the research as characterization of the isolates infecting birds and searching for genetic markers for bird-specific isolates. Was there a more general hypothesis or a larger question to answer behind the decision to undertake this research? If so, explicitly stating it would help to provide the larger context for the research.
Two methodological processes would benefit greatly from additional detail. Most people that routinely work with archival sequence data know that it is necessary to apply quality parameters to sort out bad sequence from good. 1) I think it is a good idea to provide genome sequence quality data in the manuscript, at minimum the average sequence quality (AvgQuality), N50, number of contigs (NrContigs), length of assembled genome, the congruent alleles by both assembly-based and assembly-free algorithms (NrConsensus), and the CorePercent. These should be available from the software generating the assemblies. It should be explicitly stated if assembly-free algorithms were not used.
2) When doing SNP analysis for creating phylogenetic trees it is best practice to use only high quality SNPs. Please include a brief explanation how high quality SNPs were identified and used.
Genome sequences should be archived in an appropriate sequence repository and accession numbers provided in the manuscript.
There is an instance in lines 160-162 where you state that the data you provide "implies" that a specific process is occurring. "It is noted that all bird-carried isolates contain four copies of tRNA-Gly, implying that V. parahaemolyticus gene mutation occurs after the bacterial strain gets into birds' bodies." As there is currently no data to support this, it would definitely be better to state this as an hypotheses. At a later point you do suggest ways to test the hypothesis, which is very good.
I would be interested in seeing the insertion sites of the different tRNA-Gly instances. Is the additional site "new" and not seen in isolates from sources other than birds Or are there already four (or more) insertion sites and only two to three are occupied in isolates from sources other than birds while four are occupied only in isolates from birds?
Authors have shown the genetic feature of V. parahaemolyticus isolated from birds with large number of isolate collection (n=124). The analysis of bird origin by PCR marker of bird species was comprehensive. Geographical distribution of bird isolates source was well discussed. Main findings included the presence of extra copy tRNA-Gly and qnrD gene in bird isolates. However, there are few questions/issues are concerned as following: 1.In material and method (line 309), "The 25g fecal samples were homogenized..." Was the 25g of sample obtained from pooled fecal samples of different birds or from one single bird? Please clarify.
2.In material and method (line 313-314), how many isolate was selected from each fecal sample? Assumably, it was one isolate from one sample (?). Then, what was the criteria of choosing an isolate among other purple Vp colonies in the CHROMagar plate?
3.In material and method (line 328), the accession numbers of genome assemblies of 124 isolates are absent from Table S1. Please double check. Without including other worldwide bird isolates, the statement "...implying that Vp gene mutation occurs after the bacterial strain gets into bird's body..." (line 160-163) is quite skeptical. In fact, the tRNA-Gly was also detected in non-bird isolates (15.09%) in this study although it was much smaller number that those in the bird isolates. In this way, the worldwide bird Vp are indeed necessary to be incorporated into the analysis to be strongly suggest that the extra copy of tRNA-Gly is potentially a gene marker for bird Vp. 5.The tdh+ and/or trh+ Vp were not detected from this study. However, previous studies have reported the tdh+ and /or trh+ Vp from bird although the number of isolates were smaller compared to the isolates in this study (Miyasaka, 2006, DOI: 10.1017/S0950268805005674; Muangnapoh, 2022, DOI:10.1128/spectrum.00886-22). The discussion for this part should be added for more understanding about the situation facilitating transmission of pathogenic Vp by birds. For example, the seasonal factor probably affect in the absence of pathogenic Vp in this study as the sample were collected during the cold month(?).
Normally, the pathogenic Vp favor warmer growth temperature compared to the non-pathogenic Vp.
6.The template of the references list is inconsistency (e.g. use of capital/small letter, journal abbreviation, italics where needed). It should be revised carefully.

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Introduction (lines 87-92): "To further explore the V. parahaemolyticus spreading mechanisms from marine, to the migratory birds, and finally to the food table, this study aims to characterize bird-carried V. parahaemolyticus strains for genetic diversity, potentially informative gene marker, and the presence of antimicrobial resistance and virulence factors. Specifically, we collected the fecal samples of migratory birds in ten cities (11 sampling sites) of four provinces in China." Discussion (lines 226-230): "To further explore how the V. parahaemolyticus spreads between humans, animals, and the environment, this study represents comprehensive research on the characterization of V. parahaemolyticus isolates from the fecal samples of migratory birds towards the genetic diversity, potentially bird-carried V. parahaemolyticus informative gene marker, and presence of antibiotic-resistant profiles." Two methodological processes would benefit greatly from additional detail. Most people that routinely work with archival sequence data know that it is necessary to apply quality parameters to sort out bad sequence from good. 1) I think it is a good idea to provide genome sequence quality data in the manuscript, at minimum the average sequence quality (AvgQuality), N50, number of contigs (NrContigs), length of assembled genome, the congruent alleles by both assembly-based and assembly-free algorithms (NrConsensus), and the CorePercent. These should be available from the software generating the assemblies. It should be explicitly stated if assembly-free algorithms were not used.
Response: We have added the data quality information in the method and results sections of our manuscript: Materials and methods (lines 376-386): "Both assembly-based and assembly-free algorithms were used to improve SNP calling accuracy. Specifically, we identified SNPs by integrating assembly-based method MUMmer version 3.23 (38) and assembly-free method Snippy (39). The SNPs identified by both methods were considered as high-quality SNPs and used for further phylogenetic analysis. As a result, reads with average Phred quality score less than 20 were removed and the average Phred quality scores of filtered reads were higher than 30. N50, number of contigs (NrContigs), length of the assembled genome: the average N50 is 460,970, the average number of contigs and the average size of assemblies were 90 and 5,123,946 bp, respectively. The average number of congruent alleles (NrConsensus) is 2,254. A total of 690,261 high-quality SNPs were identified, with the CorePercent of 97.4%. The detail information is provided in Table  S6. " 2) When doing SNP analysis for creating phylogenetic trees it is best practice to use only high quality SNPs. Please include a brief explanation how high quality SNPs were identified and used.
