AmpCENTA: A rapid assay for detecting AmpC-hyperproduction in Enterobacter spp. and Klebsiella aerogenes

AmpCENTA: A rapid assay for detecting AmpC-hyperproduction in Enterobacter spp. and Klebsiella aerogenes | 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 AmpCENTA: A rapid assay for detecting AmpC-hyperproduction in Enterobacter spp. and Klebsiella aerogenes Paula Gómez Estévez, Marta Rodríguez-Rodríguez, Javier Dorado Pardo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9212347/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Early identification of AmpC-hyperproducing clinical isolates is crucial for optimizing empirical antibiotic therapy and preventing the use of inappropriate antibiotics. For this reason, a visual colorimetric method, namely AmpCENTA, was developed to detect AmpC hyperproduction in Enterobacter spp. and Klebsiella aerogenes within 30 minutes. The performance of AmpCENTA was evaluated by using 133 clinical isolates, including 76 AmpC hyperproducers (34 Enterobacter spp. and 42 K. aerogenes ), 27 non-AmpC hyperproducers (22 Enterobacter spp. and 5 K. aerogenes ), and 30 producers of β-lactamases other than AmpC (3 Enterobacter spp., 18 Escherichia coli , and 9 K. pneumoniae ). The AmpCENTA showed a sensitivity of 98.7% (n = 75/76 AmpC hyperproducers) and a specificity of 100% (27/27 non-AmpC hyperproducers). Moreover, the method did not show any cross-reaction with any of the 30 isolated producing β-lactamase other than AmpC. This method constitutes a diagnostic tool that enables rapid, reliable, and low-cost detection of AmpC β-lactamase hyperproduction in Enterobacter spp. and K. aerogenes . Enterobacter spp. Klebsiella aerogenes AmpC CENTA rapid assay colorimetric resistance Figures Figure 1 Figure 2 BACKGROUND In the last years, Enterobacter spp. and Klebsiella aerogenes have acquired relevance among the microorganisms causing healthcare-related infections due to their ability to develop antimicrobial resistances and its association with the nosocomial infections ( 1 ). A multicentre prospective study found that 95.1% of Enterobacter spp. bacteraemia cases were healthcare-related, with inappropriate empirical antibiotic therapy in 41.1% of patients, a mean hospital stay of 34.3 days, and a crude 30-day mortality of 19.6%, which rose to 39.7% in other studies ( 1 – 3 ). The presence of an inducible chromosomal AmpC β-lactamase confers extended resistance to 3rd generation cephalosporins (such as ceftazidime, ceftriaxone, or cefotaxime) and piperacillin/tazobactam, when it is overexpressed, limiting the therapeutic options for patients ( 4 ). Cefepime and meropenem are the antibiotics of choice to treat infections by this kind of microorganisms due to their low susceptibility to hydrolysis by AmpC β-lactamases. ( 5 ). Although overall cefepime resistance rates in Enterobacter spp. and K. aerogenes are low (< 10%) ( 6 ), when only AmpC-hyperproducing isolates are considered, cefepime resistance increases to 15.6% ( 6 ). In contrast, meropenem resistance remains stable (< 1%). These findings highlight the need for a diagnostic test capable of distinguishing AmpC hyperproducers (constitutively derepressed isolates carrying irreversible mutations in regulatory genes such as ampD or ampR) from non-hyperproducers (isolates with inducible AmpC that may transiently develop reversible AmpC hyperproduction), enabling more targeted therapeutic decisions between cefepime and meropenem. Therefore, we have developed a rapid and cost-effective test, the AmpCENTA, for Enterobacter spp. and K. aerogenes , which might be used worldwide, regardless of the technical level of the laboratory. The AmpCENTA assay has been designed to detect AmpC hyperproduction and differentiate with those producing a β-lactamases other than AmpC based on the enzymatic hydrolysis of CENTA (chromogenic cephalosporin) and the use of cloxacillin as inhibitor. METHODS Bacterial isolates and resistance phenotype A total of 133 clinical isolates including 76 AmpC-hyperproducers (34 Enterobacter spp. and 42 K. aerogenes ), 27 non-AmpC hyperproducers (22 Enterobacter spp. and 5 K. aerogenes ) and 30 β-lactamase ≠ AmpC producers (3 Enterobacter spp., 18 Escherichia coli , and 9 Klebsiella pneumoniae ), collected from different types of clinical samples (abscesses, wound exudates, blood, sputum, bronchoalveolar lavages, tracheobronchial aspirates, and urine) at the University Hospital Virgen del Rocío (Seville, Spain) were used in the development and evaluation of the AmpCENTA test. Isolate identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) (Bruker, Germany). The initial susceptibility profile was determined using the semiautomated Microscan WalkAway system (Beckman Coulter, USA), following the EUCAST 2025 clinical breakpoints. Those isolates resistant to third-generation cephalosporins (ceftazidime and/or cefotaxime) were later tested for AmpC hyperproduction. This resistance mechanism was phenotypically confirmed using a cloxacillin disc and detecting synergy with cefotaxime or ceftazidime, in accordance with EUCAST recommendations ( 7 ). This method was designated as the gold standard. Nevertheless, due to the low proportion of clinical isolates of Enterobacter spp. and K. aerogenes producing β-lactamases other than AmpC, clinical isolates of E. coli and K. pneumoniae producing extended spectrum β-lactamases (ESBL) were also included, with the aim of demonstrating how the test would perform in the presence of β-lactamase-producing isolates unrelated to AmpC. Bacterial preparation To identify hyperproduction or non-hyperproduction of AmpC, as well as the production of β-lactamases distinct from AmpC, two different experimental conditions were evaluated based on the type of inoculum. The first use bacterial biomass collected with a sterile 1-µL loop from a culture in agar plate, while the second used 40 µL of a bacterial suspension adjusted to 0.5 McFarland standard. The experimental condition using 0.5 McFarland bacterial suspension enabled the differentiation between AmpC hyperproducing and non-hyperproducing isolates. However, isolates producing β-lactamases other than AmpC could not be detected under low inoculum conditions (0.5 McFarland). Therefore, the assay was extended to include a high-inoculum condition using bacterial biomass. AmpCENTA test and interpretation of the result For the assay, two wells were prepared for each type of inoculum (low and high) as followed: 100 µL of the protein extraction reagent (B-PER; Thermo Scientific, Massachusetts, USA) and other 100 µL of B-PER supplemented with cloxacillin (0.1 mg/mL) (Reig Jofre, Sant Joan Despí, Barcelona, Spain) were added