==== Front PLoS One PLoS One plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0243630 PONE-D-20-20807 Research Article Biology and Life Sciences Microbiology Microbial Control Antimicrobial Resistance Medicine and Health Sciences Pharmacology Antimicrobial Resistance Biology and Life Sciences Organisms Bacteria Klebsiella Klebsiella Pneumoniae Biology and Life Sciences Microbiology Medical Microbiology Microbial Pathogens Bacterial Pathogens Klebsiella Klebsiella Pneumoniae Medicine and Health Sciences Pathology and Laboratory Medicine Pathogens Microbial Pathogens Bacterial Pathogens Klebsiella Klebsiella Pneumoniae People and Places Geographical Locations Asia Japan Biology and Life Sciences Population Biology Population Dynamics Geographic Distribution Medicine and Health Sciences Pharmacology Pharmacologic Analysis Antibiotic Susceptibility Testing Earth Sciences Geography Medicine and Health Sciences Medical Conditions Infectious Diseases Nosocomial Infections Medicine and Health Sciences Public and Occupational Health Geographical distribution of Enterobacterales with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: An analysis using comprehensive national surveillance data on antimicrobial resistance Distribution of IMP-6 phenotype and its association with antimicrobial usehttps://orcid.org/0000-0003-3115-0645Hirabayashi Aki Data curationFormal analysisInvestigationMethodologyVisualizationWriting – original draftWriting – review & editing1* Yahara Koji ConceptualizationData curationFormal analysisMethodologyProject administrationSupervisionValidationVisualizationWriting – review & editing1* Kajihara Toshiki ResourcesValidationWriting – review & editing1 Sugai Motoyuki ConceptualizationResourcesSupervisionValidationWriting – review & editing1 Shibayama Keigo Funding acquisitionProject administrationSupervisionWriting – review & editing12 1 Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Tokyo, Japan 2 Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, Japan Karunasagar Iddya Editor Nitte University, INDIA Competing Interests: The authors have declared that no competing interests exist. * E-mail: akihira@nih.go.jp (AH); k-yahara@nih.go.jp (KY) 17 12 2020 2020 15 12 e02436306 7 2020 24 11 2020 © 2020 Hirabayashi et al2020Hirabayashi et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Enterobacterales resistant to carbapenems, a class of last-resort antimicrobials, are ranked as an “urgent” and “critical” public health hazard by CDC and WHO. IMP-type carbapenemase-containing Enterobacterales are endemic in Japan, and blaIMP-6 is one of the notable carbapenemase genes responsible for the resistance. The gene is plasmid-encoded and confers resistance to meropenem, but not to imipenem. Therefore, IMP-6-producing Enterobacterales isolates are occasionally overlooked in clinical laboratories and are referred to as ‘stealth-type’. Since previous reports in Japan were confined only to some geographical regions, their distribution across prefectures and the factors affecting the distribution remain unclear. Here, we revealed the dynamics of the geographical distribution of Enterobacterales with IMP-6 phenotype associated with antimicrobial use in Japan. We utilized comprehensive national surveillance data of all routine bacteriological test results from more than 1,400 hospitals in 2015 and 2016 to enumerate Escherichia coli and Klebsiella pneumoniae isolates with the antimicrobial susceptibility pattern (phenotype) characteristic of IMP-6 (imipenem susceptible, meropenem resistant), and to tabulate the frequency of isolates with the phenotype for each prefecture. Isolates were detected in approximately half of all prefectures, and combined analysis with the national data of antimicrobial usage revealed a statistically significant association between the frequency and usage of not carbapenems but third-generation cephalosporins (p = 0.006, logistic mixed-effect regression) and a weaker association between the frequency and usage of fluoroquinolones (p = 0.043). The usage of third-generation cephalosporins and fluoroquinolones may select the strains with the IMP-6 phenotype, and contribute to their occasional spread. We expect the findings will promote antimicrobial stewardship to reduce the spread of the notable carbapenemase gene. http://dx.doi.org/10.13039/100009619Japan Agency for Medical Research and DevelopmentJP19fk0108061Shibayama Keigo This study was supported by Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) under grant number JP19fk0108061. Data AvailabilityAll data to replicate the