Monocentric observational cohort study to investigate the transmission of third-generation cephalosporin-resistant Enterobacterales in a neonatal intensive care unit in Heidelberg, Germany

ABSTRACT Third-generation cephalosporin-resistant Enterobacterales is a major threat for newborns in neonatal intensive care units (NICUs). The route of acquisition in a non-outbreak setting should be investigated to implement adequate infection prevention measures. To identify risk factors for colonization with and to investigate the transmission pattern of third-generation cephalosporin-resistant Enterobacterales in a NICU setting. This monocentric observational cohort study in a tertiary NICU in Heidelberg, Germany, enrolled all hospitalized neonates screened for cephalosporin-resistant Enterobacterales. Data were collected from 1 January 2018 to 31 December 2021. Weekly screening by rectal swabs for colonization with third-generation cephalosporin-resistant Enterobacterales was performed for all newborns until discharge. Whole-genome sequencing was performed for molecular characterization and transmission analysis. In total, 1,287 newborns were enrolled. The median length of stay was 20 (range 1–250) days. Eighy-eight infants (6.8%) were colonized with third-generation cephalosporin-resistant Enterobacterales. Low birth weight [<1500 g (adjusted odds ratio, 5.1; 95% CI 2.2–11.5; P < 0.001)] and longer hospitalization [per 30 days (adjusted odds ratio, 1.7; 95% CI 1.5–2.0; P < 0.001)] were associated with colonization or infection with drug-resistant Enterobacterales in a multivariate analysis. Enterobacter cloacae complex was the most prevalent third-generation cephalosporin-resistant Enterobacterales detected, 64.8% (59 of 91). Whole-genome sequencing, performed for the available 85 of 91 isolates, indicated 12 transmission clusters involving 37 patients. This cohort study suggests that transmissions of third-generation cephalosporin-resistant Enterobacterales in newborns occur frequently in a non-outbreak NICU setting, highlighting the importance of surveillance and preventive measures in this vulnerable patient group. IMPORTANCE Preterm newborns are prone to infections. Therefore, infection prevention should be prioritized in this vulnerable patient group. However, outbreaks involving drug-resistant bacteria, such as third-generation resistant Enterobacterales, are often reported. Our study aims to investigate transmission and risk factors for acquiring third-generation cephalosporin-resistant Enterobacterales in a non-outbreak NICU setting. Our data indicated that premature birth and low birth weight are significant risk factors for colonization/infection with third-generation cephalosporin-resistant Enterobacterales. Furthermore, we could identify putative transmission clusters by whole-genome sequencing, highlighting the importance of preemptive measures to prevent infections in this patient collective.

A conclusion is not identified at the end of the discussion and only that of the summary, which should be evaluated "This cohort study suggests that transmissions" transmission is low, we must take advantage of the analysis of genomes (molecular epidemiology, Inc groups of plasmids, mechanisms of resistance to b-lactams and other antibiotics, etc.).
Data availability: it must contain the access numbers to GenBank, review the data presented and it does not work correctly.
Reviewer #2 (Public repository details (Required)): Acession numbers for genomic data were given but all of them were not accessible and were not consistent with the description in Methods.
Reviewer #2 (Comments for the Author): Nurjadi et al. investigated risk factors and nosocomial transmission tracing of third-generation resistant Enterobacterales in a NICU in Germany.I read the manuscript with interests.Although this type of data are strongly limited (or biased) by the study setting of the hospital investigated, data described here is very important to understand nosocomial transmission and appropriate treatment strategies in the absence of similar reports.However, the manuscript contains descriptive errors/mistakes in manuscript which compromise the entire quality of the manuscript (especially microbiological descriptions).The authors need careful checking of the entire manuscript.I recommend the manuscript to be checked by microbiologists.Some technical methods used in genomic analysis are unclear or not compliant with the current standard thus need clarification/explanations (especially phylogenomic analysis).Almost all third-generation cephalosporin resistant Enterobacterales isolates found in this study were species that have intrinsic AmpC resistance mechanisms without other acquired resistance mechanisms.Generally, these species, being part of gut microflora, are not considered as antimicrobial resistance organisms.Neonatal setting may slightly different from these understandings because nosocomial acquisition of these organisms can occur and can be threat to empirical treatment failure.The authors need to discuss to justify their study design.To support authors' design, I strongly recommend two points.First, include antimicrobial susceptibility data to describe these resistant organisms.Second, add outcomes of colonized/infected patients vs. non-infected patients to define impacts of colonization infection by these organisms.
