Simplified Carbapenem Inactivation Method (sCIM) as a Reliable Tool for Detecting OXA-48- Producing Enterobacterales in Clinical Practice

Simplified Carbapenem Inactivation Method (sCIM) as a Reliable Tool for Detecting OXA-48- Producing Enterobacterales in Clinical Practice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Simplified Carbapenem Inactivation Method (sCIM) as a Reliable Tool for Detecting OXA-48- Producing Enterobacterales in Clinical Practice Ana Collazos Blanco, Ana Belén García Saéz, Inés María Marcos Palomino, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9430307/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Carbapenemase-producing Enterobacterales (CPE) are among the most concerning antimicrobial-resistant pathogens due to their limited treatment options and the potential for widespread transmission. This study aimed to evaluate the sensitivity of the simplified carbapenem inactivation method (sCIM) for detecting OXA-48-producing CPE isolates. A total of 150 non-duplicated CPE strains, were collected from clinical specimens at the Hospital Central de la Defensa “Gómez-Ulla” in Spain. The isolates were confirmed as OXA-48 producers using immunochromatographic assays or PCR-based methods. Antimicrobial susceptibility was assessed, and the sCIM was compared to standard diagnostic methods. Results showed that the sCIM method successfully detected all 150 OXA-48-producing strains, with no indeterminate results, offering a sensitivity of 100% (95% CI: 97.6–100%).. The method provided a rapid and easy-to-interpret result within 16–18 hours. The study suggests that sCIM is a reliable, simplified alternative to traditional carbapenemase detection methods, especially in regions with high prevalence of OXA-48 carbapenemase-producing enterobacterales. sCIM carbapenemase OXA-48 Figures Figure 1 Importance The rapid detection of OXA-48-producing Enterobacterales remains a major diagnostic challenge due to their frequently low carbapenem MICs and subtle phenotypic expression. This study demonstrates that sCIM provides a simple, reliable, and highly sensitive method for detecting these isolates, even in cases with borderline susceptibility profiles. Given its low cost and ease of implementation, sCIM represents a valuable tool for routine clinical laboratories, particularly in high-prevalence or resource-limited settings. Introduction Carbapenem-resistant Enterobacterales (CRE) are among the highest-priority antimicrobial-resistant pathogens, being associated with limited therapeutic options and poor clinical outcomes( 1 ). Resistance to carbapenems is most commonly mediated by carbapenemases, which are classified into Ambler classes A, B, and D according to their molecular structure. Among these, carbapenemase-producing Enterobacterales (CPE) represent a major clinical concern due to their high potential for transmission between patients. In addition, carbapenemase genes are frequently located on mobile genetic elements such as plasmids, facilitating horizontal gene transfer and contributing to multispecies outbreaks. Therefore, rapid and accurate detection of CPE in clinical laboratories is critical for infection control and appropriate antimicrobial therapy. Several phenotypic methods are currently available for the detection of CPE. The Carba NP test (bioMérieux) allows rapid identification of carbapenemase activity within 30 minutes by detecting pH changes ( 2 ). In addition, immunochromatographic assays such as NG-Test Carba 5 (NG Biotech) and RESIST-5 O.K.N.V.I (Coris) provide high sensitivity and specificity, enabling the detection of the most common carbapenemase families within 15 minutes ( 3 ). The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST ) introduced the modified carbapenem inactivation method (mCIM), based on the original carbapenem inactivation method (CIM). Although mCIM is reliable for detecting a wide range of carbapenemases, its requirement for prolonged incubation limits its practicality in routine laboratory workflows. To overcome these limitations, the simplified carbapenem inactivation method (sCIM) has been proposed, eliminating the broth incubation step and offering a faster and more practical alternative ( 4 ) Most validation studies of these assays have focused on isolates producing the most prevalent carbapenemases, such as KPC, VIM, and NDM. However, data on OXA-48-like carbapenemases remain limited. This is particularly relevant as OXA-48 enzymes often confer low-level resistance and may be more difficult to detect using conventional phenotypic methods, potentially leading to underdiagnosis ( 5 , 6 ) Therefore, the aim of this study was to evaluate the susceptivity of the sCIM method for the detection of OXA-48-producing CPE isolates. Material and Methods Strain collection A total of 150 non-duplicate clinical isolates exhibiting decreased susceptibility to carbapenems (defined as reduced susceptibility to at least one of ertapenem, imipenem, or meropenem) were included in this study. All isolates were confirmed as OXA-48-producing carbapenemase-producing Enterobacterales (CPE) and were recovered from patients during routine clinical diagnostics at Hospital Central de la Defensa “Gómez-Ulla” (CSVE). Susceptibility testing and MIC determination All isolates were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany). Antimicrobial susceptibility testing (AST) was performed by broth microdilution in accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, using the MicroScan WalkAway Plus system (Beckman Coulter). Quality control was performed in each experimental setting using Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603. Carbapenemase production was determined using the RESIST-5 O.K.N.V.I immunochromatographic assay (Coris) ( 6 ) and the Xpert® Carba-R assay ( 7 ). Simplified carbapenem inactivation method (sCIM) The simplified carbapenem inactivation method (sCIM) was performed as previously described, based on the modified carbapenem inactivation method (mCIM)( 4 ). A 0.5 McFarland suspension of Escherichia coli ATCC 