study’s findings are fully available at https://github.com/bioprojects/IMP-6_phenotype_distribution.Data Availability All data to replicate the study’s findings are fully available at https://github.com/bioprojects/IMP-6_phenotype_distribution. ==== Body Introduction Carbapenems have broad spectrum activity against Gram-positive, Gram-negative, and anaerobic bacteria and are considered agents of last-resort for complicated bacterial infections. Carbapenem-resistant Enterobacterales (CRE) are becoming increasingly prevalent and are a global public health concern. Genes encoding carbapenemases including blaKPC, blaIMP, blaVIM, blaNDM, blaOXA-48, and blaOXA-181 are encoded on plasmids, which are transmissible between the same and different genera via conjugation [1]. A notable carbapenemase gene is blaIMP-6, which confers a unique susceptibility pattern to carbapenems. Bacteria harboring blaIMP-6 are generally resistant to the newer carbapenem, meropenem, but are susceptible to imipenem [2,3]. The kcat/Km value, as a measure of catalytic efficiency of the IMP-6 protein, is approximately 7-fold higher against meropenem than against imipenem [4] owing to the one point mutation (640-Adenine replaced by Guanine), leading to a single amino acid substitution (Serine-196 replaced by Glycine) compared to IMP-1 [4]. Imipenem has often been used as a representative carbapenem for antimicrobial susceptibility testing. IMP-6-producing Enterobacterales isolates are occasionally overlooked in clinical laboratories, and are referred to as a stealth-type [5]. Strains with blaIMP-6 are resistant to all other beta-lactam antimicrobials [2]. Thus, spread of these strains within and across genera poses a serious threat to public health. The IMP-6 carbapenemase was first isolated from Serratia marcescens in Japan in 1996 [4], following the isolation of the IMP-1 enzyme from S. marcescens in Japan in 1991 [6]. IMP-producing Enterobacterales have been reported mainly in Japan, Taiwan, other Asian countries, and Australia, but have seldom been reported in European countries [7,8]. Enterobacterales producing IMP-6 have so far been reported only from Japan [2,3,9–12]. Pseudomonas aeruginosa producing IMP-6 have also been reported sporadically from South Korea and China [13,14]. This is not the case for Enterobacterales. There have been sporadic reports of CRE carrying blaIMP-6 from hospitals located mainly in West Japan [2,3,9,10]. The most frequently reported bacterial species harboring blaIMP-6 are E. coli and K. pneumoniae, although the number of publications is small [15,16]. Since 2014, CRE infection has been included in the list of mandatory reporting of all symptomatic cases in Japan. The National Epidemiological Surveillance of Infectious Diseases (NESID) is a national surveillance program for symptomatic cases [17]. In 2018, 2289 CRE cases were reported and 1684 CRE isolates were analyzed [18], which revealed that the blaIMP gene was the most common (254, 85.5%) out of the 297 isolates positive for carbapenemase genes, and blaIMP-6 and blaIMP-1 are two dominant genes in CRE in Japan (52.0% and 46.3% among 123 genome-sequenced isolates with blaIMP genes). The incidence of infections caused by CRE with blaIMP-6 varied by region in the report. However, previous reports did not examine the geographical distribution of IMP-6 in each prefecture. In addition, the NESID system involves only symptomatic cases reported by physicians. Thus, the entire geographical distribution of IMP-6 including asymptomatic carriers remains unknown. Another national surveillance program, the Japan Nosocomial Infections Surveillance (JANIS), has been comprehensively collecting data of all bacteria isolated from all sample types of both symptomatic and asymptomatic patients from clinical laboratories of the participating hospitals since 2000. The data include results of bacterial culture and antimicrobial susceptibility testing that are routinely conducted in the hospitals. The number of participating hospitals as of January 2020 is 2223. The data covered more than 8.2 million specimens and over 5.8 million isolates in 2018. The data stored in a national database are available for analyses in the public interest, and will be useful to explore the entire geographical distribution of IMP-6 at phenotype level including asymptomatic carriers. In this study, we aim to determine the geographical distribution of E. coli and K. pneumoniae isolates showing a pattern indicative of the IMP-6 phenotype (imipenem susceptible, meropenem resistant), using the JANIS database since the