Global emergence of double and multi-carbapenemase producing organisms: epidemiology, clinical significance, and evolutionary benefits on antimicrobial resistance and virulence

ABSTRACT Redundant carbapenemase-producing (RCP) bacteria, which carry double or multiple carbapenemases, represent a new and concerning phenomenon. The objective of this study is to conduct a comprehensive analysis of the epidemiology and genetic mechanisms of RCP strains to support targeted surveillance and control measures. A retrospective analysis was conducted using surveillance data from 277 articles. Statistical analysis was performed to determine and evaluate species prevalence, proportions of carbapenemases, antibiotic susceptibility profiles, sample information, and patient outcomes. Complete plasmid sequencing data were utilized to investigate potential antimicrobial resistance or virulence advantages that strains may gain from acquiring redundant carbapenemases. RCP bacteria are widely distributed globally, and their prevalence is increasing over time. Several countries, including China, India, Iran, Turkey, and South Korea, have reported more than 100 RCP strains. The most commonly reported RCP species are Klebsiella pneumoniae and Acinetobacter baumannii, which exhibit varying proportions of carbapenemase combinations. Certain species-carbapenemase combinations, such as K. pneumoniae carrying New Delhi metallo-β-lactamase (NDM) + oxacillinase (OXA) (56.76%) and K. pneumoniae carbapenemase (KPC) + Verona integron-encoded metallo-β-lactamase (VIM) (50.00%) carbapenemases, are associated with high mortality rates. In patients with RCP strains isolated from the bloodstream and respiratory system, the mortality rates are 58.70% and 69.23%, respectively. Analysis of plasmids from RCP strains suggests that they may acquire additional antibiotic resistance phenotypes and virulence factors. Carbapenem-resistant bacteria carrying redundant carbapenemases pose a significant global health threat. This study provides valuable insights into the epidemiology and genetic mechanisms of these bacteria, supporting the development of effective control and prevention strategies to mitigate their transmission. IMPORTANCE This study examined the global distribution patterns of 1,780 bacteria with double or multiple carbapenemases from 277 articles and assessed their clinical impact. The presence of multiple carbapenemases increases the chances of co-resistance to other classes of antibiotics and more virulence factors, further complicating the clinical management of infections.

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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.Lines 97-98 -please add citation supporting prolonged inpatient stays, healthcare cost and increased morbidity/mortality Lines 107-129 -Please address in your discussion if any of the geographic or species distribution differences could be explained by reporting biases (e.g., a study on African isolates that was solely focused on Acinetobacter).
Lines 279-281 -Please comment on the potential impact of more widely available and affordable sequencing technologies could have impacted the reporting trends in you analysis Lines 300-305, 336-340 -Please comment on the impact variable antimicrobial treatment choices could have on your clinical outcome data, especially in light of variability in access to novel beta-lactam/beta-lactamase inhibitor combination agents (ceftazidime/avibactam, meropenem/vaborbactam, sulbactam/durlobactam) and cefiderocol.
Line 349 -Your discussion must include content related to the limitations of your study Line 358 -Please add information regarding what efforts your team undertook to prevent inclusion of duplicate data (e.g., multiple publications reporting data on the same or overlapping isolate sets).
Lines 359-366 -Please address how your search strategy addressed finding dual/multiple carbapenemase producing isolates that were included in publications that either were not focused on this issue or did not identify they contained this data in the abstract Lines 363-366 -Please clarify why GES was not included in the search terms above but is noted as part of the carbapenemase definition?
Line 368 -Please address your decision to include OXA-51 which is a chromosomal enzyme in Acinetobacter vs the other predominantly plasmid mediated enzymes?Lines 390-393 -Please justify the use of this strategy vs excluding isolates without a clear isolation date?It is easy to imagine articles you included could have potentially included 5-10+ years of isolates and with your strategy the isolation dates would be significantly mis-categorized.