25922, prepared by the direct colony suspension method, was inoculated onto Müller–Hinton agar (MHA) plates according to standard disk diffusion procedures and allowed to dry for 3–10 min. One to three colonies of each test isolate grown on blood agar were then smeared onto one side of an imipenem disk (10 µg) to achieve uniform coverage. The inoculated side of the disk was immediately placed onto the previously inoculated MHA plate. Klebsiella pneumoniae ATCC BAA-1705 (KPC producer) and Klebsiella pneumoniae ATCC 700603 were used as positive and negative controls, respectively. Plates were incubated at 35°C for 16–18 h in ambient air. Carbapenemase-producing isolates hydrolyze imipenem, thereby allowing growth of the susceptible indicator strain around the imipenem disk. The presence of an inhibition zone measuring 6–20 mm in diameter, or the observation of satellite growth of Escherichia coli ATCC 25922 around the disk, was interpreted as a positive result for carbapenemase production. An inhibition zone of ≥ 26 mm was interpreted as negative, whereas a zone of 20–25 mm was considered indeterminate. Results All 150 isolates were collected at our institution and comprised 120 Klebsiella pneumoniae (80.0%), 13 Escherichia coli (8.7%), 10 Proteus mirabilis (6.7%), 4 Citrobacter freundii (2.7%), 2 Klebsiella oxytoca (1.3%), and 1 Morganella morganii (0.7%). Isolates were recovered from a variety of clinical specimens, including urine (n = 90), surveillance samples (n = 30), respiratory samples (n = 13), wounds (n = 9), and abscesses (n = 8). All isolates exhibited decreased susceptibility to β-lactams, including carbapenems. High rates of resistance were observed for cefepime (87.3%) and ciprofloxacin (88.6%), suggesting the presence of extended-spectrum β-lactamases (ESBLs). In contrast, ceftazidime/avibactam showed high activity, with 98.0% of isolates remaining susceptible. Results for the three most prevalent species are summarized in Table 1 . Table 1 Antimicrobial susceptibility profile of the most frequently isolated Enterobacterales species. Specie strain (n) Antimicrobial Number and % of isolates Susceptible (S) or Susceptible Increase Exposure (I) K. pneumoniae (120) Amikacin 108 (90.0) Gentamicin 53 (44.2) Ciprofloxacin 7 (5.8) Cefepime 7 (5.8) Imipenem 78 (65.0) Meropenem 90 (75.0) Ceftazidime/avibactam 119 (99.2) Ceftolozane /tazobactam 18(15.0) Sulfametoxazol/trimetoprim 45 (37.5) E.coli (13) Amikacin 13 (100.0) Gentamicin 9 (69.2) Ciprofloxacin 6 (46.1) Cefepime 6 (46.1) Imipenem 11(84.6) Meropenem 13 (100.0) Ceftazidime/avibactam 13 (100.0) Ceftolozane /tazobactam 11(84.6) Sulfametoxazol/trimetoprim 9(69.2) P.mirabilis ( 10 ) Amikacin 8 (80.0) Gentamicin 8 (80.0) Ciprofloxacin 2 (20.0) Cefepime 4 (40.0) Imipenem 0 (0.0) Meropenem 5 (50.0) Ceftazidime/avibactam 10 (100.0) Ceftolozane /tazobactam 6 (60.0) Sulfametoxazol/trimetoprim 2 (20.0) Using the sCIM method, all 150 OXA-48-producing isolates were correctly identified, yielding a sensitivity of 100% (95% CI: 97.6–100%) according to predefined cutoff criteria. Two isolates were identified as co-producers of KPC and NDM carbapenemases respectively. No carbapenemase-negative isolates were included; therefore, specificity could not be assessed. Most isolates exhibited complete absence of an inhibition zone (0 mm). In contrast, isolates belonging to the Morganellaceae family displayed atypical inhibition patterns, characterized by a double-zone appearance consisting of an inner growth zone, likely associated with swarming behavior, and an outer clear zone. Despite this atypical pattern, inhibition diameters remained below the established cutoff values (Fig. 1). The influence of bacterial inoculum size on sCIM performance was assessed by testing different inoculum conditions (one, two, three, and five colonies). No significant differences in inhibition zone diameters were observed among the different inoculum sizes, indicating that the method is robust to variations in bacterial load. Additionally, no significant differences in inhibition zone diameters were observed among isolates classified as resistant, susceptible, or susceptible with increased exposure to carbapenems. Discussion Our findings demonstrate that the simplified carbapenem inactivation method (sCIM) is a highly sensitive approach for the detection of OXA-48 carbapenemase producing Enterobacterales (CPE), achieving 100% sensitivity in our isolate collection, with no indeterminate results. These findings support the reliability of sCIM as a practical alternative to more time-consuming phenotypic methods. Notably, to our knowledge, this represents one of the largest evaluations specifically focused on OXA-48-producing isolates, addressing a relevant gap in the current literature. Given the clinical and epidemiological importance of carbapenem-resistant Enterobacterales (CRE), rapid and accurate detection methods are essential for guiding antimicrobial therapy and implementing effective infection control measures ( 8 ). While polymerase chain reaction (PCR) remains the reference standard for carbapenemase identification, immunochromatographic assays provide a rapid and cost-effective alternative without requiring specialized laboratory infrastructure ( 2 ). The modified carbapenem inactivation method (mCIM), currently recommended for phenotypic detection, demonstrated high sensitivity in initial validation studies ( 5 ). However, subsequent reports have highlighted false-negative results, particularly in the detection of metallo-β-lactamases ( 6 ). In addition, mCIM presents practical limitations, including the need for a broth incubation step followed by overnight incubation, which may delay result reporting. The simplified carbapenem inactivation method (sCIM), developed to address these limitations, eliminates the 4–5 h broth incubation step and facilitates integration into routine laboratory workflows ( 6 , 8 ). Previous studies have demonstrated good performance of sCIM, particularly for metallo-β-lactamases, however, most validation studies have included only a limited number