susceptibility pattern of blaIMP-6-positive strains is unique. Additionally, the national data of antimicrobial usage were examined to explore a potential association between the frequency and usage of carbapenems, fluoroquinolones, and third-generation cephalosporins, which potentially causes selection of the strains with the IMP-6 phenotype. Materials and methods Data set All inpatient data fields were extracted between January 2015 and December 2016 from the JANIS database, which stores both culture-positive and -negative test diagnostic results with all antimicrobial susceptibility testing results. Patient identifiers are de-identified by each hospital before data submission to JANIS. Approval for extraction and use of the data was granted by the Ministry of Health, Labour and Welfare (Approval no. 1010–5). We also used national data of antimicrobial usage in 2015 and 2016 tabulated for each prefecture that are publicly available at the website of AMR Clinical Reference Center in National Center for Global Health and Medicine (http://amrcrc.ncgm.go.jp/surveillance/010/20181128172333.html) (The data tabulated for each prefecture are not available after 2016). From the data, we selected total usage of carbapenems, fluoroquinolones, and third-generation cephalosporins measured as DID (Defined Daily Doses/1000 inhabitants/day). Data tabulation We used a Java toolkit to extract aggregated data (stratified by specimen types) of the number of E. coli and K. pneumoniae isolates susceptible or resistant to imipenem and meropenem from the raw data in accordance with CLSI 2012 criteria [19]. De-duplication was conducted according to JANIS [20] to remove repeated isolates of the same species isolated from a patient within 30 days, regardless of specimen type, but considering the antimicrobial resistance phenotype. The de-duplication procedure selects and counts isolates with significantly different drug susceptibility as different isolates, even if they were isolated within 30 days from the same patient. A subsequent isolate is selected and counted if it shows change from susceptible to resistant (or vice versa) or a 4-fold or more comparison of the minimum inhibitory concentration value for a specific antimicrobial when compared to a previous isolate from the same patient within the 30-day period. We used an in-house Perl script to tabulate the aggregated data for each prefecture. We then calculated frequency of the IMP-6 phenotype as the number of isolates susceptible to imipenem but resistant to meropenem divided by the total number of isolates subject to antimicrobial susceptibility testing of the two antimicrobials. We also used the Java toolkit to extract aggregated data of the number of E. coli and K. pneumoniae isolates susceptible or resistant to imipenem, meropenem, ceftazidime, piperacillin, fosfomycin and amikacin. The aggregated data are available at https://github.com/bioprojects/IMP-6_phenotype_distribution. Statistical analyses Descriptive statistical analyses and univariate association analyses were performed with JMP Pro version 14 (SAS Institute, Cary, NC, USA). The aggregated data of the frequency of IMP-6 phenotype in each prefecture was visualized as a map using R version 3.6.1 and leaflet package. A statistical test of association between the usage of antimicrobials (either carbapenems, fluoroquinolones, and third-generation cephalosporins) and frequency of IMP-6 phenotype was conducted by logistic mixed-effect regression using glmer function of lme4 package [21] in R. In the univariate association analyses and statistical tests, we combined the aggregated data of E. coli and K. pneumoniae to calculate frequency of IMP-6 phenotype, given that plasmids encoding blaIMP-6 can transfer between genera. Results Geographical distribution of frequency of E. coli and K. pneumoniae isolates with the IMP-6 phenotype is shown as prefectural maps in Fig 1. The isolates continued to be found in some prefectures, but were not in approximately half of the prefectures (white in Fig 1). The percentages of prefectures where the isolate with IMP-6 phenotype is undetectable were 42.6% and 55.3% for E. coli in 2015 and 2016, and 42.6% for K. pneumoniae in both 2015 and 2016, respectively. 