into two different wells. Then, biomass collected with a sterile 1-µL loop from an agar plate, and 40 µL of a bacterial suspension adjusted to 0.5 McFarland were added to their respective wells. Following a 5-minute room temperature incubation, 30 µL of CENTA solution (1 mg/mL) was added (Fig. 1 A). Finally, the test is incubated for 30 minutes at room temperature. During this period, hydrolysis of CENTA by AmpC leads to a measurable color change from pale yellow to bright yellow. The addition of cloxacillin, which suppresses the color change by inhibiting AmpC activity confirms that the observed activity is attributable to AmpC. After incubation, the results are interpreted: wells A and B (bacterial biomass) are used to identify isolates producing β-lactamases other than AmpC, while wells C and D (0.5 McFarland) allow the differentiation between AmpC hyperproducers and non-hyperproducers isolates. Thus, the appearance of yellow coloration in wells A and B indicates the production of β-lactamases other than AmpC. If the first well turns yellow while the second well does not, the isolate could correspond to either AmpC hyperproducer or non-hyperproducer isolate. To confirm the result, wells C and D are analyzed. The appearance of yellow coloration in well C indicates AmpC-hyperproduction. Conversely, the absence of yellow coloration in well C indicates that the isolate has not an hyperproduction of AmpC (Fig. 1 A, 1 B and 1 D). For automation purposes, the reaction mixture color change was also followed by measuring absorbance at 405 nm, which is the absorption maximum of the hydrolyzed form of CENTA, using a Multiskan microplate reader (Thermo Fisher Scientific). Measurements were recorded at time 0 and subsequently every 5 minutes for a total duration of 30 minutes. The increase in absorbance (ΔAbs) was calculated as the difference in absorbance between wells A vs B (for high inoculum) and C vs D (for low inoculum) (ΔAbs = Abs cloxacilina− - Abs cloxacilina+ ). At a high inoculum, after 10 minutes of incubation, it is possible to distinguish between AmpC hyperproducers/non-hyperproducers and isolates producing β-lactamases other than AmpC. AmpC hyperproducers and non-AmpC hyperproducers showed a high ΔAbs value, as well A turned yellow while well B remained pale yellow. However, isolates producing β-lactamases other than AmpC showed a low ΔAbs value, since both wells A and B turned yellow, resulting in similarly high absorbance values in both cases (Fig. 2 A). At a low inoculum, after 5 minutes of incubation, a significant difference in ΔAbs was observed between AmpC hyperproducing and non-hyperproducing isolates. Non-hyperproducing isolates exhibited low and similar absorbance values in wells C and D, whereas AmpC hyperproducing isolates showed high absorbance in well C and a low absorbance in well D, resulting in a high ΔAbs value (Fig. 2 A). Relative expression of ampC In order to quantify the expression of the ampC gene ( bla ACT ), total RNA from 15 AmpC-hyperproducing and 14 non-hyperproducing isolates were extracted using the RNeasy Mini Kit (Qiagen, Germany), followed by quantitative real-time PCR (RT-qPCR). The RT-qPCR assay was performed using the One-Step NZYSpeedy RT-qPCR Green kit (NZYtech, Portugal). The following primers for the bla ACT gene, targeting conserved regions of this gene, and for the housekeeping rpoB gene were used, respectively: bla ACT (Fw: 5’-CGG ATG AGG TCA CGG ATA AC-3’; Rv: 5’-TGG CGT TGG CGT AAA GA-3’) ( 8 ) and rpoB (Fw: 5’-GAT CAA CTC CCT GTC CGT GT-3’; Rv: 5’-GAG TTC GCC TGA GCG ATA AC-3’) (this study). The obtained cycle threshold (Ct) values were analyzed by the 2 −ΔΔCT method (Fold change) ( 9 ). Relative expression levels were calculated by comparison with the sample with the lower level of expression. Determination of the MIC profiles The susceptibility of the isolates to ceftazidime was assessed by broth microdilution (MHB) following the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Clinical breakpoints were determined according to the EUCAST guidelines (7). The E. coli ATCC 25922 strain was used as a standard control. Statistical tests Sensitivity and specificity were determined from AmpCENTA results obtained for 133 clinical isolates, using cloxacillin synergy with cefotaxime or ceftazidime as the reference standard. Sensitivity was defined as the proportion of AmpC hyperproducers correctly identified, and specificity as the proportion of non-hyperproducers correctly classified. Additionally, 95% confidence intervals (CI), positive predictive value (PPV), and negative predictive value (NPV) were also calculated. Significant differences in expression levels and ΔAbs were analyzed by one-way analysis of variance (ANOVA) in GraphPad Prism 10 software. A p-value < 0.05 was considered statistically significant. RESULTS AND DISCUSSION The performance of AmpCENTA was assessed in 133 clinical isolates: 76 AmpC hyperproducers (34 Enterobacter spp. and 42 K. aerogenes ), 27 non-AmpC hyperproducers (22 Enterobacter spp. and 5 K. aerogenes ) and 30 ESBLs producers (3 Enterobacter spp., 18 Escherichia coli , and 9 Klebsiella pneumoniae ). Previously, all isolates were tested using the MicroScan system WalkAway and phenotypically confirmed using a cloxacillin diffusion disk and detecting synergy with cefotaxime or ceftazidime as the gold standard technique. Within the collection of isolates tested by the gold standard assay, 76 were AmpC hyperproducers, 27 were non-hyperproducers and 30 were β-lactamases-producers other than AmpC. The AmpCENTA showed a sensitivity of 98.7% (75/76 AmpC-hyperproducers (95% CI: 92.9–99.8) and a specificity of 100% (27/27 non-AmpC-hyperproducers) (95% CI: 87.5–100). The positive predictive value (PPV) was 100% (95% CI: 95.1–100), whereas the negative predictive value (NPV) was 96.4% (95% CI: 82.3–99.4). No false positives (0%) were observed (95% CI: 0–12.5), while the false-negative rate was 1.3% (95% CI: 0.2–7.1). Moreover, the method accurately detected all 30 isolated β-lactamase–producing strains that were different from AmpC. Although the rapid test was designed for visual reading, its automation was also explored by measuring absorbance at 405 nm and interpreting the results accordingly. As shown in Fig. 2 A, at high inoculum, both non-AmpC and AmpC hyperproducers showed high and comparable ΔAbs values after 30 minutes. However, the β-lactamase producers other than AmpC, maintained low ΔAbs values at high inoculum, which remained significantly different from those observed in both AmpC hyperproducers and non-hyperproducers. In this way, it is possible to differentiate and identify isolates producing β-lactamases