of OXA-48-producing isolates, leaving an important gap in the evaluation of this method for this clinically relevant carbapenemase ( 6 , 8 ). In this context, our results are consistent with those reported by Schaffarczyk et al., who observed 100% sensitivity across a large and diverse isolate collection ( 6 ). Conversely, Hosoda et al. reported substantially lower sensitivity (54.9%) ( 5 ), likely reflecting differences in carbapenemase distribution. While our study focused exclusively on OXA-48 producers, their cohort predominantly included metallo-β-lactamase-producing isolates, with all false-negative results associated with IMP-1. Methodological factors such as bacterial inoculum have also been suggested to influence sCIM performance ( 8 ). However, in our study, no significant differences were observed when varying inoculum size, supporting the robustness of the method under routine conditions. Similarly, the absence of indeterminate results in our cohort contrasts with previous reports (6–36%) ( 5 , 6 , 9 ). This discrepancy may be explained by differences in enzyme hydrolytic activity. For instance, Hamprecht et al. described Proteus mirabilis isolates harboring carbapenemases with weak activity, such as OXA-23 and OXA-58, which may contribute to indeterminate phenotypes ( 9 ). Importantly, none of the isolates in our study yielded indeterminate results, including those with low hydrolytic activity and MIC values within EUCAST susceptibility breakpoints. This finding highlights the ability of sCIM to reliably detect carbapenemase production even in isolates with borderline phenotypic profiles. An additional observation was the atypical inhibition pattern in Morganellaceae strains, characterized by relatively large inhibition zones despite confirmed carbapenemase production but with inhibition diameters remained below the established cutoff values. This may be related to the species-specific characteristics such as swarming, and should be considered when interpreting results. This observation may represent a relevant practical consideration for laboratories applying sCIM in routine settings. The “satellite phenomenon” described by Wan et al. ( 10 ) provides a mechanistic explanation based on limited enzyme diffusion and local antibiotic degradation. Although previously reported, this phenomenon was not observed in our cohort, suggesting that its occurrence may depend on specific bacterial or enzymatic factors. Detection of carbapenemases in Proteus mirabilis remains particularly challenging due to low MIC values despite enzyme production ( 9 ). Notably, all P. mirabilis isolates in our study were correctly identified by sCIM, including those within the susceptible range, further supporting the sensitivity of the method in clinically challenging scenarios. Although both meropenem and imipenem disks have been used for sCIM, imipenem has been associated with improved sensitivity in certain organisms ( 10 ). Based on this, imipenem disks were selected in our study. This study has some limitations. First, it was conducted using isolates from a single geographic setting, which may limit the generalizability of the findings. Second, sCIM lacks strict standardization of bacterial inoculum, potentially introducing operator-dependent variability. Despite these limitations, our study includes a large and well-characterized collection of OXA-48-producing isolates, a group for which data remain limited. Furthermore, all experiments were performed in duplicate, supporting the reproducibility of the method under routine clinical laboratory conditions. From a clinical perspective, sCIM represents a rapid, cost-effective, and reliable method for detecting OXA-48-producing isolates. By eliminating the incubation step required for mCIM, it offers a practical alternative that can significantly improve workflow efficiency. These findings support the use of sCIM as a frontline phenotypic screening tool in clinical microbiology laboratories, particularly in settings with high OXA-48 prevalence and limited access to molecular or immunochromatographic methods. Declarations Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contribution Author ContributionsAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by A.C.B, A.; AB.G.S; C.P.C and IM.M.P; The first draft of the manuscript was written by A.C.B, M.Z.C and M.S.S. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Paul M, Carrara E, Retamar P, Tängdén T, Bitterman R, Bonomo RA et al (2022) European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines for the treatment of infections caused by multidrug-resistant Gram-negative bacilli (endorsed by European society of intensive care medicine). Clin Microbiol Infect 28(4):521–547. 10.1016/j.cmi.2021.11.025 Yıldız SS, Kaşkatepe B, Avcıküçük H, Öztürk Ş (2017) Performance of CarbaNP and CIM tests in OXA-48 carbapenemase-producing Enterobacteriaceae. Acta Microbiol Immunol Hung 64(1):9–16. 10.1556/030.64.2017.009 El Kettani A, Maaloum F, Nzoyikorera N, Khalis M, Katfy K, Belabbes H et al (2021) Evaluation of the Performances of the Rapid Test RESIST-5 O.O.K.N.V Used for the Detection of Carbapenemases-Producing Enterobacterales. Antibiotics 10(8):953. 10.3390/antibiotics10080953 Jing X, Zhou H, Min X, Zhang X, Yang Q, Du S et al (2018) The Simplified Carbapenem Inactivation Method (sCIM) for Simple and Accurate Detection of Carbapenemase-Producing Gram-Negative Bacilli. Front Microbiol 9:2391. 10.3389/fmicb.2018.02391 Hosoda T, Doi Y, Suzuki M (2021) Comparison of sCIM and Other Phenotypic Detection Methods for Carbapenemase-Producing Enterobacterales. She RC, editor. Microbiol Spectr. ;9(3):e01608-21. 10.1128/Spectrum.01608-21 Schaffarczyk L, Noster J, Stelzer Y, Sattler J, Gatermann S, Hamprecht A (2024) Detection of rare carbapenemases in Enterobacterales—comparison of two colorimetric and three CIM-based carbapenemase assays. Babiker A, editor. Microbiol Spectr. ;12(2):e03015-23. 