10.1371/journal.pone.0243630.g001Fig 1 Prefectural maps of frequency distribution of isolates with the IMP-6 phenotype in 2015 and 2016. Prefectures where isolates with the IMP-6 phenotype were not isolated are colored in white, and others are colored in six different colors (with bin size 0.2%) in the red range. More quantitatively, boxplots and histograms of the frequency distribution are shown in Fig 2. The mean of total number of antimicrobial susceptibility tests as denominator of the frequency was 4241 for E. coli and 2185 for K. pneumoniae. The distribution was obviously skewed, with a higher frequency in some prefectures. The highest frequency was 1.7% (28/1644) for E. coli and 1.6% (13/790) for K. pneumoniae. 10.1371/journal.pone.0243630.g002Fig 2 Boxplots and histograms of frequency distribution of isolates with the IMP-6 phenotype in 2015 and 2016. In the box plot, the left and right of the box indicate 25th and 75th percentile, respectively, the horizontal line indicates the median, the middle of the diamond indicates the mean, the right outliers are above the 75th percentile + 1.5 interquartile range, and the red horizontal line at the top left indicates the shortest range in which half of the data was distributed. The total number of E. coli isolates with the IMP-6 phenotype after de-duplication was 221 in 2015 and 242 in 2016. The corresponding numbers for K. pneumoniae were 189 in 2015 and 201 in 2016. A breakdown of the number of isolates according to specimen types is shown in Table 1. For E. coli, the isolates were most frequently from urine samples (49.3% in 2015 and 52.9% in 2016), followed by stool and respiratory samples. For K. pneumoniae, the isolates were almost equally frequent in urine, stool, and respiratory samples. A breakdown of the number of isolates without the de-duplication is also shown in S1 Table. No statistically significant difference in the proportion among the four specimen types was found between the two conditions (with and without de-duplication) in 2015 and 2016 for E. coli (p = 0.204 and p = 0.795, respectively), and for K. pneumoniae (p = 0.862 and p = 0.478, respectively) with chi-square test. 10.1371/journal.pone.0243630.t001Table 1 The number and proportion of isolates with the IMP-6 phenotype according to specimen types after de-duplication. E. coli K. pneumoniae 2015 2016 2015 2016 Blood 7 (3.2%) 9 (3.7%) 17 (9.0%) 12 (6.0%) Respiratory 46 (20.8%) 47 (19.4%) 72 (38.1%) 59 (29.4%) Urine 109 (49.3%) 128 (52.9%) 48 (25.4%) 70 (34.8%) Stool 59 (26.7%) 58 (24.0%) 52 (27.5%) 60 (29.9%) Total 221 (100%) 242 (100%) 189 (100%) 201 (100%) The relationship between the frequency and antimicrobial usage (DID) (Fig 3) clearly revealed almost no relationship between the usage of carbapenems and the frequency of the IMP-6 phenotype, while a positive relationship with third-generation cephalosporins was evident. Logistic mixed-effect regression analysis accounting for the longitudinal correlation revealed the association was statistically significant in the E coli and K. pneumoniae group (p = 0.006). The association was still statistically significant in K. pneumoniae (p = 0.002) and was suggested in E. coli (p = 0.072). The association between frequency of isolates with the IMP-6 phenotype and antimicrobial usage was also evident for fluoroquinolones, although the significance was weaker (p = 0.043 for the combined group of E. coli and K. pneumoniae) than that of third-generation cephalosporins. In the isolates with the IMP-6 phenotype, resistance rate of ceftazidime, as a representative third-generation cephalosporins, was 94.4% of the E. coli and 93.0% of the K. pneumoniae isolates across the prefectures. 10.1371/journal.pone.0243630.g003Fig 3 Association between antimicrobial usage and frequency of isolates with the IMP-6 phenotype. The frequency in the bottom of the figure was calculated after summing the denominator (total number of antimicrobial susceptibility testing) between E. coli and K. pneumoniae and that of the numerator (number of isolates with the IMP-6 phenotype). A linear regression line with a 95% credibility interval is illustrated for 2015 (blue) and 2016 (red). Discussion Presently, using data of antimicrobial susceptibility testing of all isolates in more than 1,400 hospitals in Japan, isolates with the phenotype unique to IMP-6 were enumerated for each prefecture. Such a utilization of the phenotypic data of bacterial culture and antimicrobial susceptibility testing that are routinely conducted in hospitals throughout a country is complementary and cost-effective compared to genetic testing by sequencing to detect blaIMP-6, which requires additional time and cost and currently cannot be extended to potential asymptomatic carriers across Japan. The frequency distribution of the isolates was depicted as a map (Fig 1) and quantitatively as boxplots and histograms (Fig 2). Overall, the longitudinal