Title.Change Enterobacteriaceae to Enterobacterales, as the authors used in the text.Line 30.Please clarify "cephalosporin" here is "third-generation cephalosporin".It is important to maintain consistency of the study objective, methods, results, and discussion.In Methods (line 30, 32), carbapenem-resistant Enterobacterales were investigated but the corresponding results were not shown.Running titile is "MDRO" whereas third-generation cephalosporin resistance were used in other places.I suggest to limit the abstract to "third-generation cephalosporin resistance".Descriptions for CRE can be placed in the text.Line 31.Did the authors use stool as a screening specimen?I think this is important and should be described in Abstract.Line 37-38.There were other significant risk factors in the text.Why these two?Furthermore, the ORs here were different from those described in the text.I think here results from multivariate analysis (low birthweight and longer LOS) should be presented.Line 77.Italicize "Staphylococcus aureus" Line 106. Figure 1 does not describe risk factors.Line 108.Results for CRE are missing.Antimicrobial susceptibility data should be described (the testing methods are described in Methods).Line 115.In Figure 1, these 3 isolates which did not undergo WGS are not described.Line 118.blaTEM-1 is not ESBL.Line 122."hqSNP" is not commonly used term.Needs definition and explanations.It is very unclear why the authors defined <= 15 SNPs as transmissions.Scientific evidence to support this is needed, especially in the setting that the authors did not use core SNP approach, which have been reported and validated in many studies.Lien 129.Do not italicize "complex".Figure 3. Capitalize "K"lebsiella.Line 131.Does this mean Enterobacter cloacae subsp.cloacae?Line 134.AmpC should be "ampC" if this meant gene name.Line 215.Missing dots between genus and species.Line 254.Did the authors used the checklist for STROBE?At least title does not contain term indicating study design.Line 271. Isolation and contact precautions were applied only when both cephalosporin-resistant and carbapenem-resistant organisms were detected?Line 275.Please give a specific product name of medium.Line 278.MALDI should be MALDI-TOF mass spectrometry.Line 279.Please clarify if interpretive criteria for cephalosporin or carbapenem resistance changed during the study period.Line 294.Please clarify criteria for species identification (cutoff value etc.) and supporting evidence, as this method is not the standard approach (ANI).Please disclose all reference genomes used.Line 294-296.This approach is different from the current standard of the core SNP approach.Please briefly explain the differences and merits of choosing this approach with supporting evidence.Clarify reference genomes for each species and these should be included in the phylogenetic trees.Line 308.The Bioproject number here is different from those described in Supplementary data.Furthermore, these numbers were not accessible so that I cannot assess the validity of genomic analysis.

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It is described that the identification by MALDI-TOF was used, and the results are not shown and if they agree with the species identified by WGS.Response: the comparison of MALDI-TOF MS performance with species identification was not the focus of the study.It is generally known that the discriminatory power of MALDI-TOF MS is not as good as WGS.In general, the species identification was concordant.As expected, MALDI-TOF is limited in distinguishing the species level in the Enterobacter cloacae complex.However, this is generally acknowledged, and we believe that this is not relevant to the study.If the results were discordant (Gram-positive wrongly identified as Gram-negative), this would be omitted from the analysis.
The antimicrobial susceptibility obtained with VITEK2 does not correlate with the resistome obtained.Response: the discrepancy between Vitek and genotypic resistance is also known and anticipated.Resistance mechanisms can be mediated by the presence or absence of a particular gene/resistance determinant but also be caused by overexpression of resistance determinants or efflux pumps.Therefore, it is plausible that phenotypic and genotypic resistance may not correlate.