10.1128/spectrum.03015-23 Gu D, Yan Z, Cai C, Li J, Zhang Y, Wu Y et al (2023) Comparison of the NG-Test Carba 5, Colloidal Gold Immunoassay (CGI) Test, and Xpert Carba-R for the Rapid Detection of Carbapenemases in Carbapenemase-Producing Organisms. Antibiotics 12(2):300. 10.3390/antibiotics12020300 Yamada K, Sasaki M, Murakami H, Aoki K, Morita T, Ishii Y et al (2021) Evaluation of the simplified carbapenem inactivation method as a phenotypic detection method for carbapenemase-producing Enterobacterales. J Microbiol Methods 187:106273. 10.1016/j.mimet.2021.106273 Hamprecht A, Sattler J, Noster J, Stelzer Y, Fuchs F, Dorth V et al (2023) Proteus mirabilis – analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection. Clin Microbiol Infect 29(9):1198. 10.1016/j.cmi.2023.05.032 Wan D, Jing X, Zhou H, Min X, Zhang X, Wu T et al (2020) Differences between meropenem and imipenem disk to detect carbapenemase in gram-negative bacilli using simplified carbapenem inactivation method. J Infect Chemother 26(6):636–639. 10.1016/j.jiac.2020.02.012 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 15 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9430307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629141445,"identity":"742be887-bf44-4b33-bec9-826b73cdf80d","order_by":0,"name":"Ana Collazos Blanco","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYHACxgMPQJQEcwOIkgMREBE84EACWAsjWIsxQoRYLYlgEp8W/vbDBw4k1NTJyc9ubPxcUGOTPj/s8EOgIXZyug3YtUicSUs4kHCMzdjgzsFm6RnH0nI33k4zAGpJNjY7gMOaGzxABWw8iRskEhukeRsO526cnQDSciBxGw4t8mAt/yQS589IbP4N1JJuODv9A14tBiAtiW0GiQ03EttAtiTIS+fgt8UQ5JfEvgSQX9qseY6lGW6Qzik4kGCA2y9yxw8ffPDhGyjEmg/f5qmxkZefnb75w4cKOzmc3sd0KlilAbHKQUC+gRTVo2AUjIJRMBIAAPuHaAkFInszAAAAAElFTkSuQmCC","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Collazos","lastName":"Blanco","suffix":""},{"id":629141446,"identity":"7d18bf10-1e64-419e-80a8-229baae1ca68","order_by":1,"name":"Ana Belén García Saéz","email":"","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Belén García","lastName":"Saéz","suffix":""},{"id":629141447,"identity":"48b2bb45-8e80-424a-b3d8-48c4a052e2a1","order_by":2,"name":"Inés María Marcos Palomino","email":"","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":false,"prefix":"","firstName":"Inés","middleName":"María Marcos","lastName":"Palomino","suffix":""},{"id":629141448,"identity":"baccb3a5-04fe-4330-aa28-72ebc56a01e7","order_by":3,"name":"Carolina Plaza Cristobal","email":"","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"Plaza","lastName":"Cristobal","suffix":""},{"id":629141449,"identity":"d467d4c4-185f-4c54-b137-772799e6dba9","order_by":4,"name":"María Isabel Zamora Cintas","email":"","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Isabel Zamora","lastName":"Cintas","suffix":""},{"id":629141450,"identity":"11bf9300-4a9f-45fc-8f38-540c756c3102","order_by":5,"name":"María Simón Sacristán","email":"","orcid":"","institution":"Hospital Central de la Defensa Gómez Ulla","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Simón","lastName":"Sacristán","suffix":""}],"badges":[],"createdAt":"2026-04-15 18:25:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9430307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9430307/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107900191,"identity":"493c73a2-5556-445d-9ca5-d1cd27675321","added_by":"auto","created_at":"2026-04-27 11:27:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113956,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9430307/v1/98025c128d60cf6aa4c70fbb.png"},{"id":108803713,"identity":"3671472a-552d-442b-957f-d3a4ebadc365","added_by":"auto","created_at":"2026-05-08 15:04:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":310648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9430307/v1/d7da15c9-e641-4de4-bbb9-b6c4c79988a8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSimplified Carbapenem Inactivation Method (sCIM) as a Reliable Tool for Detecting OXA-48- Producing Enterobacterales in Clinical Practice\u003c/p\u003e","fulltext":[{"header":"Importance","content":"\u003cp\u003eThe rapid detection of OXA-48-producing Enterobacterales remains a major diagnostic challenge due to their frequently low carbapenem MICs and subtle phenotypic expression. This study demonstrates that sCIM provides a simple, reliable, and highly sensitive method for detecting these isolates, even in cases with borderline susceptibility profiles. Given its low cost and ease of implementation, sCIM represents a valuable tool for routine clinical laboratories, particularly in high-prevalence or resource-limited settings.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCarbapenem-resistant Enterobacterales (CRE) are among the highest-priority antimicrobial-resistant pathogens, being associated with limited therapeutic options and poor clinical outcomes(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Resistance to carbapenems is most commonly mediated by carbapenemases, which are classified into Ambler classes A, B, and D according to their molecular structure. Among these, carbapenemase-producing Enterobacterales (CPE) represent a major clinical concern due to their high potential for transmission between patients. In addition, carbapenemase genes are frequently located on mobile genetic elements such as plasmids, facilitating horizontal gene transfer and contributing to multispecies outbreaks. Therefore, rapid and accurate detection of CPE in clinical laboratories is critical for infection control and appropriate antimicrobial therapy.