other than AmpC. On the other hand, the low inoculum graph illustrates that ΔAbs was significantly higher in the AmpC-hyperproducing isolates than that observed in non-AmpC-hyperproducers from 5 minutes of incubation onwards. Although significant differences were already observable after 5 minutes of incubation, a total incubation time of 30 minutes is recommended, as some isolates require longer to hydrolyse CENTA (Fig. 2 A). Additionally, ampC gene ( bla ACT ) expression was quantified in a total of 29 isolates (15 AmpC-hyperproducers and 14 non-AmpC-hyperproducers). As shown in Fig. 2 B, significant differences were detected, allowing two large groups of ampC expression to be distinguished. Regarding the results, the isolates with higher ampC expression were those that also showed a positive result for AmpC hyperproduction. However, among the isolates with lower ampC expression, one isolate was previously recognised as AmpC-hyperproducer according to our gold standard, although it was detected as non-AmpC hyperproducer in the AmpCENTA test (Fig. 2 B). These results support the hypothesis that the isolate may possess a resistance mechanism different to AmpC, possibly involving reduced membrane permeability and/or efflux pumps that limits antibiotic entry. Furthermore, the minimum inhibitory concentration (MIC) for ceftazidime was correlated with the AmpC expression (2 −ΔΔCT method) observed in AmpC hyperproducing isolates and non-AmpC-hyperproducers (Fig. 2 C). As observed, higher AmpC expression is associated with increased MIC values, indicating that AmpC production directly contributes to elevated resistance levels. Automated technologies in antimicrobial susceptibility testing have replaced many conventional phenotypic methods, especially in the routine of clinical microbiology laboratories. A clear example is the use of the MicroScan WalkAway system, which is based on broth microdilution. However, these methods do not allow rapid susceptibility susceptibility testing, as antimicrobial susceptibility results are available after 16–18 hours ( 10 ). In the last years, rapid phenotypic methods such as the βLACTA test have been commercialized for the detection of extended spectrum β-lactamases; however, this assay is useful only for ESBLs and is not suitable for detecting AmpC hyperproduction ( 11 ). On the other hand, genotypic approaches, based on PCR, have also been developed. Nevertheless, in Enterobacter spp. and K. aerogenes these molecular methods are not appropriate for detecting AmpC hyperproduction, as they cannot differentiate basal production from true AmpC hyperproduction ( 12 ). AmpC β-lactamase remains challenging to identify, as there is currently no consensus on the most effective laboratory method for its detection. This highlights the need for the development of a reliable test capable of accurately detecting AmpC hyperproduction. The introduction of the AmpCENTA test into clinical practice would have clinical implications, being essential to establish an adequate antibiotic treatment (Fig. 1 C). CONCLUSIONS The AmpCENTA assay is a rapid, low-cost, and straightforward method that yields results within 30 minutes and demonstrates excellent sensitivity and specificity. The ability of AmpCENTA to quickly detect whether an isolate is an AmpC hyperproducer is essential for guiding appropriate empirical antibiotic treatment in infections caused by Enterobacter spp. and/or K. aerogenes , since the activity of cefepime may be compromised in AmpC-hyperproducing isolates. Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE The study was conducted in accordance with the Declaration of Helsinki. The bacterial isolates were obtained as part of the routine clinical diagnostic procedures and were fully anonymized prior to this study. The research protocol was approved by the Ethics Committee of the University Hospital Virgen del Rocío of Seville, Spain (approval number 0019-N-23). The consent to participate was not necessary due to this study did not involve human participants, human material, or animals. CONSENT FOR PUBLICATION Not required. This study did not involve human participants, human material, or animals. COMPETING INTERESTS The authors declare that they have no competing interests. AUTHOR´ CONTRIBUTIONS PGE: Formal analysis, Investigation, Methodology, Writing – original draft; MRR: Investigation, Methodology, Writing – review and editing; JDP: ; PDP: ; JMC: Resources, Supervision; JAL: Resources, Writing – review and editing; ARV: Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Resources Writing-review and editing, JMOR: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – review and editing. All authors read and approved the final manuscript. FUNDING This work was funded by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain, and co-funded by the European Union (PI22/01464, PI25/00217 and PI23/01760). PGE is supported by the Subprograme PFIS, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain, and co-funded by the European Union (FI24/00301). M.R.R is supported by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain, and co-funded by the European Union (PI22/01464). JMOR is supported by the Subprograme Miguel Servet, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain, and co-funded by the European Union (CP24/00137). A.R.V. is supported by the Subprograma Juan Rodés, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain, y co-funded by de European Union (JR20/00023) and by a Clinical Investigator contract (Acción B-Clínicos Investigadores) from the Servicio Andaluz de Salud/Consejería de Sanidad, Presidencia y Emergencias, Junta de Andalucía (B-0042-2020). Author Contribution PGE: Formal analysis, Investigation, Methodology, Writing – original draft; MRR: Investigation, Methodology, Writing – review and editing; JDP: ; PDP: ; JMC: Resources, Supervision; JAL: Resources, Writing – review and editing; ARV: Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Resources Writing-review and editing, JMOR: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – review and editing. All authors read and approved the final manuscript. ACKNOWLEDGEMENTS Not applicable. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Álvarez-Marín R, Navarro-Amuedo D, Gasch-Blasi O, et al. A prospective, multicenter case control study of risk factors for acquisition and mortality in Enterobacter species bacteremia. J Infect. 2020;80:174–81. Ye Y et al. Enterobacter bacteremia: clinical features, risk factors for multiresistance and mortality in a Chinese Universitary Hospital. Infection. 2006. Deal EN et al. Predictors of in-hospital mortality for bloodstream infections caused by Enterobacter species. Pharmacotherapy. 