Lines 401-408 -This approach is not appropriate for demonstrating the impact of multiple carbapenemase enzymes in varying organisms.A more appropriate analysis here would require reporting of all MIC values for organisms expressing single and multiple enzymes of interest.Your analysis approach also does not address the potential impact of enzyme copy number on phenotypic MIC values.Given the available retrospective data, your publication should likely restrict itself to a simple descriptive and non-comparative presentation of the MIC data.
Reviewer #2 (Comments for the Author): Summary of Key Findings Thank you for the opportunity to review this work.In this manuscript, Yuan et al. performed a retrospective analysis to establish the prevalence of redundant carbapenemase producing (RCP) bacteria, and used sequencing data to evaluate their clinical impact and virulence factors.Key findings include a summary of 1,780 reported RCPs globally, with over 93% harboring two carbapenemases.Reports of up to six carbapenemases have been reported from different Enterobacterales isolates, and the incidence of these reports have increased over time.It is possible that infection with strains harboring multiple carbapenemases may be associated with more severe disease, as well a increased risk of co-resistance.
Major Concerns 1.In the search strategy section, the timeframe of searched publications is not provided.Please include this. 2. Lines 401-403: I am concerned that the exclusion of ambiguous MIC values (e.g., those that are non-discrete) results in the exclusion of important information.However, this is difficult to ascertain when the interpretive criteria (CLSI, EUCAST)/breakpoints used is unknown.For example, any MIC that came from an automated system and uses current CLSI breakpoints with have a resistant meropenem MIC of >=4.While comparing MICs is not necessarily the point of this study, I think further explanation regarding why discrete MICs were the only included (and what type of information this leaves out) would be helpful.3. Lines 179-184: The interpretation of these data are challenging given that other clinical context is not available.Was analysis done to account for other factors that may be associated with death?If not, perhaps this can simply be re-worded to say that patients who died more commonly were infected with strains harboring "xyz" resistance mechanism.4. I do not see a limitations section in this paper, and suggest that on be included.Topics to note in this section include those mentioned above, the inability to determine whether the prevalence of mechanisms has been increasing over time or technology has become more able to detect these (or both), any challenges related to being unable to clearly say the infection caused death (elaborate on how clinical information was adjudicated or how analyses were done).
Minor Concerns 1. Lines 378-379: the authors note that antibiotic susceptibility profiles were included in the data that were extracted from published studies.It is important to note in the limitations that there is significant variability in the way laboratories perform susceptibility testing, including the use of interpretive breakpoints, which directly impacts the way minimum inhibitory concentrations are interpreted.2. Line 34: The text states that this study aims to analyze the epidemiology and genetic mechanisms of RCP strains with the goal of facilitating targeted surveillance and control measures.I feel that it might be more appropriate to say something like "support targeted surveillance and control measures."Unless there is a pipeline in place that can support the implementation of actions this study recommends, I suggest making this change.3. Throughout the manuscript, antimicrobial is hyphenated and should not be.4. Line 42, should be "100 RCP strains" 5. Line 48, please provide a citation for the mortality rates mentioned 6. Lines 96-98, while it is well known that AMR contributes to poor clinical outcomes, I think that a citation is needed here 7. Lines 147-148: consider re-wording to something like, "Consider re-wording to something like: "to investigate the association between different carbapenemase combinations and bacterial species."8. Line 185-186: consider re-wording, specimens and sources are not responsible for death.9. Lines 230-232: Is this hypothesis accepted in the scientific community?Or is this a hypothesis the authors are making?Please clarify.If this is something that has been published on, please provide a citation.10.Lines 314-316 state, "Our findings also indicate that the specimen type of RCP bacteria isolation can significantly influence the severity of the infection and patient prognosis."Consider re-wording.I think what is meant here is that the severity of infection differs by site of infection (as indicated by where the specimens came from, or specimen type).