The ideal way to identify bacterial species is the average nucleotide identity analysis; Therefore, it is suggested to use this program for the different species and correlate them with the MALDI-TOF result; Since if the MALDI-TOF database is not updated, it does not specifically identify the species of complexes.Response: species identification based on mash screening, which is equivalent to ANI as it evaluates through both genomes the number of shared hashes and the overall nucleotide identity, was performed.This was not mentioned explicitly, but instead, we referred to our previous studies to avoid redundancy and due to word limitation.The different species of Enterobacter cloacae complex are elaborated in lines 135-138.With MALDI-TOF MS alone, definitive species designation is not possible, as correctly mentioned by the reviewer.Introduction; detail more other studies from your hospital and what has changed.Response: we disagree with this suggestion since we do not see any added benefits in listing previous studies done in our hospital.The relevant changes are mentioned already.Screening measures were implemented, but molecular typing was performed with PFGE or MLST.In this study, we implemented systematic genomic surveillance to incorporate strain typing using WGS into the routine screening.All relevant details were already included in the introduction (line 74-82).

Results.
Line 95-96, colonization or infection; it is not clear.Response; this can be colonization or infection, or both.For the risk factors, detecting cephalosporin-resistant Enterobacterales refer to colonization and infection.We have replaced "or" with "and" for better clarity.
It would be necessary to describe and more to discuss the interesting data of figure 1.Why these species and how was it during the time?Response: Figure 1 provides an overview of the species detected over the study period.The species are discussed in the respective subheadings in the results section.The species were not selected by any criteria, but these were all third-generation cephalosporin-resistant Enterobacterales.Enterobacter hormaechei was the most common, which could be considered a potential outbreak.We have added this sentence to lines 134-135 in the revised manuscript.
The Supplementary Figure S1 should be more described in results and correlate with ST and something that is not discussed and determined are the incompatibility groups of the plasmids.See an example.Duran-Bedolla J, Rodríguez-Medina N, Dunn M, Mosqueda-García D, Barrios-Camacho H, Aguilar-Vera A, Aguilar-Vera E, Suárez-Rodríguez R, Ramírez-Trujillo JA, Garza-Ramos U. Plasmids of the incompatibility group FIBK occurs in Klebsiella variicola from diverse ecological niches.Int Microbiol.2023 Mar 27. doi: 10.1007/s10123-023-00346-0.Epub ahead of print.PMID: 36971854.Response: Supplementary Figure 1 was meant to show a global distribution of the AMR genes between bacterial species.The Sequence Type and the plasmid incompatibility types are presented in Figures 2 and 3 for the most prevalent species.As we did not observe evidence of plasmid transfer between strains in those groups, we do not see the relevance of repeating this information in the supplementary Figure 1.
Little transmission of bacteria is identified, what is not clear is the phylogenetic relationship (clones) between the identified bacteria that were transmitted.This makes sense to the discussion that the measures taken during the study helped so much.Compare the transmission of bacteria in an ICU with the % identified in this study.Response: we did not anticipate a lot of transmission since this study was performed in a non-outbreak setting.This is also the strength of the study, since most studies are performed only in outbreak settings, thus exaggerating the magnitude of transmission in "normal" settings.I am afraid that the reviewer misunderstood the aim of the study; this was a non-interventional study, so no effect on measures/interventions can be evaluated.This study was observational to see how many transmissions occur in a non-outbreak setting when systematic genomic surveillance is implemented.This study could deliver the necessary evidence to conduct an intervention study if transmission rates are too high and more rigorous infection prevention and control measures are needed.
To emphasize the epidemiology of the molecules of the identified bacterial species, they are practically not mentioned in the study.The ST must be determined for the isolates that were not performed.Response: the MLST were provided in the supplementary table 1; the missing MLST were due to mutations in some loci and are now provided with the closest known ST, new ST or none when there is no scheme in Pubmlst.The phylogenetic relationship between AMR genes and the strains is already described in Figures 2-3 and supplementary Figure 1.
Almost all third-generation cephalosporin resistant Enterobacterales isolates found in this study were species that have intrinsic AmpC resistance mechanisms without other acquired resistance mechanisms.Generally, these species, being part of gut microflora, are not considered as antimicrobial resistance organisms.Neonatal setting may slightly different from these understandings because nosocomial acquisition of these organisms can occur and can be threat to empirical treatment failure.Response: from the microbiological perspective, this is true.However, according to the national guideline (commission for hospital hygiene and infection prevention, KRINKO, of the Robert Koch Institute in Germany), isolates with third-generation cephalosporin phenotypic resistance (the so-called 2-MRGN, multidrug-resistant gram-negatives with resistance towards broad-spectrum penicillin and third-generation cephalosporins) should be considered are relevant bacteria in these neonates (especially those with low-birth weight).The recommendations do not differentiate between mobile/transferable or intrinsic resistance mechanisms but are based solely on phenotypic resistance.