2007. Tamma PD, Doi Y, Bonomo RA, et al. Antibacterial Resistance Leadership Group. A Primer on AmpC β-Lactamases: Necessary Knowledge for an Increasingly Multidrug-resistant World. Clin Infect Dis. 2019;69:1446–55. Tamma PD, Aitken SL, Bonomo RA, et al. Infectious Diseases Society of America Guidance on the Treatment of AmpC β-Lactamase-Producing Enterobacterales, Carbapenem-Resistant Acinetobacter baumannii, and Stenotrophomonas maltophilia Infections. Clin Infect Dis. 2022;74:2089–114. Boattini M, et al. Enterobacterales carrying chromosomal AmpC β-lactamases in Europe (EuESCPM): Epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020–2022. Int J Antimicrob agents vol. 2024;63(5):107115. European Commitee on Antimicrobial Susceptibility Testing. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance 2017. David M, Livermore D, Jamrozy S, Mushtaq, Wright W, Nichols K, Young N, Woodford. AmpC β-lactamase induction by avibactam and relebactam. J Antimicrob Chemother Volume. December 2017;72(12):3342–8. Pfaffl MW. May, A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res, 29, Issue 9, 1 2001, Page 45. Salam MA, Al-Amin MY, Pawar JS, Akhter N. Irine Banu Lucy, Conventional methods and future trends in antimicrobial susceptibility testing. Saudi J Biol Sci Volume. 2023;30(3):103582. Morosini MI, García-Castillo M, Tato M, Gijón D, Valverde A, Ruiz-Garbajosa P, Cantón R. Rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae by use of the new chromogenic βLacta test. J Clin Microbiol. 2014;52(5):1741–4. Rodríguez-Guerrero E, Callejas-Rodelas JC, Navarro-Marí JM, Gutiérrez-Fernández J. Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC . Microorganisms. 2022;10(3):611. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9212347","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627455066,"identity":"e1395ae3-07b2-4dc5-b470-a0abf128d75e","order_by":0,"name":"Paula Gómez Estévez","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Gómez","lastName":"Estévez","suffix":""},{"id":627455069,"identity":"0239bbfb-b0a5-43df-a374-d08cb8875036","order_by":1,"name":"Marta Rodríguez-Rodríguez","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Rodríguez-Rodríguez","suffix":""},{"id":627455074,"identity":"4fa53c23-76c4-4db3-ad11-080d7188d8dd","order_by":2,"name":"Javier Dorado Pardo","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"Dorado","lastName":"Pardo","suffix":""},{"id":627455078,"identity":"7b8d8db7-cd22-49da-9432-cfac421644a1","order_by":3,"name":"Pilar Durán Parejo","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"Pilar","middleName":"Durán","lastName":"Parejo","suffix":""},{"id":627455083,"identity":"f00fd9d7-d2cc-487f-ad6a-e88a60faa016","order_by":4,"name":"José Miguel Cisneros","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Miguel","lastName":"Cisneros","suffix":""},{"id":627455085,"identity":"9dcbc1be-9c1b-4fc5-b59e-45e5c25442ba","order_by":5,"name":"José Manuel Ortiz de la Rosa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3QsUrEMBzH8V8IxOU0a0pBn0BoKRTE+iQuOYR26hscZ1ziou6+iDimBJwKXW/McS9wt+kiplVwauvokO8QSMiHPwkQCv3TDEBxAhB3UP2e3JlpQL8J80v6rCA8UbMEP4TFxwMZ5o7H465p3nF5yo4Mza5ei/X5vfVTVsX1GImebmAEqowtJNnVbSnydunJW1mrEZK0/i0J7FJD0qzWVuTGE6LsNJGwt5o7Fl/0pNv+gRhYyYRkMenJZmZK9ECTRqFKtXA0fdRl9LLxU+TEW/iC7A7+x844l8R96ILnXbV1+1UxSobI56D3vydy6nooFAqFZvsCI7FZzwgfs30AAAAASUVORK5CYII=","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":true,"prefix":"","firstName":"José","middleName":"Manuel Ortiz de la","lastName":"Rosa","suffix":""},{"id":627455087,"identity":"43fc71d7-1d27-4b84-bfad-b2b65a0cb02f","order_by":6,"name":"José Antonio Lepe","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Antonio","lastName":"Lepe","suffix":""},{"id":627455091,"identity":"f0293bd6-c1f3-43b3-bd67-582e8dbcff1d","order_by":7,"name":"Ángel Rodríguez-Villodres","email":"","orcid":"","institution":"University Hospital Virgen del Rocío","correspondingAuthor":false,"prefix":"","firstName":"Ángel","middleName":"","lastName":"Rodríguez-Villodres","suffix":""}],"badges":[],"createdAt":"2026-03-24 13:10:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9212347/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9212347/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109096826,"identity":"6027fd77-330d-4e57-befd-f4d0a9d6d47c","added_by":"auto","created_at":"2026-05-12 14:05:55","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10816862,"visible":true,"origin":"","legend":"\u003cp\u003eA) AmpCENTA Testing: Methodology and Interpretation. B) Possible results of the AmpCENTA test. C) Flowchart of the possible application in clinical practice. D) Algorithm for interpretation of AmpCENTA test results based on well color change.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9212347/v1/42545dea197a2b41afcf8d89.jpeg"},{"id":109096671,"identity":"082fda5b-35e4-4b42-a41e-3ec41411e4c5","added_by":"auto","created_at":"2026-05-12 14:04:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2090953,"visible":true,"origin":"","legend":"\u003cp\u003eA) Increase in absorbance (ΔAbs) at 405 nm over incubation time (0–30 minutes) for low and high inoculum conditions in AmpC hyperproducers, non-AmpC hyperproducers and β-lactamases other than AmpC. B) Relative expression of the \u003cem\u003eampC\u003c/em\u003e gene (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e). C) MIC and relative expression of the \u003cem\u003eampC\u003c/em\u003e gene in AmpC hyperproducers and non-AmpC hyperproducers. *, P ≤ 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9212347/v1/3ae34f16738f57fd71fd7958.png"},{"id":109097947,"identity":"e4b4bd1a-5b69-4ea2-8613-e6eeb8aac943","added_by":"auto","created_at":"2026-05-12 14:11:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12073381,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9212347/v1/af4a8f6f-75ff-4148-9c47-6ebd601f62c2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"AmpCENTA: A rapid assay for detecting AmpC-hyperproduction in Enterobacter spp. and Klebsiella aerogenes","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eIn the last years, \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e have acquired relevance among the microorganisms causing healthcare-related infections due to their ability to develop antimicrobial resistances and its association with the nosocomial infections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). A multicentre prospective study found that 95.1% of \u003cem\u003eEnterobacter spp.