While we are willing to consider a revised version of this paper at Spectrum, it would be in your best interest to improve the writing.I recommend that you ask a colleague of yours who is a native English speaker to read and provide you some feedback on the writing.You are also welcome to use one of the services here: https://journals.asm.org/content/language-editing-servicesGlobal 1 Emergence of Double and Multi-Carbapenemase Producing Organisms: Epidemiology, Clinical Significance, and Evolutionary Benefits on AMR and Virulence.

Summary of Key Findings (200-250 words)
Thank you for the opportunity to review this work.In this manuscript, Yuan et al. performed a retrospective analysis to establish the prevalence of redundant carbapenemase producing (RCP) bacteria, and used sequencing data to evaluate their clinical impact and virulence factors.Key findings include a summary of 1,780 reported RCPs globally, with over 93% harboring two carbapenemases.Reports of up to six carbapenemases have been reported from different Enterobacterales isolates, and the incidence of these reports have increased over time.It is possible that infection with strains harboring multiple carbapenemases may be associated with more severe disease, as well a increased risk of co-resistance.
Major Concerns (at most 5-6): 1.In the search strategy section, the timeframe of searched publications is not provided.Please include this. 2. Lines 401-403: I am concerned that the exclusion of ambiguous MIC values (e.g., those that are non-discrete) results in the exclusion of important information.However, this is difficult to ascertain when the interpretive criteria (CLSI, EUCAST)/breakpoints used is unknown.For example, any MIC that came from an automated system and uses current CLSI breakpoints with have a resistant meropenem MIC of >=4.While comparing MICs is not necessarily the point of this study, I think further explanation regarding why discrete MICs were the only included (and what type of information this leaves out) would be helpful.3. Lines 179-184: The interpretation of these data are challenging given that other clinical context is not available.Was analysis done to account for other factors that may be associated with death?If not, perhaps this can simply be re-worded to say that patients who died more commonly were infected with strains harboring "xyz" resistance mechanism.4. I do not see a limitations section in this paper, and suggest that on be included.
Topics to note in this section include those mentioned above, the inability to determine whether the prevalence of mechanisms has been increasing over time or technology has become more able to detect these (or both), any challenges related to being unable to clearly say the infection caused death (elaborate on how clinical information was adjudicated or how analyses were done).
Minor Concerns (at most 5-20 in bullet points): 1. Lines 378-379: the authors note that antibiotic susceptibility profiles were included in the data that were extracted from published studies.It is important to note in the limitations that there is significant variability in the way laboratories perform susceptibility testing, including the use of interpretive breakpoints, which directly impacts the way minimum inhibitory concentrations are interpreted.
2. Line 34: The text states that this study aims to analyze the epidemiology and genetic mechanisms of RCP strains with the goal of facilitating targeted surveillance and control measures.I feel that it might be more appropriate to say something like "support targeted surveillance and control measures."Unless there is a pipeline in place that can support the implementation of actions this study recommends, I suggest making this change.3. Throughout the manuscript, antimicrobial is hyphenated and should not be.4. Line 42, should be "100 RCP strains" 5. Line 48, please provide a citation for the mortality rates mentioned 6. Lines 96-98, while it is well known that AMR contributes to poor clinical outcomes, I think that a citation is needed here 7. Lines 147-148: consider re-wording to something like, "Consider re-wording to something like: "to investigate the association between different carbapenemase combinations and bacterial species."8. Line 185-186: consider re-wording, specimens and sources are not responsible for death.9. Lines 230-232: Is this hypothesis accepted in the scientific community?Or is this a hypothesis the authors are making?Please clarify.If this is something that has been published on, please provide a citation.10.Lines 314-316 state, "Our findings also indicate that the specimen type of RCP bacteria isolation can significantly influence the severity of the infection and patient prognosis."Consider re-wording.I think what is meant here is that the severity of infection differs by site of infection (as indicated by where the specimens came from, or specimen type).
Thank you for submitting your paper to Spectrum.Please clarify that aztreonam is active against isolated NDM producers, but that organisms often have multiple mechanisms of resistance present (e.g., ESBL, other carbapenemases, porin/efflux mutations) that will render aztreonam inactive.