The authors need to discuss to justify their study design.To support authors' design, I strongly recommend two points.First, include antimicrobial susceptibility data to describe these resistant organisms.

Response:
We think including all phenotypic susceptibility data is unnecessary.As you can see from the resistome, most of the bacterial species found in this patient group were multi-susceptible.Therefore, for clarity purposes, we only displayed the figures' genotypic resistance.The sequenced isolates were selected based on their resistance towards third-generation cephalosporin.
Second, add outcomes of colonized/infected patients vs. non-infected patients to define impacts of colonization infection by these organisms.Response: we think it is unnecessary; colonized children are not necessarily sick, and we do not believe that assessing the clinical outcome based on colonization is clinically meaningful.In our cohort, only 9 children had an infection, all of which were colonized, so no meaningful statistical analysis could be performed comparing the colonized/infected and non-colonized/infected groups.
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Point-by-point response R1
Thank you for giving us the opportunity to revise and improve our manuscript.We have tried to the best of our ability to address the reviewers' comments and hope to have done so to your satisfaction.

Reviewer comments:
Reviewer #1 (Comments for the Author): The study describes the characterization of colonizing isolates resistant to third-generation cephalosporins in a neonatal ICU.
Around the document they erroneously describe "third-generation cephalosporin Enterobacterales" Response: this comment is unclear since the reviewer did not elaborate on why this term should be incorrect.In the study, we focused on third-generation cephalosporin and species belonging to the order Enterobacterales so that we are now aware of what " erroneous " meant in this context.If the reviewer refers to the term "Enterobacteriaceae" versus "Enterobacterales", this is now harmonized throughout the manuscript.
The title should be modified by eliminating the study period; including the country.That must be very well specified in M and M.
Response: we have now changed the title to "Monocentric observational cohort study to investigate transmission of third-generation cephalosporin-resistant Enterobacterales in a neonatal intensive care unit, Heidelberg, Germany.",following the recommendation of both reviewers.
The bacteria are isolated from the rectum of children; explaining colonization; however, whether there were infections in children is not described.It is not disputed how much it affected colonization and thus the results of the prevention measures taken during the study; in the same way the possible infections.
Response: we have now included the data on infections.There were 9 children with various infections, bloodstream infections, meningitis and wound infections.All of the 9 children with 3rd gen cephalosporin-resistant Enterobacterales were also colonized.This was not an intervention study or to evaluate the infection prevention measure so we would not speculate on the efficacy of specific measures with the small number of infected children in our setting.Furthermore, the infection prevention measures implemented are not targeted to prevent endogenous infection.Since the reviewer's question was unclear, we hope to have understood the question correctly and answered it adequately.
It is described that the identification by MALDI-TOF was used, and the results are not shown and if they agree with the species identified by WGS.
Response: the comparison of MALDI-TOF MS performance with species identification was not the focus of the study.It is generally known that the discriminatory power of MALDI-TOF MS is not as good as WGS.In general, the species identification was concordant.As expected, MALDI-TOF is limited in distinguishing the species level in the Enterobacter cloacae complex.However, this is generally acknowledged, and we believe that this is not relevant to the study.If the results were discordant (Gram-positive wrongly identified as Gram-negative), this would be omitted from the analysis.
The antimicrobial susceptibility obtained with VITEK2 does not correlate with the resistome obtained.
Response: the discrepancy between Vitek and genotypic resistance is also known and anticipated.Resistance mechanisms can be mediated by the presence or absence of a particular gene/resistance determinant but also be caused by overexpression of resistance determinants or efflux pumps.Therefore, it is plausible that phenotypic and genotypic resistance may not correlate.
The ideal way to identify bacterial species is the average nucleotide identity analysis; Therefore, it is suggested to use this program for the different species and correlate them with the MALDI-TOF result; Since if the MALDI-TOF database is not updated, it does not specifically identify the species of complexes.
Response: species identification based on mash screening, which is equivalent to ANI as it evaluates through both genomes the number of shared hashes and the overall nucleotide identity, was performed.This was not mentioned explicitly, but instead, we referred to our previous studies to avoid redundancy and due to word limitation.The different species of Enterobacter cloacae complex are elaborated in lines 135-138.With MALDI-TOF MS alone, definitive species designation is not possible, as correctly mentioned by the reviewer.
