Mobile class A β-lactamase gene bla GMA-1

ABSTRACT The first reported bla GMA-1 gene encoding gammaproteobacterial mobile class A β-lactamase (GMA-1) was identified in a recently defined mobile DNA element, a strand-biased circularizing integrative element (SE). Increased genomic data revealed the presence of bla GMA-1 in marine bacteria, including pathogenic species such as Vibrio parahaemolyticus and Photobacterium damselae. Herein, we address the substrate range of GMA-1 and how frequently bla GMA-1 was acquired by the chromosomes or plasmids via SEs using sequences in a publicly available database. An Escherichia coli strain carrying bla GMA-1 exhibited resistance to amoxicillin, piperacillin, and carbenicillin, but it remained susceptible to cephalosporins, monobactam, and carbapenems, indicating that GMA-1 belongs to functional group 2c, narrow-spectrum β-lactamases. bla GMA-1-flanking sequence analysis for sequences in the RefSeq/GenBank database revealed a total of eight distinct SE-mediated bla GMA-1 acquisition events and six SE-independent bla GMA-1 acquisition events, including bla GMA-1 alone translocation, without involving a specific insertion sequence, or integron. Thus, this study shows that GMA-1 is specialized for penicillin degradation and is mainly disseminated by SEs; however, SE is not the only genetic mechanism transmitting bla GMA-1. IMPORTANCE Despite increasing reports, class A β-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family β-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A β-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in β-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.


1) Introduction
As SE elements have been identified only very recently, clearly describing their characteristics would be helpful to readers e.g., move some information from Fig 2 legend to the main text and/or refer to this figure on the Introduction?Also see comments of Fig. 2 below.Lines 64, 237 -att sites seem to be mentioned only on Line 237 and in Figure 2 legend -it is not clear how these relate to the inverted repeats.Line 36 -"transporters" "porins" "of beta-lactamase genes" Line 38 -suggest "beta-lactamases of clinical concern include.." ESBLs are not carbapenemases, so this needs clarifying.Line 45 -"in class 1 integrons" Line 49 "and carbapenems" Line 50 -suggest "several Vibrio spp."Line 52 -should this be aquaculture?Line 53 -suggest "reported three conjugative... plasmids from".second "three" is not needed.Line 57-8 -"Among class A beta-lactamases" not really needed.Suggest "both over the whole sequence and at motif level".Line 61 -"E. coli transconjugants containing..." Line 62 -"plasmid" is not needed before pAQU1 -the "p" indicates that it is a plasmid (also lines 92, 95,100 109, 112, 142, 174, 243 etc) -and the species could be indicated in parentheses.Line 64 -"their termini".Lines 65-7 -this could be explained more clearly.Line 70 -"resistome development"?
2) Characterization of GMA-1 -Lines 76-90, I have only a few suggestions for rewording here.Lines 82-4 -strictly speaking, it is the antibiotic that has the MIC against the isolate, so this should be reworded.Line 78 -"of 2c beta-lactamases" -no "the" needed -also start of line 85.Line 79 -"from P. damselae"?Line 80 -"low-copy vector"?Line 87 -"cephalosporins, but not 2nd, 3rd or 4th generation" Fig. 1 legend -the tree is for the whole proteins or the motifs?It's not clear what is meant by "shown by their terminal nodes"."Functional groups"?