\u003c/em\u003e bacteraemia cases were healthcare-related, with inappropriate empirical antibiotic therapy in 41.1% of patients, a mean hospital stay of 34.3 days, and a crude 30-day mortality of 19.6%, which rose to 39.7% in other studies (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The presence of an inducible chromosomal AmpC β-lactamase confers extended resistance to 3rd generation cephalosporins (such as ceftazidime, ceftriaxone, or cefotaxime) and piperacillin/tazobactam, when it is overexpressed, limiting the therapeutic options for patients (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Cefepime and meropenem are the antibiotics of choice to treat infections by this kind of microorganisms due to their low susceptibility to hydrolysis by AmpC β-lactamases. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Although overall cefepime resistance rates in \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eK. aerogenes\u003c/em\u003e are low (\u0026lt;\u0026thinsp;10%) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), when only AmpC-hyperproducing isolates are considered, cefepime resistance increases to 15.6% (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In contrast, meropenem resistance remains stable (\u0026lt;\u0026thinsp;1%). These findings highlight the need for a diagnostic test capable of distinguishing AmpC hyperproducers (constitutively derepressed isolates carrying irreversible mutations in regulatory genes such as ampD or ampR) from non-hyperproducers (isolates with inducible AmpC that may transiently develop reversible AmpC hyperproduction), enabling more targeted therapeutic decisions between cefepime and meropenem. Therefore, we have developed a rapid and cost-effective test, the AmpCENTA, for \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eK. aerogenes\u003c/em\u003e, which might be used worldwide, regardless of the technical level of the laboratory. The AmpCENTA assay has been designed to detect AmpC hyperproduction and differentiate with those producing a β-lactamases other than AmpC based on the enzymatic hydrolysis of CENTA (chromogenic cephalosporin) and the use of cloxacillin as inhibitor.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates and resistance phenotype\u003c/h2\u003e \u003cp\u003eA total of 133 clinical isolates including 76 AmpC-hyperproducers (34 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 42 \u003cem\u003eK. aerogenes\u003c/em\u003e), 27 non-AmpC hyperproducers (22 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 5 \u003cem\u003eK. aerogenes\u003c/em\u003e) and 30 β-lactamase\u0026thinsp;\u0026ne;\u0026thinsp;AmpC producers (3 \u003cem\u003eEnterobacter\u003c/em\u003e spp., 18 \u003cem\u003eEscherichia coli\u003c/em\u003e, and 9 \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e), collected from different types of clinical samples (abscesses, wound exudates, blood, sputum, bronchoalveolar lavages, tracheobronchial aspirates, and urine) at the University Hospital Virgen del Roc\u0026iacute;o (Seville, Spain) were used in the development and evaluation of the AmpCENTA test. Isolate identification was carried out by matrix-assisted laser desorption ionization\u0026ndash;time of flight mass spectrometry (MALDI-TOF) (Bruker, Germany). The initial susceptibility profile was determined using the semiautomated Microscan WalkAway system (Beckman Coulter, USA), following the EUCAST 2025 clinical breakpoints. Those isolates resistant to third-generation cephalosporins (ceftazidime and/or cefotaxime) were later tested for AmpC hyperproduction. This resistance mechanism was phenotypically confirmed using a cloxacillin disc and detecting synergy with cefotaxime or ceftazidime, in accordance with EUCAST recommendations (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This method was designated as the gold standard.\u003c/p\u003e \u003cp\u003eNevertheless, due to the low proportion of clinical isolates of \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eK. aerogenes\u003c/em\u003e producing β-lactamases other than AmpC, clinical isolates of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e producing extended spectrum β-lactamases (ESBL) were also included, with the aim of demonstrating how the test would perform in the presence of β-lactamase-producing isolates unrelated to AmpC.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial preparation\u003c/h3\u003e\n\u003cp\u003eTo identify hyperproduction or non-hyperproduction of AmpC, as well as the production of β-lactamases distinct from AmpC, two different experimental conditions were evaluated based on the type of inoculum. The first use bacterial biomass collected with a sterile 1-\u0026micro;L loop from a culture in agar plate, while the second used 40 \u0026micro;L of a bacterial suspension adjusted to 0.5 McFarland standard. The experimental condition using 0.5 McFarland bacterial suspension enabled the differentiation between AmpC hyperproducing and non-hyperproducing isolates. However, isolates producing β-lactamases other than AmpC could not be detected under low inoculum conditions (0.5 McFarland). Therefore, the assay was extended to include a high-inoculum condition using bacterial biomass.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eAmpCENTA test and interpretation of the result\u003c/b\u003e\u003c/div\u003e \u003cp\u003eFor the assay, two wells were prepared for each type of inoculum (low and high) as followed: 100 \u0026micro;L of the protein extraction reagent (B-PER; Thermo Scientific, Massachusetts, USA) and other 100 \u0026micro;L of B-PER supplemented with cloxacillin (0.1 mg/mL) (Reig Jofre, Sant Joan Desp\u0026iacute;, Barcelona, Spain) were added into two different wells. Then, biomass collected with a sterile 1-\u0026micro;L loop from an agar plate, and 40 \u0026micro;L of a bacterial suspension adjusted to 0.5 McFarland were added to their respective wells. Following a 5-minute room temperature incubation, 30 \u0026micro;L of CENTA solution (1 mg/mL) was added (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Finally, the test is incubated for 30 minutes at room temperature. During this period, hydrolysis of CENTA by AmpC leads to a measurable color change from pale yellow to bright yellow. The addition of cloxacillin, which suppresses the color change by inhibiting AmpC activity confirms that the observed activity is attributable to AmpC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter incubation, the results are interpreted: wells A and B (bacterial biomass) are used to identify isolates producing β-lactamases other than AmpC, while wells C and D (0.5 McFarland) allow the differentiation between AmpC hyperproducers and non-hyperproducers isolates. Thus, the appearance of yellow coloration in wells A and B indicates the production