The whole-genome molecular epidemiology of sequential isolates of Acinetobacter baumannii colonizing the rectum of patients in an adult intensive care unit of a tertiary hospital

ABSTRACT Acinetobacter baumannii is a nosocomial multidrug-resistant pathogen that specifically colonizes and infects patients in intensive care units (ICUs). Genome plasticity of A. baumannii may contribute to the rapid development of A. baumannii resistance during patient treatment. There is little clarity on the dynamics of colonization of A. baumannii in humans. We studied 269 serial isolates of A. baumannii colonizing the rectum of 32 adult patients in an ICU; 5 to 16 isolates were obtained from each patient over a period of weeks to months. The isolates were sequenced using the Illumina platform. Multi-locus sequence typing revealed genetic diversity with 13 sequence types (STs); the dominant ST2 accounted for 202 of the 269 isolates and was isolated from each patient. A core genome comparison of isolates was used to elucidate a detailed understanding of the relationship among isolates within an ST and the genomic relationship among STs. One to three STs were observed in the isolates from each patient. Instances of unchanged colonization, sequential lineage colonization, and colonization with multiple lineages of A. baumannii were seen. The number of resistance genes carried by the isolates varied from 2 to 17. The predominant genes corresponded to those encoding resistance to β-lactam and aminoglycoside antibiotics. Generally, there was a strong correlation between ST and a particular antimicrobial resistance gene profile. This study makes an important baseline contribution to information that will help us understand the role of A. baumannii colonization in the development of opportunistic infections in ICU patients. IMPORTANCE Acinetobacter baumannii is a multidrug-resistant nosocomial pathogen that colonizes and infects debilitated patients in the ICU. There is very little information on the genomic characteristics of colonizing strains. This information is important to understand the evolution of lineages of A. baumannii that develop resistance while patients receive antibiotic treatment in the ICU. Our study demonstrated different patterns of colonization of the rectum of ICU patients with different STs of A. baumannii while one ST colonized all patients. Some STs carried more antibiotic resistance genes compared to others. However, there was a correlation between ST and a particular resistance gene profile. Our results further elucidate the dynamics of enteric colonization of this opportunistic pathogen.

I have a few queries and suggestions on the presentation of the results 1.It appears (but is not completely clear) that the same cohort of patients and isolates have been used in previous studies : Al-Hashem G, Rotimi VO, Albert MJ.Genetic relatedness of serial rectal isolates of Acinetobacter baumannii in an adult intensive care unit of a tertiary hospital in Kuwait.PLoS354 One.2020;15: e0230976.Al-Hashem G, Rotimi VO, Albert MJ.Antimicrobial resistance of serial isolates of Acinetobacter baumannii colonizing the rectum of adult intensive care unit patients in a teaching hospital in Kuwait. Microb Drug Resist. 2021;27:64-72 It would be good to understand how the present study builds up on the previous observations 2. Did the antimicrobial resistance genes in a particular background strain type increase with time?For patients in whom the no recolonization or mixed infection was suspected, it would be good to see whether sequential isolates had increased acquisition of AMR genes? 3. The methodology for phylogenetic analysis has not be described adequately.The methods for this needs to be expanded.
4. If the clinical cohort is the same as the previously described cohort (see point 1) then having the genotype to phenotype correlation for the antibiotics is essential and should be presented.
5.The sequences pf/reference for the gyB gene primer sets would be good to include 6.It would be good to have a figure showing the sampling timepoint -for example instead of denoting samples are the 5th sample it would be good to know that the sample was collected 20 days from the initial sampling time point.7. Lines 271-275 of Discussion: Given that single colony sampling was predominantly used, this may have led to an underestimate of colonization with multiple STs.Further investigation of simultaneous colonization would require the characterization of multiple isolates from each swab sample In their previous work, the authors have suggested that the morphological features of the colony could suggest genotype differences -can this be used as a measure in the current study to understand the genetic diversity/heterogeneity that the authors suggest here?8.The study was conducted in a single hospital, was there a spatial-epidemiological clustering of the strains in any way?9. Is it possible that the presence of ST2 in majority of the isolates has more to do with the single site -I.e the presence and persistence of this strain in the hospital rather than the more general claim that this is best colonizer?Did all mixed infections become predominantly ST2 at the end of the study?10.The phylogenetic trees could be presented better -probably by collapsing some branches, adding bootstrap support values (if trees are maximum likelihood trees), adding/overlaying metadata of country, time and resistance profiles on the tree itself.
11. Were all individuals sampled the same number of times?How much of the Strain distribution is impacted by differences in sampling?
12. The authors report that -"Within STs, there were often groups of isolates with the same antimicrobial resistance gene profile."-it would be good to see this summarized in a figure with cumulative frequencies for example. 1 shows the distribution of STs detected in each patient."-It would be good to also see this as a frequency distribution and also changes for a person over time 15.Did the authors perform any analysis on mutations acquired by a strain in a person over time?