\u003c/p\u003e \u003cp\u003eSeveral phenotypic methods are currently available for the detection of CPE. The Carba NP test (bioM\u0026eacute;rieux) allows rapid identification of carbapenemase activity within 30 minutes by detecting pH changes (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In addition, immunochromatographic assays such as NG-Test Carba 5 (NG Biotech) and RESIST-5 O.K.N.V.I (Coris) provide high sensitivity and specificity, enabling the detection of the most common carbapenemase families within 15 minutes (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eClinical and Laboratory Standards Institute (CLSI)\u003c/em\u003e and European Committee on Antimicrobial Susceptibility Testing \u003cem\u003e(EUCAST\u003c/em\u003e) introduced the modified carbapenem inactivation method (mCIM), based on the original carbapenem inactivation method (CIM). Although mCIM is reliable for detecting a wide range of carbapenemases, its requirement for prolonged incubation limits its practicality in routine laboratory workflows. To overcome these limitations, the simplified carbapenem inactivation method (sCIM) has been proposed, eliminating the broth incubation step and offering a faster and more practical alternative (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eMost validation studies of these assays have focused on isolates producing the most prevalent carbapenemases, such as KPC, VIM, and NDM. However, data on OXA-48-like carbapenemases remain limited. This is particularly relevant as OXA-48 enzymes often confer low-level resistance and may be more difficult to detect using conventional phenotypic methods, potentially leading to underdiagnosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study was to evaluate the susceptivity of the sCIM method for the detection of OXA-48-producing CPE isolates.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain collection\u003c/h2\u003e \u003cp\u003eA total of 150 non-duplicate clinical isolates exhibiting decreased susceptibility to carbapenems (defined as reduced susceptibility to at least one of ertapenem, imipenem, or meropenem) were included in this study. All isolates were confirmed as OXA-48-producing carbapenemase-producing Enterobacterales (CPE) and were recovered from patients during routine clinical diagnostics at Hospital Central de la Defensa \u0026ldquo;G\u0026oacute;mez-Ulla\u0026rdquo; (CSVE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSusceptibility testing and MIC determination\u003c/h3\u003e\n\u003cp\u003eAll isolates were identified by matrix-assisted laser desorption ionization\u0026ndash;time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany). Antimicrobial susceptibility testing (AST) was performed by broth microdilution in accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, using the MicroScan WalkAway Plus system (Beckman Coulter). Quality control was performed in each experimental setting using \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ATCC 700603. Carbapenemase production was determined using the RESIST-5 O.K.N.V.I immunochromatographic assay (Coris) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and the Xpert\u0026reg; Carba-R assay (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSimplified carbapenem inactivation method (sCIM)\u003c/h3\u003e\n\u003cp\u003eThe simplified carbapenem inactivation method (sCIM) was performed as previously described, based on the modified carbapenem inactivation method (mCIM)(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). A 0.5 McFarland suspension of \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, prepared by the direct colony suspension method, was inoculated onto M\u0026uuml;ller\u0026ndash;Hinton agar (MHA) plates according to standard disk diffusion procedures and allowed to dry for 3\u0026ndash;10 min. One to three colonies of each test isolate grown on blood agar were then smeared onto one side of an imipenem disk (10 \u0026micro;g) to achieve uniform coverage. The inoculated side of the disk was immediately placed onto the previously inoculated MHA plate. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ATCC BAA-1705 (KPC producer) and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ATCC 700603 were used as positive and negative controls, respectively. Plates were incubated at 35\u0026deg;C for 16\u0026ndash;18 h in ambient air.\u003c/p\u003e \u003cp\u003eCarbapenemase-producing isolates hydrolyze imipenem, thereby allowing growth of the susceptible indicator strain around the imipenem disk. The presence of an inhibition zone measuring 6\u0026ndash;20 mm in diameter, or the observation of satellite growth of \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 around the disk, was interpreted as a positive result for carbapenemase production. An inhibition zone of \u0026ge;\u0026thinsp;26 mm was interpreted as negative, whereas a zone of 20\u0026ndash;25 mm was considered indeterminate.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAll 150 isolates were collected at our institution and comprised 120 \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (80.0%), 13 \u003cem\u003eEscherichia coli\u003c/em\u003e (8.7%), 10 \u003cem\u003eProteus mirabilis\u003c/em\u003e (6.7%), 4 \u003cem\u003eCitrobacter freundii\u003c/em\u003e (2.7%), 2 \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (1.3%), and 1 \u003cem\u003eMorganella morganii\u003c/em\u003e (0.7%).\u003c/p\u003e \u003cp\u003eIsolates were recovered from a variety of clinical specimens, including urine (n\u0026thinsp;=\u0026thinsp;90), surveillance samples (n\u0026thinsp;=\u0026thinsp;30), respiratory samples (n\u0026thinsp;=\u0026thinsp;13), wounds (n\u0026thinsp;=\u0026thinsp;9), and abscesses (n\u0026thinsp;=\u0026thinsp;8). All isolates exhibited decreased susceptibility to β-lactams, including carbapenems. High rates of resistance were observed for cefepime (87.3%) and ciprofloxacin (88.6%), suggesting the presence of extended-spectrum β-lactamases (ESBLs). In contrast, ceftazidime/avibactam showed high activity, with 98.0% of isolates remaining susceptible. Results for the three most prevalent species are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial susceptibility profile of the most frequently isolated Enterobacterales species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecie strain (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntimicrobial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber and % of isolates Susceptible (S) or Susceptible Increase Exposure (I)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e (120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e108 (90.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53 (44.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (5.