3) Identification of SE I would suggest combining information on Lines 116-129 and 133-40 and placing after Lines 91-106, first describing the SE identified here, before going on to talk about the patterns.Lines 95-8 -I would suggest making it clear here that Refs 13 and 15 are your previous work and this part was essentially to finalize analysis of 04Ya311.Lines 102-6 -if there are papers associated with the accession numbers they should be cited, at least in Table S1.Lines 121-7 -the nucleotide sequences of various SE appear highly related, so I would suggest referring to these rather than "gene product sequences" here -if the nucleotide sequences are highly related it follows that the protein products will be too.Line 123 -what are these cargo genes and where are they in relation to blaGMA-1?This might be most easily shown on a figure (see comments on Figure 2 below).pSEA-1 Table S1 lists NZ_LC081338.1 as a duplicate of NZ_AP024167.1, with both plasmids named as pSEA1, but the sequences are slightly different lengths.Are both correct?If not, maybe the incorrect one could be removed from GenBank to reduce confusion.Line 125 -are the SE names correct here?Table S1 and elsewhere in the text have VpaV493, not VdaVS492, and SE-VcVS12C, not SE-VcVS12B.Fig. 2 -I would suggest changing this to better show the relationships between all SE analysed here and incorporate the information on Lines 133-40.Diagrams are not really needed for the sequences without SE -the accessions could just be stated.Diagrams for the previously-characterized SE also analysed here could be included, with all diagrams aligned and SE names added.Variants of the intA, tfp intB and srap genes could each be shown e.g. in different shades of the same colour, as the different WP_ accessions are not easy to distinguish at a glance.The att site alignments for the new SE could be shown separately.Fig. 3A -I find this format very difficult to interpret and I don't think it is useful to include the non-SE blaGMA-1 contexts herethis figure is not really needed if the suggested changes to Fig. 2 are made and Fig. 3B could be shown separately or maybe with Fig. 2 or Fig. 4? Legend, Line 389 -"similarity" and "identity" have specific meanings in relation to proteins Line 91 -suggest "SE are the main type of mobile element..." Lines 93, 172 -not clear what is meant by "primal" here?Line 98 -suggest "to investigate this" Line 100 -this is unclear -the only copy is on pAQU1?Line 103 -suggest ">5 kb on either side of blaGMA-1" if this is what is meant?Lines 103-4 -"13 entries including pAQU1" might work better.Lines 104-6 -if all species are listed then "included" should be removed.4) blaGMA-1 contexts I would suggest combining information on Lines 107-116, 130-33 and 141-165 to describe the different "patterns".This information is currently presented in a way that I think is overly complicated and confusing.From Table S1, "patterns" 1-6 just seem to correspond to different SE and this could be made much clearer in the text once all of the SE have been described Lines 109-111 -these are all identical SE-6945, so it is not clear why they are considered two "patterns"?Lines 109-112 -are the whole plasmids and chromosome closely related or just the blaGMA-1 contexts?Lines 112-6 -similarly these are all SE-Pda04Ya311 and this could just be stated.Lines 118-29 -if this information is moved, as suggested above, patterns 4-6 could just be equated with the relevant SE.Also consider if the "patterns" are really different between highly related SE with some different segments?Lines 130-33 -see comments on Fig. 3 above.Lines 141-55 -I think that this analysis has some problems.It is not clear why integron components might be expected and the fact that IS and Tn were searched for should also be mentioned before describing the elements that were found.I am not sure that the presence of any of these transposases is actually relevant -the whole IS elements need to be identified and checked for TSD etc, to see if they are just simple insertions.A quick look suggests that some are not close to blaGMA-1 and maybe associated with different resistance genes.Are the IS actually ISShrf9, ISVsa5 and ISKpn13 or relatives (cut off is 95% identical nt sequences in ISFinder).More information about the types of plasmids and any relationships between then should also be included.Lines 159-63 -it might be good to make it clearer here that one IR is identical in all sequences but the other has differences.Fig. 4 -it is not immediately clear what these six sequences represent, without referring back to other figures, or why only these sequences are included -using only the accessions to identify them is not informative.Could the extra T in the motif in CP007004 be an error?If the reads are available, it would be possible to check this by read mapping.The legend should explain what * indicate.I would suggest using dots to mark every 10th base rather than numbering all positions.How were the promoter motifs identified?I couldn't find thsi in methods.The -10 motif seems extended?Line 132 -suggest "six of these nine locations are associated with SE" Line 133 -is there sufficient evidence to generalise beyond the current dataset?Line 146 -not clear what "respectively" refers to here?Line 165 -how would this supress cost? 5) Methods Line 178 -this is one strain?If so "...Japan) was cultured" Lines 193-7 -primer sequences are usually given in uppercase.Lines 200-2 -how was this determined, i.e. was the recombinant plasmid sequenced?If so, this should be stated.Line 208 -"is not included" "by standard microdilution" Line 210 -"on three" Line 222 -delete "of".Line 225, 232 -the listed names are of genes, not proteins.Line 235 -"the termini of SE were"? 6) Suggested minor wording changes to improve clarity etc in other parts of the manuscript Lines 12-3 -genomic data presumably revealed the gene, not the protein?Lines 14-6 might be better before Lines 12-3.Lines 17,30 -suggest "in sequences in..." Line 20 -"...2c, narrow spectrum beta-lactamases."Also see comments above on number of "patterns" Line 27 -suggest delete "on class A beta-lactamases" Line 167 -"demonstrates", again see comments above on number of patterns, Lines 169-70 -suggest "the chromosome" Line 171 -"hitchhiking on" References -Line 279 -the whole aac gene names should be in italics."beta" in several reference titles probably should be changed to the beta symbol.Line 364 -"except for carbenicillin" 7) Table S1 Column B -heading should be "strain/plasmid"?Column C -what does "not open to public" mean -not listed in GenBank?Can this information be obtained from an associated paper or is it suggested by the submitter's location?Column G "franks" should be "flanks".