"Table
16.The methods have not been described in enough detail for the analyses presented to be replicated.) Reviewer #2 (Comments for the Author): Bulach and colleagues performed a longitudinal genomic epidemiology investigation in the Intensive Care Unit (ICU) of the Mubarak Al Kabir Hospital (Kuwait).They inlcuded in their study the patients who showed a persistent colonization of Acinetobacter baumannii, performing longitudinal rectal swabs and WGS-based typing of the isolates.The most frequency Sequence Type (ST) they found was ST2 (not surprisingly), but other STs were also isolated.They also found patients colonized by multiple lineage of the bacterium.Correctly they stated at the end of the manuscript that this aspect is very interesting and that further investigations are necessary (e.g. using third generation sequencing approach).
The experimental design in sound and the manuscript well written.The results are interesting for the scientific community.
I can only suggest some minor changes in the text.In particular I suggest to capitalize the text indicated by the acronyms e.g.LINE 40: intensive care units have to be capitalized (the same in all occurrences in the manuscript) LINE 45: Multilocus sequence typing should be "Multi-locus Sequence Typing" LINE 46: sequence types should be capitalized I also suggest replacing "development" with "selection" at LINE 87.

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27/8/2023
Professor Varadharajan Sundaramurthy Editor, Microbiology Spectrum Re: The manuscript en tled "Whole genome molecular epidemiology of sequen al isolates of Acinetobacter baumannii colonizing the rectum of pa ents in an adult intensive care unit of a ter ary hospital" (Spectrum02191-23) Dear Dr. Sundaramurthy, Thank you for forwarding the reviewers' comments.Our responses to the comments are addressed below.Please refer to the "Marked-Up" manuscript to view the changes.

Reviewer 1
Thank you for the opportunity to review -"Whole genome molecular epidemiology of sequen al isolates of Acinetobacter baumannii colonizing the rectum of pa ents in an adult intensive care unit of a ter ary hospital".In this manuscript, the authors present genomic analysis of 269 sequen al isolates of A. baumanni from 32 ICU pa ents at a ter ary care hospital.A majority of the isolates (202/269) belonged to a single strain type (ST2).All isolates carried an bio c resistance genes (2-17) -par cularly those conferring resistance to beta-lactam and aminoglycoside an bio cs.

Response
We thank the referee for this kind assessment.
I have a few queries and sugges ons on the presenta on of the results 1.It appears (but is not completely clear) that the same cohort of pa ents and isolates have been used in previous studies: Al-Hashem G, Ro mi VO, Albert MJ.Gene c relatedness of serial rectal isolates of Acinetobacter baumannii in an adult intensive care unit of a ter ary hospital in Kuwait.PLoS354 One.2020;15: e0230976.Al-Hashem G, Ro mi VO, Albert MJ.An microbial resistance of serial isolates of Acinetobacter baumannii colonizing the rectum of adult intensive care unit pa ents in a teaching hospital in Kuwait. Microb Drug Resist. 2021;27:64-72 It would be good to understand how the present study builds up on the previous observa ons

True (L 101-102).
To understand how the present study builds up on the previous observa ons, please see L 315-329.
2. Did the an microbial resistance genes in a par cular background strain type increase with me?For pa ents in whom no recoloniza on or mixed infec on was suspected, it would be good to see whether sequen al isolates had increased acquisi on of AMR genes?
3. The methodology for phylogene c analysis has not been described adequately.The methods for this need to be expanded.
4. If the clinical cohort is the same as the previously described cohort (see point 1) then having the genotype to phenotype correla on for the an bio cs is essen al and should be presented.