of β-lactamases other than AmpC. If the first well turns yellow while the second well does not, the isolate could correspond to either AmpC hyperproducer or non-hyperproducer isolate. To confirm the result, wells C and D are analyzed. The appearance of yellow coloration in well C indicates AmpC-hyperproduction. Conversely, the absence of yellow coloration in well C indicates that the isolate has not an hyperproduction of AmpC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eFor automation purposes, the reaction mixture color change was also followed by measuring absorbance at 405 nm, which is the absorption maximum of the hydrolyzed form of CENTA, using a Multiskan microplate reader (Thermo Fisher Scientific). Measurements were recorded at time 0 and subsequently every 5 minutes for a total duration of 30 minutes. The increase in absorbance (ΔAbs) was calculated as the difference in absorbance between wells A vs B (for high inoculum) and C vs D (for low inoculum) (ΔAbs\u0026thinsp;=\u0026thinsp;Abs\u003csup\u003ecloxacilina\u0026minus;\u003c/sup\u003e - Abs\u003csup\u003ecloxacilina+\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eAt a high inoculum, after 10 minutes of incubation, it is possible to distinguish between AmpC hyperproducers/non-hyperproducers and isolates producing β-lactamases other than AmpC. AmpC hyperproducers and non-AmpC hyperproducers showed a high ΔAbs value, as well A turned yellow while well B remained pale yellow. However, isolates producing β-lactamases other than AmpC showed a low ΔAbs value, since both wells A and B turned yellow, resulting in similarly high absorbance values in both cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt a low inoculum, after 5 minutes of incubation, a significant difference in ΔAbs was observed between AmpC hyperproducing and non-hyperproducing isolates. Non-hyperproducing isolates exhibited low and similar absorbance values in wells C and D, whereas AmpC hyperproducing isolates showed high absorbance in well C and a low absorbance in well D, resulting in a high ΔAbs value (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRelative expression of\u003c/b\u003e \u003cb\u003eampC\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to quantify the expression of the \u003cem\u003eampC\u003c/em\u003e gene (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e), total RNA from 15 AmpC-hyperproducing and 14 non-hyperproducing isolates were extracted using the RNeasy Mini Kit (Qiagen, Germany), followed by quantitative real-time PCR (RT-qPCR). The RT-qPCR assay was performed using the One-Step NZYSpeedy RT-qPCR Green kit (NZYtech, Portugal). The following primers for the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e gene, targeting conserved regions of this gene, and for the housekeeping rpoB gene were used, respectively: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e (Fw: 5\u0026rsquo;-CGG ATG AGG TCA CGG ATA AC-3\u0026rsquo;; Rv: 5\u0026rsquo;-TGG CGT TGG CGT AAA GA-3\u0026rsquo;) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and \u003cem\u003erpoB\u003c/em\u003e (Fw: 5\u0026rsquo;-GAT CAA CTC CCT GTC CGT GT-3\u0026rsquo;; Rv: 5\u0026rsquo;-GAG TTC GCC TGA GCG ATA AC-3\u0026rsquo;) (this study). The obtained cycle threshold (Ct) values were analyzed by the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method (Fold change) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Relative expression levels were calculated by comparison with the sample with the lower level of expression.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDetermination of the MIC profiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe susceptibility of the isolates to ceftazidime was assessed by broth microdilution (MHB) following the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Clinical breakpoints were determined according to the EUCAST guidelines (7). The \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 strain was used as a standard control.\u003c/p\u003e\n\u003ch3\u003eStatistical tests\u003c/h3\u003e\n\u003cp\u003eSensitivity and specificity were determined from AmpCENTA results obtained for 133 clinical isolates, using cloxacillin synergy with cefotaxime or ceftazidime as the reference standard. Sensitivity was defined as the proportion of AmpC hyperproducers correctly identified, and specificity as the proportion of non-hyperproducers correctly classified. Additionally, 95% confidence intervals (CI), positive predictive value (PPV), and negative predictive value (NPV) were also calculated. Significant differences in expression levels and ΔAbs were analyzed by one-way analysis of variance (ANOVA) in GraphPad Prism 10 software. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe performance of AmpCENTA was assessed in 133 clinical isolates: 76 AmpC hyperproducers (34 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 42 \u003cem\u003eK. aerogenes\u003c/em\u003e), 27 non-AmpC hyperproducers (22 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 5 \u003cem\u003eK. aerogenes\u003c/em\u003e) and 30 ESBLs producers (3 \u003cem\u003eEnterobacter\u003c/em\u003e spp., 18 \u003cem\u003eEscherichia coli\u003c/em\u003e, and 9 \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e). Previously, all isolates were tested using the MicroScan system WalkAway and phenotypically confirmed using a cloxacillin diffusion disk and detecting synergy with cefotaxime or ceftazidime as the gold standard technique. Within the collection of isolates tested by the gold standard assay, 76 were AmpC hyperproducers, 27 were non-hyperproducers and 30 were β-lactamases-producers other than AmpC.\u003c/p\u003e \u003cp\u003eThe AmpCENTA showed a sensitivity of 98.7% (75/76 AmpC-hyperproducers (95% CI: 92.9\u0026ndash;99.8) and a specificity of 100% (27/27 non-AmpC-hyperproducers) (95% CI: 87.5\u0026ndash;100). The positive predictive value (PPV) was 100% (95% CI: 95.1\u0026ndash;100), whereas the negative predictive value (NPV) was 96.4% (95% CI: 82.3\u0026ndash;99.4). No false positives (0%) were observed (95% CI: 0\u0026ndash;12.5), while the false-negative rate was 1.3% (95% CI: 0.2\u0026ndash;7.1). Moreover, the method accurately detected all 30 isolated β-lactamase\u0026ndash;producing strains that were different from AmpC.\u003c/p\u003e \u003cp\u003eAlthough the rapid test was designed for visual reading, its automation was also explored by measuring absorbance at 405 nm and interpreting the results accordingly. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, at high inoculum, both non-AmpC and AmpC hyperproducers showed high and comparable ΔAbs values after 30 minutes. However, the β-lactamase producers other than AmpC, maintained low ΔAbs values at high inoculum, which remained significantly different from those observed in both AmpC hyperproducers and non-hyperproducers. In this way, it is possible to differentiate and identify isolates producing β-lactamases other than AmpC.\u003c/p\u003e \u003cp\u003eOn the other hand, the low inoculum graph illustrates that ΔAbs was significantly higher in the AmpC-hyperproducing isolates than that observed in non-AmpC-hyperproducers from 5 minutes of incubation onwards. Although significant differences were already observable after 5 minutes of incubation, a total incubation time of 30 minutes is recommended, as some isolates require longer to hydrolyse CENTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAdditionally, \u003cem\u003eampC\u003c/em\u003e gene (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u003c/sub\u003e) expression was quantified in a total of 29 isolates (15 AmpC-hyperproducers and 14 non-AmpC-hyperproducers). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, significant differences were detected, allowing two large groups of \u003cem\u003eampC\u003c/em\u003e expression to be distinguished. Regarding the results, the isolates with higher \u003cem\u003eampC\u003c/em\u003e expression were those that also showed a positive result for AmpC hyperproduction. However, among the isolates with lower \u003cem\u003eampC\u003c/em\u003e expression, one isolate was previously recognised as AmpC-hyperproducer according to our gold standard, although it was detected as non-AmpC hyperproducer in the AmpCENTA test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results support the hypothesis that the isolate may possess a resistance mechanism different to AmpC, possibly involving reduced membrane permeability and/or efflux pumps that limits antibiotic entry.\u003c/p\u003e \u003cp\u003eFurthermore, the minimum inhibitory concentration (MIC) for ceftazidime was correlated with the AmpC expression (2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method) observed in AmpC hyperproducing isolates and non-AmpC-hyperproducers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). As observed, higher AmpC expression is associated with increased MIC values, indicating that AmpC production directly contributes to elevated resistance levels.\u003c/p\u003e \u003cp\u003eAutomated technologies in antimicrobial susceptibility testing have replaced many conventional phenotypic methods, especially in the routine of clinical microbiology laboratories. A clear example is the use of the MicroScan WalkAway system, which is based on broth microdilution. However, these methods do not allow rapid susceptibility susceptibility testing, as antimicrobial susceptibility results are available after 16\u0026ndash;18 hours (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In the last years, rapid phenotypic methods such as the βLACTA test have been commercialized for the detection of extended spectrum β-lactamases; however, this assay is useful only for ESBLs and is not suitable for detecting AmpC hyperproduction (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, genotypic approaches, based on PCR, have also been developed. Nevertheless, in \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eK. aerogenes\u003c/em\u003e these molecular methods are not appropriate for detecting AmpC hyperproduction, as they cannot differentiate basal production from true AmpC hyperproduction (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmpC β-lactamase remains challenging to identify, as there is currently no consensus on the most effective laboratory method for its detection. This highlights the need for the development of a reliable test capable of accurately detecting AmpC hyperproduction. The introduction of the AmpCENTA test into clinical practice would have clinical implications, being essential to establish an adequate antibiotic treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe AmpCENTA assay is a rapid, low-cost, and straightforward method that yields results within 30 minutes and demonstrates excellent sensitivity and specificity. The ability of AmpCENTA to quickly detect whether an isolate is an AmpC hyperproducer is essential for guiding appropriate empirical antibiotic treatment in infections caused by \u003cem\u003eEnterobacter\u003c/em\u003e spp. and/or \u003cem\u003eK. aerogenes\u003c/em\u003e, since the activity of cefepime may be compromised in AmpC-hyperproducing isolates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/h2\u003e \u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki. The bacterial isolates were obtained as part of the routine clinical diagnostic procedures and were fully anonymized prior to this study. The research protocol was approved by the Ethics Committee of the University Hospital Virgen del Roc\u0026iacute;o of Seville, Spain (approval number 0019-N-23). The consent to participate was not necessary due to this study did not involve human participants, human material, or animals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCONSENT FOR PUBLICATION\u003c/strong\u003e \u003cp\u003eNot required. This study did not involve human participants, human material, or animals.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAUTHOR\u0026acute; CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003ePGE: Formal analysis, Investigation, Methodology, Writing \u0026ndash; original draft; MRR: Investigation, Methodology, Writing \u0026ndash; review and editing; JDP: ; PDP: ; JMC: Resources, Supervision; JAL: Resources, Writing \u0026ndash; review and editing; ARV: Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Resources Writing-review and editing, JMOR: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing \u0026ndash; review and editing. All authors read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work was funded by the Instituto de Salud Carlos III, Subdirecci\u0026oacute;n General de Redes y Centros de Investigaci\u0026oacute;n Cooperativa, Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Spain, and co-funded by the European Union (PI22/01464, PI25/00217 and PI23/01760). PGE is supported by the Subprograme PFIS, Instituto de Salud Carlos III, Subdirecci\u0026oacute;n General de Redes y Centros de Investigaci\u0026oacute;n Cooperativa, Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Spain, and co-funded by the European Union (FI24/00301). M.R.R is supported by the Instituto de Salud Carlos III, Subdirecci\u0026oacute;n General de Redes y Centros de Investigaci\u0026oacute;n Cooperativa, Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Spain, and co-funded by the European Union (PI22/01464). JMOR is supported by the Subprograme Miguel Servet, Instituto de Salud Carlos III, Subdirecci\u0026oacute;n General de Redes y Centros de Investigaci\u0026oacute;n Cooperativa, Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Spain, and co-funded by the European Union (CP24/00137). A.R.V. is supported by the Subprograma Juan Rod\u0026eacute;s, Instituto de Salud Carlos III, Subdirecci\u0026oacute;n General de Redes y Centros de Investigaci\u0026oacute;n Cooperativa, Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Spain, y co-funded by de European Union (JR20/00023) and by a Clinical Investigator contract (Acci\u0026oacute;n B-Cl\u0026iacute;nicos Investigadores) from the Servicio Andaluz de Salud/Consejer\u0026iacute;a de Sanidad, Presidencia y Emergencias, Junta de Andaluc\u0026iacute;a (B-0042-2020).