Reviewer #2 (Comments for the Author): The manuscript "Mobile class A β-lactamase gene blaGMA-1" by Yano et al. describes the gammaproteobacterial mobile class A B-lactamase (GMA) gene and how it relates to resistance.The authors also characterize this target through a few approaches.The manuscript has good grammatical structure and details an interesting area of antimicrobial resistance research.
Opportunities: This manuscript is framed around the genomic/gene components of antimicrobial resistance, however the research detailed in the methods section does not reflect this.I found myself looking for specific information on multiple genomes/isolates and could only identify 1 that was sequenced.
In addition, many of the questions answered in the manuscript are reported as defining features that are already known in the introduction.While this confirms the results, there should be a greater scope to support the research.
There are some interesting areas to explore and the overall manuscript could be strengthened with a broader scope.

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Re: Spectrum02589-23 (Mobile class A β-lactamase gene bla GMA-1 ) Dear Dr. Hirokazu Yano: Thank you for submitting your manuscript to Microbiology Spectrum.Your study was reviewed by two experts, and I would now like you to revise your manuscript in line with their feedback.
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The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.This manuscript has a few different aspects, describing experiments to characterize a recently identified beta-lactamase named GMA-1 first found on a strand-biased circularizing integrative element (SE) and analysis of available contexts of this gene, identifying additional SE that carry it and also contexts outside SE.The data are of interest, the experiments to characterize GMA-1 appear sound and the conclusions are reasonable.However, I found both the text and figures relating to the results of sequence analyses difficult to follow.From a quick look at some of the sequences, I think these parts are unnecessarily complicated and could be simplified -I have given some suggestions on this in my comments below, including for some reorganization -describing characterization of GMA at the start works, but then describing the new SE, then the "patterns" might work better.Only sequences up to September 2022, which is now almost a year ago, are included in the analysis and checking for updates might add to the study.NCBI also now lists a blaGMA-2 variant, which could also be looked at or at least commented on.Some minor rewording would also improve clarity, with suggestions given below.(Response) We thank reviewer 1 for their interest in this work and suggestions.We increased the dataset and split the comparative genomics parts into two sections: (i) SE-mediated blaGMA-1 acquisition and (ii) SE-independent blaGMA-1 acquisition.The second part was also explained further.A new putative blaGMA-1 translocation event was discovered, which was accompanied by the 5-bp TSD generation.The message that "the SE are the main type of mobile element transmitting bla GMA-1 " remains the same.
We searched for a bla GMA-2 variant in the RefSeq/GenBank database; however, there was only one DNA molecule carrying bla GMA-2 , and bla GMA-2 was not associated with SE.In this study, we stated that "RefSeq/GenBank files encoding GMA-2, another GMA variant that differed from GMA-1 by six aminoacids, were also searched for in the NCBI database.The only GMA-2 hit was the chromosomal contig (RefSeq ID: NZ_VXDD01000003.1) of Vibrio fortis S7-72, which was not associated with SE.This was not further analyzed due to the lack of information for bla GMA-2 movement analysis."(line 289 in the revised manuscript) 1) Introduction As SE elements have been identified only very recently, clearly describing their characteristics would be helpful to readers e.g., move some information from Fig 2 legend to the main text and/or refer to this figure on the Introduction?Also see comments of Fig. 2 below.(Response) We thank reviewer 1 for their suggestions.In the revised manuscript, we explained the genetic organization and 6-bp flanking sequences of SE, and mentioned terms, such as attL, using Fig. 2 in the Introduction section.The previous Fig. 2 was updated as the new Fig. 3. Lines 64, 237 -att sites seem to be mentioned only on Line 237 and in Figure 2 legend -it is not clear how these relate to the inverted repeats.(Response) The following sentence was added to the Introduction section: "The border regions between SE and the host genome, including inverted repeats, are called attL and attR.The C and C' joint on the circular SE copy is termed attS, whereas the target location in the host genome is termed attB.The exact attL/attR/attS/attB length required for recombination is still unknown."(line 72-75) Line 36 -"transporters" "porins" "of beta-lactamase genes" (Response) We modified the words as suggested.(line 41) Line 38 -suggest "beta-lactamases of clinical concern include.." ESBLs are not carbapenemases, so this needs clarifying.
(Response) We thank reviewer 1 for bringing this issue to our notice.We have rephrased the sentence as follows: "Several clinically significant β-lactamases fall into class A group 2be (commonly known as extended-spectrum β-lactamases: ESBLs), class A group 2f (carbapenemases), and class B group 3a or 3b metallo-β-lactamases (MBLs) capable of degrading most of the currently available synthetic β-lactams." • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred : spectrum@asmusa.orgReviewercomments:Reviewer #1 (Comments for the Author):