Response This correla on is shown (L196-205 & L303-309).
5.The sequences pf/reference for the gyB gene primer sets would be good to include

Response Included (L116).
6.It would be good to have a figure showing the sampling mepoint -for example instead of deno ng samples are the 5th sample it would be good to know that the sample was collected 20 days from the ini al sampling me point.S1) with the reference.

Response This informa on was included in a previous publica on in a table (Al-Hashem et al 2020, PLOS ONE). To avoid duplica on, we have included a modified table (Table
7. Lines 271-275 of Discussion: Given that single colony sampling was predominantly used, this may have led to an underes mate of coloniza on with mul ple STs.Further inves ga on of simultaneous coloniza on would require the characteriza on of mul ple isolates from each swab sample In their previous work, the authors have suggested that the morphological features of the colony could suggest genotype differences -can this be used as a measure in the current study to understand the gene c diversity/heterogeneity that the authors suggest here?
8. The study was conducted in a single hospital, was there a spa al-epidemiological clustering of the strains in any way?

Response
This was not inves gated.9. Is it possible that the presence of ST2 in majority of the isolates has more to do with the single site -I.e the presence and persistence of this strain in the hospital rather than the more general claim that this is best colonizer?Did all mixed infec ons become predominantly ST2 at the end of the study?
Mixed infec ons did not become predominantly ST2 at the end of the study.Please see Table S1.
10.The phylogene c trees could be presented be er -probably by collapsing some branches, adding bootstrap support values (if trees are maximum likelihood trees), adding/overlaying metadata of country, me, and resistance profiles on the tree itself.11.Were all individuals sampled the same number of mes?How much of the Strain distribu on is impacted by differences in sampling?
12. The authors report that -"Within STs, there were o en groups of isolates with the same an microbial resistance gene profile."-it would be good to see this summarized in a figure with cumula ve frequencies for example.

Response
Please see L213-216. 1 shows the distribu on of STs detected in each pa ent." -It would be good to also see this as a frequency distribu on and also changes for a person over me 15.Did the authors perform any analysis on muta ons acquired by a strain in a person over me?
16.The methods have not been described in enough detail for the analyses presented to be replicated.

Response
This has been already dealt with.
Reviewer #2 (Comments for the Author): Bulach and colleagues performed a longitudinal genomic epidemiology inves ga on in the Intensive Care Unit (ICU) of the Mubarak Al Kabir Hospital (Kuwait).They included in their study the pa ents who showed a persistent coloniza on of Acinetobacter baumannii, performing longitudinal rectal swabs and WGS-based typing of the isolates.The most frequency Sequence Type (ST) they found was ST2 (not surprisingly), but other STs were also isolated.They also found pa ents colonized by mul ple lineage of the bacterium.Correctly they stated at the end of the manuscript that this aspect is very interes ng and that further inves ga ons are necessary (e.g. using third genera on sequencing approach).
The experimental design in sound and the manuscript well wri en.The results are interes ng for the scien fic community.
14. Discussion lines 202-203: The genome characteristics of our A. baumannii isolates were like those published previously (Park et al 2011; Liu et al 2014; Michiels et al 2016; Wibberg et al 2018).Can the authors clarify what these features are?
• 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 Addi onal relevant informa on has been added to the taxon label for bothFig 1 and Fig S1.
lines 202-203: The genome characteris cs of our A. baumannii isolates were like those published previously (Park et al 2011; Liu et al 2014; Michiels et al 2016; Wibberg et al 2018).Can the authors clarify what these features are?Response Please see L240-242.