correlation of the frequency between 2015 and 2016 was high, but not complete (Spearman’s nonparametric correlation coefficient: 0.61 in E. coli and 0.72 in K. pneumoniae). The findings reflected that the isolates with the IMP-6 phenotype tended to be continuously found in specific prefectures, although an occasional increase in the frequency in one year was not necessarily repeated in another year. The results are consistent with the previous report of NESID that the isolates with blaIMP-6 are detected only in some prefectures [18]. The statistically significant association suggests that third-generation cephalosporins, rather than carbapenems, might contribute to the selection of strains with the IMP-6 phenotype. The absence of an association for carbapenems might reflect that their usage is mainly confined to injections that are strictly controlled by monitoring and notification systems among the hospitals participating in the national surveillance JANIS. On the other hand, fluoroquinolones and third-generation cephalosporins are available as oral formulations that more easily administered without the involvement of the notification system, as well as injectables. As indicated by the X-axis of Fig 3, the amount of usage (measured by DID) of fluoroquinolones and third-generation cephalosporins was much larger than that of carbapenems. It is known that a strain carrying blaIMP-6 is resistant to third-generation cephalosporins, including ceftazidime [4]. This is confirmed by the ≥93% resistance to ceftazidime among the hundreds of isolates in our study, suggesting the phenotype corresponds well to the presence of blaIMP-6, however, the phenotype-genotype correspondence is not absolute. The remaining 5.6% of E. coli and 7.0% of K. pneumoniae isolates examined in our study are unlikely to carry blaIMP-6, contrary to the phenotype. Although JANIS is the comprehensive national surveillance of antimicrobial resistance, the targets thus far have been confined to bacterial culturing and antimicrobial susceptibility testing. Actual isolates and their genetic resistance determinants are not included. Further studies are warranted to elucidate the distribution of the blaIMP-6 gene across geographical regions, and to determine the extent to which the presence of this gene corresponds to the observed antimicrobial susceptibility patterns. A weaker association between the frequency of isolates with the IMP-6 phenotype and antimicrobial usage was evident for fluoroquinolones (Fig 3). This finding suggests that fluoroquinolone resistance might also contribute to selection of isolates with the IMP-6 phenotype. Fluoroquinolone resistance in Enterobacterales mainly involves chromosomal genes. The most commonly detected resistance mechanisms are mutations in chromosomal genes encoding gyrase and/or topoisomerase IV, such as gyrA and parC, which result in weakened quinolone-enzyme interactions and cause high-level quinolone resistance [22]. Less frequently, plasmid-encoded quinolone resistance genes, such as qnr, aac(6’)-Ib-cr, and qep, are observed, although plasmid-mediated quinolone resistance determinants confer only low-level resistance to quinolones [2,23]. Linkage of these chromosomal mechanisms of fluoroquinolone resistance with plasmid-encoded blaIMP-6 is expected if the plasmid harboring blaIMP-6 is transmitted into extended-spectrum beta-lactamase (ESBL) producing E. coli or K. pneumoniae clones, such as the worldwide pandemic clone, ST131, in E. coli [24,25], which harbors point mutations in chromosomal gyrA and parC genes [26–28]. However, the linkage is conceivably weaker than that of blaIMP-6 and ESBL genes encoded in the same plasmid, which could be a cause of the weaker association between frequency of isolates with the IMP-6 phenotype and usage of fluoroquinolones. Treatment of isolates carrying blaIMP-6 is problematic. Although isolates are susceptible to imipenem in vitro, the effectiveness of imipenem in vivo has not been established. It is known that a strain carrying blaIMP-6 shows susceptibility to piperacillin due to the single amino acid substitution, compared to IMP-1 [4]. However, in previous reports, isolates with blaIMP-6 almost always had ESBL genes, such as blaCTX-M [2,3,9,12], which renders resistance to all beta-lactams including piperacillin, except for imipenem. A report from Hiroshima, where studies on strains with blaIMP-6 have been ongoing since 2009 [5], described sequence analyses of Enterobacterales isolates harboring blaIMP-6. The analyses revealed that in 12/14 K. pneumoniae, 1/1 K. oxytoca, and 3/6 