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePGE: Formal analysis, Investigation, Methodology, Writing \u0026ndash; original draft; MRR: Investigation, Methodology, Writing \u0026ndash; review and editing; JDP: ; PDP: ; JMC: Resources, Supervision; JAL: Resources, Writing \u0026ndash; review and editing; ARV: Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Resources Writing-review and editing, JMOR: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing \u0026ndash; review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u0026Aacute;lvarez-Mar\u0026iacute;n R, Navarro-Amuedo D, Gasch-Blasi O, et al. A prospective, multicenter case control study of risk factors for acquisition and mortality in Enterobacter species bacteremia. J Infect. 2020;80:174\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe Y et al. Enterobacter bacteremia: clinical features, risk factors for multiresistance and mortality in a Chinese Universitary Hospital. Infection. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeal EN et al. Predictors of in-hospital mortality for bloodstream infections caused by Enterobacter species. Pharmacotherapy. 2007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamma PD, Doi Y, Bonomo RA, et al. Antibacterial Resistance Leadership Group. A Primer on AmpC β-Lactamases: Necessary Knowledge for an Increasingly Multidrug-resistant World. Clin Infect Dis. 2019;69:1446\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamma PD, Aitken SL, Bonomo RA, et al. Infectious Diseases Society of America Guidance on the Treatment of AmpC β-Lactamase-Producing Enterobacterales, Carbapenem-Resistant Acinetobacter baumannii, and Stenotrophomonas maltophilia Infections. Clin Infect Dis. 2022;74:2089\u0026ndash;114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoattini M, et al. Enterobacterales carrying chromosomal AmpC β-lactamases in Europe (EuESCPM): Epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020\u0026ndash;2022. Int J Antimicrob agents vol. 2024;63(5):107115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Commitee on Antimicrobial Susceptibility Testing. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid M, Livermore D, Jamrozy S, Mushtaq, Wright W, Nichols K, Young N, Woodford. AmpC β-lactamase induction by avibactam and relebactam. J Antimicrob Chemother Volume. December 2017;72(12):3342\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfaffl MW. May, A new mathematical model for relative quantification in real-time RT\u0026ndash;PCR. Nucleic Acids Res, 29, Issue 9, 1 2001, Page 45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalam MA, Al-Amin MY, Pawar JS, Akhter N. Irine Banu Lucy, Conventional methods and future trends in antimicrobial susceptibility testing. Saudi J Biol Sci Volume. 2023;30(3):103582.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorosini MI, Garc\u0026iacute;a-Castillo M, Tato M, Gij\u0026oacute;n D, Valverde A, Ruiz-Garbajosa P, Cant\u0026oacute;n R. Rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae by use of the new chromogenic βLacta test. J Clin Microbiol. 2014;52(5):1741\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Guerrero E, Callejas-Rodelas JC, Navarro-Mar\u0026iacute; JM, Guti\u0026eacute;rrez-Fern\u0026aacute;ndez J. Systematic Review of Plasmid AmpC Type Resistances in \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and Preliminary Proposal of a Simplified Screening Method for \u003cem\u003eampC\u003c/em\u003e. Microorganisms. 2022;10(3):611.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Enterobacter spp., Klebsiella aerogenes, AmpC, CENTA, rapid assay, colorimetric, resistance","lastPublishedDoi":"10.21203/rs.3.rs-9212347/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9212347/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEarly identification of AmpC-hyperproducing clinical isolates is crucial for optimizing empirical antibiotic therapy and preventing the use of inappropriate antibiotics. For this reason, a visual colorimetric method, namely AmpCENTA, was developed to detect AmpC hyperproduction in \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e within 30 minutes. The performance of AmpCENTA was evaluated by using 133 clinical isolates, including 76 AmpC hyperproducers (34 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 42 \u003cem\u003eK. aerogenes\u003c/em\u003e), 27 non-AmpC hyperproducers (22 \u003cem\u003eEnterobacter\u003c/em\u003e spp. and 5 \u003cem\u003eK. aerogenes\u003c/em\u003e), and 30 producers of β-lactamases other than AmpC (3 \u003cem\u003eEnterobacter\u003c/em\u003e spp., 18 \u003cem\u003eEscherichia coli\u003c/em\u003e, and 9 \u003cem\u003eK. pneumoniae\u003c/em\u003e). The AmpCENTA showed a sensitivity of 98.7% (n\u0026thinsp;=\u0026thinsp;75/76 AmpC hyperproducers) and a specificity of 100% (27/27 non-AmpC hyperproducers). Moreover, the method did not show any cross-reaction with any of the 30 isolated producing β-lactamase other than AmpC. This method constitutes a diagnostic tool that enables rapid, reliable, and low-cost detection of AmpC β-lactamase hyperproduction in \u003cem\u003eEnterobacter\u003c/em\u003e spp. and \u003cem\u003eK. aerogenes\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"AmpCENTA: A rapid assay for detecting AmpC-hyperproduction in Enterobacter spp. and Klebsiella aerogenes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 13:54:30","doi":"10.21203/rs.3.rs-9212347/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a47099a-0189-4783-9ac3-fb95d2fcf818","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T13:54:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 13:54:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9212347","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9212347","identity":"rs-9212347","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}