Response: We have further explained the SNP choice in the materials and methods section of our manuscript (lines 376-380): "Both assembly-based and assembly-free algorithms were used to improve SNP calling accuracy. Specifically, we identified SNPs by integrating assembly-based method MUMmer version 3.23 (37) and assembly-free method Snippy (38). The SNPs identified by both methods were considered as high-quality SNPs and used for further phylogenetic analysis." "The genome sequences were archived to the sequence repository GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and their accession numbers are listed in TABLE S1." There is an instance in lines 160-162 where you state that the data you provide "implies" that a specific process is occurring. "It is noted that all bird-carried isolates contain four copies of tRNA-Gly, implying that V. parahaemolyticus gene mutation occurs after the bacterial strain gets into birds' bodies." As there is currently no data to support this, it would definitely be better to state this as an hypothesis. At a later point you do suggest ways to test the hypothesis, which is very good. Response: We have revised this description to a hypothesis in our manuscript (lines 164-167): "It is noted that all bird-carried isolates contain four copies of tRNA-Gly. Therefore, we hypothesized that V. parahaemolyticus gene mutation occurs after the bacterial strain gets into birds' bodies." I would be interested in seeing the insertion sites of the different tRNA-Gly instances. Is the additional site "new" and not seen in isolates from sources other than birds Or are there already four (or more) insertion sites and only two to three are occupied in isolates from sources other than birds while four are occupied only in isolates from birds? Response: We have added the description of insertion sites in our manuscript (lines 167-172): "The insertion sites are the extra copies of tRNA-Gly, which are between the genes VPt086 (NC_004603.1: 2,989,075 bp -2,989,150 bp) and VPt089 (NC_004603.1: 2,989,431 bp -2,989,507 bp) in the reference genome RIMD 2210633 (FIG 3). This kind of "four copies of tRNA-Gly" pattern is not specific for bird isolates, which was also identified in non-birds isolates but with a much lower frequency (15%). Authors have shown the genetic feature of V. parahaemolyticus isolated from birds with large number of isolate collection (n=124). The analysis of bird origin by PCR marker of bird species was comprehensive. Geographical distribution of bird isolates source was well discussed. Main findings included the presence of extra copy tRNA-Gly and qnrD gene in bird isolates. However, there are few questions/issues are concerned as following: Response: Thank Reviewer #3's valuable suggestions. We have revised our manuscript according to the advice. Please see the following responses to each of the comments.
1.In material and method (line 309), "The 25g fecal samples were homogenized..." Was the 25g of sample obtained from pooled fecal samples of different birds or from one single bird? Please clarify.
Response: Revised (lines 329-331): "Briefly, 25g faces samples from each bird were homogenized with 225mL of phosphate-buffered saline (PBS) for 15-30s in a 4 mL centrifuge tube, followed by incubation at 37°C for 16 h." 2.In material and method (line 313-314), how many isolate was selected from each fecal sample? Assumably, it was one isolate from one sample (?). Then, what was the criteria of choosing an isolate among other purple Vp colonies in the CHROMagar plate?
Response: Revised: According to the pre-experimental results and operation instruction of CHROM agar Vibrio plate, above 90% of the round, semi-transparent, smooth surface and 2-3mm diameter isolates on the CHROM agar Vibrio plate were V. parahaemolyticus colonies, so we revised as follow: Lines 333-335: "One purple isolate (round, translucent, smooth surface, diameter 2-3 mm) was randomly selected as V. parahaemolyticus candidates and thus picked for PCR identification from each CHROM agar Vibrio plate." 3.In material and method (line 328), the accession numbers of genome assemblies of 124 isolates are absent from Without including other worldwide bird isolates, the statement "...implying that Vp gene mutation occurs after the bacterial strain gets into bird's body..." (line 160-163) is quite skeptical. In fact, the tRNA-Gly was also detected in non-bird isolates (15.09%) in this study although it was much smaller number that those in the bird isolates. In this way, the worldwide bird Vp are indeed necessary to be incorporated into the analysis to be strongly suggest that the extra copy of tRNA-Gly is potentially a gene marker for bird Vp.
Response: We have analyzed the worldwide bird V. parahaemolyticus strains and added the discussion in our manuscript (lines 262-276): "We further analyzed all the nine publicly available genomes from bird isolates worldwide (18,20), and six of them have the extra copy of tRNA-Gly, indicating a convergent pattern of 4ｘ tRNA-Gly copies in bird isolates in different regions. Combined with the fact that all the 124 bird isolates sequenced in this study also have 4ｘtRNA-Gly copies, the extra copy of tRNA-Gly may be useful for associating V. parahaemolyticus strains with birds as a potential source of contamination. As the temperate changes, V. parahaemolyticus varies its biological response by impacting biofilm formation (21) and gene expression (22). Therefore, the extra tRNA-Gly copy may be the biological response of V. parahaemolyticus, to survive in birds with a higher temperature than the usual environment. Future studies could interrogate whether the extra copy of tRNA-Gly increases fitness at higher temperatures through head-to-head direct competition experiments (10,23). It should be cautious that in vitro assay is a vast simplification of the environment of a bird gut; while in vivo assay would help better explore the mechanism of forming such a specifically informative gene marker." Public genomes of strains from birds.