E. coli, blaIMP-6 and blaCTX-M-2 were encoded on the same 47-kb incN plasmid. When we checked the proportion of resistance to piperacillin in isolates with the IMP-6 phenotype, the mean results across the prefectures were 81.4% for E. coli and 92.6% for K. pneumoniae. Conversely, when we similarly checked the proportion of resistance to amikacin and fosfomycin in isolates with the IMP-6 phenotype, the mean values across the prefectures were 4.0% for E. coli and 4.5% for K. pneumoniae for amikacin, and 1.0% for E. coli and 4.0% for K. pneumoniae for fosfomycin. The resistance rates to amikacin and fosfomycin were significantly lower than the rates to piperacillin, suggesting their potential for treatment. However, characteristics, transferability, and adverse effects of these drugs need to be considered. A systematic review and meta-analysis including 243 studies revealed the association of antimicrobial consumption with the development of antimicrobial resistance [29]. In the meta-analysis, a stronger link between consumption and resistance was found for countries in southern Europe. Analysis of some other cross-national database studies has revealed a correlation between antimicrobial consumption and resistant rate in Europe [30,31]. If possible, comparison with such international data are warranted to generalize our findings. However, antimicrobial consumption data is often complied differently between countries, and a careful data preparation is needed for accurate international comparison. Furthermore, Enterobacterales producing IMP-6 have so far been reported only from Japan. This was the reason for our focus on the inter-prefecture comparison in Japan. Further studies are also warranted to reveal appropriate measures reflecting such factors and to systematically collect and analyze them in addition to the antimicrobial usage data (S1 Text). In summary, the frequency distribution of the isolates showing the phenotype specific to IMP-6 was determined across prefectures in Japan. The frequency of the IMP-6 phenotype was statistically significantly associated with usage of third-generation cephalosporins and was weakly associated with usage of fluoroquinolones, rather than usage of carbapenems. The results highlight the usefulness of national surveillance data of antimicrobial susceptibility testing results to infer the epidemiological distribution of isolates with specific resistance genes or mechanisms, without the additional cost and time of actual genetic testing. Such an inference is possible for other pathogens, for example Panton-Valentine Leukocidin or Toxic Shock Syndrome Toxin-1 producing methicillin-resistant Staphylococcus aureus [32]. The approach is also useful for exploring the emergence of a new multi-drug resistance phenotype, which requires systematic analyses of combination of resistance patterns of several key antimicrobials [33]. This study provides a basis for future studies utilizing national surveillance data of antimicrobial susceptibility testing results and integrated analyses incorporating additional data, such as antimicrobial usage and genetic testing. Supporting information S1 Table The number and proportion of isolates with the IMP-6 phenotype according to specimen types without de-duplication. (DOCX) Click here for additional data file. S1 Text (DOCX) Click here for additional data file. We are grateful to all the hospitals that participated and contributed data to JANIS, and to Editage (www.editage.jp) for English language editing. 10.1371/journal.pone.0243630.r001 Decision Letter 0 Karunasagar Iddya Academic Editor © 2020 Iddya Karunasagar2020Iddya KarunasagarThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version0 28 Aug 2020 PONE-D-20-20807 Geographical distribution of Enterobacteriaceae with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: an analysis using comprehensive national surveillance data on antimicrobial resistance PLOS ONE Dear Dr. Hirabayashi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I would like to thank the editors for sending me such an interesting and relevant manuscript to review. I believe that the findings from this study are very important and have the ability not only to inform antimicrobial stewardship, but provide a good example of for other countries to investigate their own antimicrobial resistance and usage relationships. I have a few comments for the authors, which I hope are of use. General - Would the authors consider using more current taxonomy i.e. Enterobacterales as opposed to Enterobacteriaceae [https://doi.org/10.1099/ijsem.0.001485]? - Use