Introduction; detail more other studies from your hospital and what has changed.
Response: we disagree with this suggestion since we do not see any added benefits in listing previous studies done in our hospital.The relevant changes are mentioned already.Screening measures were implemented, but molecular typing was performed with PFGE or MLST.In this study, we implemented systematic genomic surveillance to incorporate strain typing using WGS into the routine screening.All relevant details were already included in the introduction (line 74-82). Results.
Line 95-96, colonization or infection; it is not clear.
Response; this can be colonization or infection, or both.For the risk factors, detecting cephalosporin-resistant Enterobacterales refer to colonization and infection.We have replaced "or" with "and" for better clarity.
It would be necessary to describe and more to discuss the interesting data of figure 1.Why these species and how was it during the time?Response: Figure 1 provides an overview of the species detected over the study period.The species are discussed in the respective subheadings in the results section.The species were not selected by any criteria, but these were all third-generation cephalosporin-resistant Enterobacterales.Enterobacter hormaechei was the most common, which could be considered a potential outbreak.We have added this sentence to lines 134-135 in the revised manuscript.
The Supplementary Figure S1 should be more described in results and correlate with ST and something that is not discussed and determined are the incompatibility groups of the plasmids.1 was meant to show a global distribution of the AMR genes between bacterial species.The Sequence Type and the plasmid incompatibility types are presented in Figures 2 and 3 for the most prevalent species.As we did not observe evidence of plasmid transfer between strains in those groups, we do not see the relevance of repeating this information in the supplementary Figure 1.
Little transmission of bacteria is identified, what is not clear is the phylogenetic relationship (clones) between the identified bacteria that were transmitted.This makes sense to the discussion that the measures taken during the study helped so much.Compare the transmission of bacteria in an ICU with the % identified in this study.Response: we did not anticipate a lot of transmission since this study was performed in a non-outbreak setting.This is also the strength of the study, since most studies are performed only in outbreak settings, thus exaggerating the magnitude of transmission in "normal" settings.I am afraid that the reviewer misunderstood the aim of the study; this was a non-interventional study, so no effect on measures/interventions can be evaluated.This study was observational to see how many transmissions occur in a non-outbreak setting when systematic genomic surveillance is implemented.This study could deliver the necessary evidence to conduct an intervention study if transmission rates are too high and more rigorous infection prevention and control measures are needed.
To emphasize the epidemiology of the molecules of the identified bacterial species, they are practically not mentioned in the study.The ST must be determined for the isolates that were not performed.Response: the MLST were provided in the supplementary table 1; the missing MLST were due to mutations in some loci and are now provided with the closest known ST, new ST or none when there is no scheme in Pubmlst.The phylogenetic relationship between AMR genes and the strains is already described in Figures 2-3 and supplementary Figure 1.
How are the species related, for example, Enterobacter cloacae complex; plasmids are being transferred Response: the phylogenetic relationship between species is display on Suppl.Figure 1.We did not find any indication of plasmid transfer between species in our setting.
A conclusion is not identified at the end of the discussion and only that of the summary, which should be evaluated "This cohort study suggests that transmissions" transmission is low, we must take advantage of the analysis of genomes (molecular epidemiology, Inc groups of plasmids, mechanisms of resistance to b-lactams and other antibiotics, etc.).Response: We disagree.The last paragraph is the conclusion.We do not explicitly state this paragraph as the conclusion since it is clear that this paragraph summarizes the findings, concluding that systematic molecular characterization can help detect transmission to guide infection prevention measures in this patient group.Data availability: it must contain the access numbers to GenBank, review the data presented and it does not work correctly.

Response:
The problem with the Bioproject was due to a Microsoft Excel mishap.All samples are in the Bioproject PRJNA954276, and somehow when copy pasting in Excel, it creates a sequence starting from PRJNA954276 to PRJNA954364.We apologize for this mistake, and it is now fixed.All biosample accession were correct.It is not currently possible to provide a reviewer link via NCBI, but if the reviewer wants to see the data, we could ask for an earlier release.
Acession numbers for genomic data were given but all of them were not accessible and were not consistent with the description in Methods.
• 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

of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. "
For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions • 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