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (5.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78 (65.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90 (75.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftazidime/avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e119 (99.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftolozane /tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18(15.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfametoxazol/trimetoprim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45 (37.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e (13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13 (100.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9 (69.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (46.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (46.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11(84.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13 (100.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftazidime/avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13 (100.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftolozane /tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11(84.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfametoxazol/trimetoprim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9(69.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cem\u003eP.mirabilis\u003c/em\u003e (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8 (80.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8 (80.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4 (40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5 (50.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftazidime/avibactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10 (100.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftolozane /tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (60.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfametoxazol/trimetoprim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUsing the sCIM method, all 150 OXA-48-producing isolates were correctly identified, yielding a sensitivity of 100% (95% CI: 97.6\u0026ndash;100%) according to predefined cutoff criteria. Two isolates were identified as co-producers of KPC and NDM carbapenemases respectively. No carbapenemase-negative isolates were included; therefore, specificity could not be assessed.\u003c/p\u003e \u003cp\u003eMost isolates exhibited complete absence of an inhibition zone (0 mm). In contrast, isolates belonging to the \u003cem\u003eMorganellaceae\u003c/em\u003e family displayed atypical inhibition patterns, characterized by a double-zone appearance consisting of an inner growth zone, likely associated with swarming behavior, and an outer clear zone. Despite this atypical pattern, inhibition diameters remained below the established cutoff values (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThe influence of bacterial inoculum size on sCIM performance was assessed by testing different inoculum conditions (one, two, three, and five colonies). No significant differences in inhibition zone diameters were observed among the different inoculum sizes, indicating that the method is robust to variations in bacterial load.\u003c/p\u003e \u003cp\u003eAdditionally, no significant differences in inhibition zone diameters were observed among isolates classified as resistant, susceptible, or susceptible with increased exposure to carbapenems.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that the simplified carbapenem inactivation method (sCIM) is a highly sensitive approach for the detection of OXA-48 carbapenemase producing Enterobacterales (CPE), achieving 100% sensitivity in our isolate collection, with no indeterminate results. These findings support the reliability of sCIM as a practical alternative to more time-consuming phenotypic methods. Notably, to our knowledge, this represents one of the largest evaluations specifically focused on OXA-48-producing isolates, addressing a relevant gap in the current literature.\u003c/p\u003e \u003cp\u003eGiven the clinical and epidemiological importance of carbapenem-resistant Enterobacterales (CRE), rapid and accurate detection methods are essential for guiding antimicrobial therapy and implementing effective infection control measures (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). While polymerase chain reaction (PCR) remains the reference standard for carbapenemase identification, immunochromatographic assays provide a rapid and cost-effective alternative without requiring specialized laboratory infrastructure (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe modified carbapenem inactivation method (mCIM), currently recommended for phenotypic detection, demonstrated high sensitivity in initial validation studies (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, subsequent reports have highlighted false-negative results, particularly in the detection of metallo-β-lactamases (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In addition, mCIM presents practical limitations, including the need for a broth incubation step followed by overnight incubation, which may delay result reporting.\u003c/p\u003e \u003cp\u003eThe simplified carbapenem inactivation method (sCIM), developed to address these limitations, eliminates the 4\u0026ndash;5 h broth incubation step and facilitates integration into routine laboratory workflows (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Previous studies have demonstrated good performance of sCIM, particularly for metallo-β-lactamases, however, most validation studies have included only a limited number of OXA-48-producing isolates, leaving an important gap in the evaluation of this method for this clinically relevant carbapenemase (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this context, our results are consistent with those reported by Schaffarczyk et al., who observed 100% sensitivity across a large and diverse isolate collection (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Conversely, Hosoda et al. reported substantially lower sensitivity (54.9%) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), likely reflecting differences in carbapenemase distribution. While our study focused exclusively on OXA-48 producers, their cohort predominantly included metallo-β-lactamase-producing isolates, with all false-negative results associated with IMP-1.