a consistent approach to reporting p-values e.g. 1 significant figure, 2 decimal places etc. Line 38: Suggest remove “This examination revealed a skewed frequency distribution.” This sentence means nothing in isolation. Line 39-40: Were high rates observed in prefectures with high levels of 3GC prescribing? If so, suggest this sentence is merged with following sentence. Line 44 and throughout: Remove “not carbapenems”. I don’t think you need to precede your 3GC observations with “not carbapenems” after the first mention. Abstract: Could you conclude the abstract with a public health/antimicrobial stewardship recommendation? I believe the findings are important and actionable. Line 53: Change “species” to “genera” Line 63-65: This is a repeated statement – you have already stated that IMP carbapenemases are plasmid-mediated etc. earlier in the Introduction. Lines 80-87: Summarise this section further. Suggest remove sentences between line 81 and 85 as you have already referenced the study and therefore detailed findings do not need to be provided. Lines 92-96. This statement would be better placed in the Discussion, to highlight the advantages of the study design. Lines 104-111: This reads more like an introduction of the methods. Suggest ending the Introduction by stating the aim(s) of your study. Line 125: Defined daily dose = DDD Line 160: Change “species” to “genera” Lines 163-166: The detail in this sentence is superfluous since you have described the dataset and study years in the Methods. Suggest the sentence is simplified. Line 168: Add “respectively” after “2016”. Line 178: Provide the actual number (since this is the frequency) with the proportion in brackets immediately afterwards. Line 179-180: This needs to precede the reporting of frequency and associated proportions. Also need to clarify what this “average” represents, and the type of “average” reported i.e. mean, median, mode? Line 185: Change “average” to “median”. Lines 191-192: Report 2015 data before 2016 (as you have done for the rest of the manuscript). Lines 199-200: It is not clear which p-value relates to which specimen type. Lines 206-210: This statement is more suited to the Discussion. Lines 211-215: Much of this is already covered in the Methods and can be removed. Lines 215-218: The wording of this goes beyond reporting the results, introducing elements of interpretation, which belongs in the Discussion. Lines 241-242: If the proportion of Enterobacter cloacae is so high, why were these isolates not included in the study? An explanation in the Introduction or Methods (depending on reason) may be necessary. Lines 242-245: This information belongs in the Results. Lines 262-265: This information belongs in the Results. Lines 269-271: Is there a reference for this? Figure 1: The category “<1” does not make sense in the legend. Figure 3: I think this data would be better presented as a table. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0243630.r002 Author response to Decision Letter 0 Submission Version1 23 Sep 2020 Please see the attached file "Response to Reviewers". Attachment Submitted filename: Response to Reviewers.docx Click here for additional data file. 10.1371/journal.pone.0243630.r003 Decision Letter 1 Karunasagar Iddya Academic Editor © 2020 Iddya Karunasagar2020Iddya KarunasagarThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version1 25 Nov 2020 Geographical distribution of Enterobacterales with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: an analysis using comprehensive national surveillance data on antimicrobial resistance PONE-D-20-20807R1 Dear Dr. Hirabayashi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Iddya Karunasagar Academic Editor PLOS ONE Additional Editor Comments (optional): All reviewer comments addressed Reviewers' comments: 10.1371/journal.pone.0243630.r004 Acceptance letter Karunasagar Iddya Academic Editor © 2020 Iddya Karunasagar2020Iddya KarunasagarThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 Dec 2020 PONE-D-20-20807R1 Geographical distribution of Enterobacterales with a carbapenemase IMP-6 phenotype and its association with antimicrobial use: an analysis using comprehensive national surveillance data on antimicrobial resistance Dear Dr. Hirabayashi: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Iddya Karunasagar Academic Editor PLOS ONE ==== Refs References 1 Nordmann P , Dortet L , Poirel L . Carbapenem resistance in Enterobacteriaceae: here is the storm ! Trends Mol Med . 2012 ;18 (5 ): 263 –272 . 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