\u003c/p\u003e \u003cp\u003eMethodological factors such as bacterial inoculum have also been suggested to influence sCIM performance (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, in our study, no significant differences were observed when varying inoculum size, supporting the robustness of the method under routine conditions.\u003c/p\u003e \u003cp\u003eSimilarly, the absence of indeterminate results in our cohort contrasts with previous reports (6\u0026ndash;36%) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This discrepancy may be explained by differences in enzyme hydrolytic activity. For instance, Hamprecht et al. described \u003cem\u003eProteus mirabilis\u003c/em\u003e isolates harboring carbapenemases with weak activity, such as OXA-23 and OXA-58, which may contribute to indeterminate phenotypes (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, none of the isolates in our study yielded indeterminate results, including those with low hydrolytic activity and MIC values within EUCAST susceptibility breakpoints. This finding highlights the ability of sCIM to reliably detect carbapenemase production even in isolates with borderline phenotypic profiles.\u003c/p\u003e \u003cp\u003eAn additional observation was the atypical inhibition pattern in \u003cem\u003eMorganellaceae\u003c/em\u003e strains, characterized by relatively large inhibition zones despite confirmed carbapenemase production but with inhibition diameters remained below the established cutoff values. This may be related to the species-specific characteristics such as swarming, and should be considered when interpreting results. This observation may represent a relevant practical consideration for laboratories applying sCIM in routine settings.\u003c/p\u003e \u003cp\u003eThe \u0026ldquo;satellite phenomenon\u0026rdquo; described by Wan et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) provides a mechanistic explanation based on limited enzyme diffusion and local antibiotic degradation. Although previously reported, this phenomenon was not observed in our cohort, suggesting that its occurrence may depend on specific bacterial or enzymatic factors.\u003c/p\u003e \u003cp\u003eDetection of carbapenemases in \u003cem\u003eProteus mirabilis\u003c/em\u003e remains particularly challenging due to low MIC values despite enzyme production (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Notably, all \u003cem\u003eP. mirabilis\u003c/em\u003e isolates in our study were correctly identified by sCIM, including those within the susceptible range, further supporting the sensitivity of the method in clinically challenging scenarios.\u003c/p\u003e \u003cp\u003eAlthough both meropenem and imipenem disks have been used for sCIM, imipenem has been associated with improved sensitivity in certain organisms (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Based on this, imipenem disks were selected in our study.\u003c/p\u003e \u003cp\u003eThis study has some limitations. First, it was conducted using isolates from a single geographic setting, which may limit the generalizability of the findings. Second, sCIM lacks strict standardization of bacterial inoculum, potentially introducing operator-dependent variability.\u003c/p\u003e \u003cp\u003eDespite these limitations, our study includes a large and well-characterized collection of OXA-48-producing isolates, a group for which data remain limited. Furthermore, all experiments were performed in duplicate, supporting the reproducibility of the method under routine clinical laboratory conditions.\u003c/p\u003e \u003cp\u003eFrom a clinical perspective, sCIM represents a rapid, cost-effective, and reliable method for detecting OXA-48-producing isolates. By eliminating the incubation step required for mCIM, it offers a practical alternative that can significantly improve workflow efficiency. These findings support the use of sCIM as a frontline phenotypic screening tool in clinical microbiology laboratories, particularly in settings with high OXA-48 prevalence and limited access to molecular or immunochromatographic methods.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by A.C.B, A.; AB.G.S; C.P.C and IM.M.P; The first draft of the manuscript was written by A.C.B, M.Z.C and M.S.S. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePaul M, Carrara E, Retamar P, T\u0026auml;ngd\u0026eacute;n T, Bitterman R, Bonomo RA et al (2022) European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines for the treatment of infections caused by multidrug-resistant Gram-negative bacilli (endorsed by European society of intensive care medicine). Clin Microbiol Infect 28(4):521\u0026ndash;547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmi.2021.11.025\u003c/span\u003e\u003cspan address=\"10.1016/j.cmi.2021.11.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYıldız SS, Kaşkatepe B, Avcık\u0026uuml;\u0026ccedil;\u0026uuml;k H, \u0026Ouml;zt\u0026uuml;rk Ş (2017) Performance of CarbaNP and CIM tests in OXA-48 carbapenemase-producing Enterobacteriaceae. Acta Microbiol Immunol Hung 64(1):9\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1556/030.64.2017.009\u003c/span\u003e\u003cspan address=\"10.1556/030.64.2017.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl Kettani A, Maaloum F, Nzoyikorera N, Khalis M, Katfy K, Belabbes H et al (2021) Evaluation of the Performances of the Rapid Test RESIST-5 O.O.K.N.V Used for the Detection of Carbapenemases-Producing Enterobacterales. Antibiotics 10(8):953. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antibiotics10080953\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics10080953\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing X, Zhou H, Min X, Zhang X, Yang Q, Du S et al (2018) The Simplified Carbapenem Inactivation Method (sCIM) for Simple and Accurate Detection of Carbapenemase-Producing Gram-Negative Bacilli. Front Microbiol 9:2391. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2018.02391\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2018.02391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHosoda T, Doi Y, Suzuki M (2021) Comparison of sCIM and Other Phenotypic Detection Methods for Carbapenemase-Producing Enterobacterales. She RC, editor. Microbiol Spectr. ;9(3):e01608-21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/Spectrum.01608-21\u003c/span\u003e\u003cspan address=\"10.1128/Spectrum.01608-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaffarczyk L, Noster J, Stelzer Y, Sattler J, Gatermann S, Hamprecht A (2024) Detection of rare carbapenemases in Enterobacterales\u0026mdash;comparison of two colorimetric and three CIM-based carbapenemase assays. Babiker A, editor. Microbiol Spectr. ;12(2):e03015-23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/spectrum.03015-23\u003c/span\u003e\u003cspan address=\"10.1128/spectrum.03015-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu D, Yan Z, Cai C, Li J, Zhang Y, Wu Y et al (2023) Comparison of the NG-Test Carba 5, Colloidal Gold Immunoassay (CGI) Test, and Xpert Carba-R for the Rapid Detection of Carbapenemases in Carbapenemase-Producing Organisms. Antibiotics 12(2):300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antibiotics12020300\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics12020300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamada K, Sasaki M, Murakami H, Aoki K, Morita T, Ishii Y et al (2021) Evaluation of the simplified carbapenem inactivation method as a phenotypic detection method for carbapenemase-producing Enterobacterales. J Microbiol Methods 187:106273. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mimet.2021.106273\u003c/span\u003e\u003cspan address=\"10.1016/j.mimet.2021.106273\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamprecht A, Sattler J, Noster J, Stelzer Y, Fuchs F, Dorth V et al (2023) Proteus mirabilis \u0026ndash; analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection. Clin Microbiol Infect 29(9):1198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmi.2023.05.032\u003c/span\u003e\u003cspan address=\"10.1016/j.cmi.2023.05.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan D, Jing X, Zhou H, Min X, Zhang X, Wu T et al (2020) Differences between meropenem and imipenem disk to detect carbapenemase in gram-negative bacilli using simplified carbapenem inactivation method. J Infect Chemother 26(6):636\u0026ndash;639. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jiac.2020.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.jiac.2020.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"sCIM, carbapenemase, OXA-48","lastPublishedDoi":"10.21203/rs.3.rs-9430307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9430307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbapenemase-producing Enterobacterales (CPE) are among the most concerning antimicrobial-resistant pathogens due to their limited treatment options and the potential for widespread transmission. This study aimed to evaluate the sensitivity of the simplified carbapenem inactivation method (sCIM) for detecting OXA-48-producing CPE isolates. A total of 150 non-duplicated CPE strains, were collected from clinical specimens at the Hospital Central de la Defensa “Gómez-Ulla” in Spain. The isolates were confirmed as OXA-48 producers using immunochromatographic assays or PCR-based methods. Antimicrobial susceptibility was assessed, and the sCIM was compared to standard diagnostic methods. Results showed that the sCIM method successfully detected all 150 OXA-48-producing strains, with no indeterminate results, offering a sensitivity of 100% (95% CI: 97.6–100%).. The method provided a rapid and easy-to-interpret result within 16–18 hours. The study suggests that sCIM is a reliable, simplified alternative to traditional carbapenemase detection methods, especially in regions with high prevalence of OXA-48 carbapenemase-producing enterobacterales.\u003c/p\u003e","manuscriptTitle":"Simplified Carbapenem Inactivation Method (sCIM) as a Reliable Tool for Detecting OXA-48- Producing Enterobacterales in Clinical Practice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 11:27:22","doi":"10.21203/rs.3.rs-9430307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T08:03:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T17:04:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106326784801855342283887148471525629668","date":"2026-04-24T16:29:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T07:45:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10346864950745002407875598691129217955","date":"2026-04-24T02:03:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300395196893673346742205826451275599781","date":"2026-04-21T11:07:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336800233476675162310514653427485958883","date":"2026-04-21T07:38:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"295946863973518617857385610157989689633","date":"2026-04-19T15:42:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-19T07:30:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T02:00:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T02:00:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","date":"2026-04-15T18:08:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"effa24ba-89a0-4f88-90e4-816bbd0aba46","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T08:03:47+00:00","index":68,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T17:04:22+00:00","index":67,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T11:27:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 11:27:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9430307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9430307","identity":"rs-9430307","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}