ESBL Detection by MIC Pattern. No Need for Confirmation.

If your institution still conducts routine ESBL screening and confirmatory testing, are you aware of the prevalence rates specific to each organism within your patient population?

A few years ago, when CLSI lowered the breakpoints for cephalosporins and carbapenems, they recommended that ESBL confirmatory testing be performed solely for epidemiological surveillance and infection prevention purposes.

For microbiology laboratories, this change provided a straightforward method to reduce turnaround time and eliminate the additional steps associated with performing the double-disk diffusion test.

A significant issue with this change was that some laboratories entirely ceased routine ESBL testing, leaving the testing decision to clinicians or infection preventionists on a case-by-case basis.

This poses a significant issue as it places reliance on a diverse group of healthcare professionals, who have varying levels of background and experience, to identify potential ESBL cases based on organism-specifics such as species, phenotypic results, and laboratory capabilities. Ideally, this process should be standardized and centralized at the laboratory level, with clear criteria guiding routine screening and confirmation.

The clinical dilemma of treating infections caused by ESBL-producing Enterobacterales underscores the critical need for robust laboratory detection and reporting. While carbapenems have long been the mainstay for serious ESBL infections, the imperative to preserve their efficacy has fueled extensive research into carbapenem-sparing alternatives.

Piperacillin/tazobactam, for instance, has shown variable success; while some studies suggest non-inferiority to carbapenems for certain infections like uncomplicated cystitis or when specific pharmacokinetic/pharmacodynamic targets are met, the landmark MERINO trial demonstrated higher mortality with piperacillin/tazobactam for ESBL bloodstream infections, cautioning against its broad use in such severe cases.

Similarly, cefepime has been explored, with its utility potentially enhanced by newer beta-lactamase inhibitors like enmetazobactam, which show promise in restoring cefepime's activity against many ESBL producers. The decision to employ these alternatives hinges on accurate ESBL identification, local susceptibility patterns, infection severity, and source.

Consequently, consistent ESBL screening and clear reporting by microbiology laboratories are paramount. This information directly drives clinicians and pharmacists to make evidence-based therapeutic choices, facilitating antimicrobial stewardship by enabling the judicious use of carbapenems and promoting alternative agents where appropriate, thereby mitigating the selection pressure for further resistance.

Resistance Against Extended Spectrum Beta-Lactamases (ESBL)

Beta-lactam resistance in Enterobacterales is one of the easiest mechanisms to recognize and classify due to its distinct phenotypic patterns. When discussing extended-spectrum beta-lactams, we typically refer to third- and fourth-generation cephalosporins such as ceftriaxone/cefotaxime, ceftazidime, and cefepime. In a hierarchical manner, beta-lactamases can be classified as follows:

●       Penicillinases (including inhibitor-resistant penicillinases): Hydrolyzing penicillin, amoxicillin, ampicillin (with and without inhibitors), piperacillin/tazobactam, and cefazolin.

●       Cephalosporinases (AmpC): Similar spectrum to penicillinases but additionally hydrolyze cefoxitin, third-generation cephalosporins, and aztreonam, and are not inhibited by traditional inhibitors such as clavulanic acid or tazobactam.

●       Extended-Spectrum Beta-Lactamases (ESBL): Similar spectrum to AmpC but are inhibited by classic inhibitors, do not hydrolyze cefoxitin, and have additional activity against cefepime.

●       Carbapenemases: Exhibit activity like the previously mentioned enzymes, showing variability depending on the enzyme subtype, but their most important activity is the hydrolysis of carbapenems.

ESBL enzymes may occur across all members of the Enterobacterales group; however, according to CLSI guidelines, detection efforts specifically focus on E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis.

SPiCE-M organisms can carry and produce ESBL enzymes; however, the concurrent presence of AmpC enzymes complicates the screening and detection process. Despite this challenge, certain protocols employing cloxacillin, cefoxitin, and cefepime/clavulanic acid have been developed to effectively differentiate between AmpC and ESBL activity.

Since E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis do not possess inducible extended-spectrum cephalosporinases like AmpC, any non-susceptible breakpoint observed against third- or fourth-generation cephalosporins may be considered indicative of acquired ESBL production. However, plasmid-mediated AmpC enzymes can also be a factor, although from a clinical and therapeutic perspective, carbapenem-based treatment is optimal for ESBL and AmpC.

Although mechanisms such as PBP mutations, porin loss, and efflux pumps can also affect resistance to third- and fourth-generation cephalosporins, their impact in Enterobacterales is typically less pronounced than in non-fermenters. This simplifies attributing resistance primarily to the presence of ESBL enzymes. Non-enzymatic mediated resistance mechanisms against beta-lactams are uncommon. These include PBP mutations conferring aztreonam resistance in specific E. coli strains and porin loss leading to reduced carbapenem susceptibility, especially in K. pneumoniae and E. cloacae complex. From this perspective, resistance to ceftriaxone, ceftazidime, or cefepime can typically be attributed to beta-lactamase-mediated mechanisms.

ESBL

There are three primary groups of genes classified as ESBL:

●       CTX-M: According to our AdventHealth data, CTX-M enzymes represent approximately 97% of all ESBL enzymes identified.

●       TEM and SHV: Originally penicillinases, certain genotypes have evolved extended activity against third- and fourth-generation cephalosporins. We do not currently have an FDA-cleared molecular method for identifying this group.

●       ESBL-like OXA: These enzymes also confer extended-spectrum beta-lactamase activity, though their exact prevalence remains unknown due to the lack of molecular method available.

These enzymes share common phenotypic profiles:

●       Resistant to penicillins, inhibition by classic beta-lactamase inhibitors such as clavulanic acid and tazobactam (with a caveat for inhibitor-resistant penicillinases).

●       Resistant to first-, third-, and fourth-generation cephalosporins, as well as aztreonam.

●       Susceptible to cefoxitin.

While there are differences in affinity for ceftriaxone and ceftazidime between CTX-M and TEM/SHV enzymes, these variations are not considered clinically significant. Therefore, from both microbiological and clinical standpoints, they should be treated as similar entities.

From a BCID perspective, CTX-M is the sole ESBL marker and the most frequently detected resistant gene among Gram-negative organisms. Its detection directly indicates ESBL production not just in E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis, but also in any member of the SPiCE-M group.

Based on our observations at AdventHealth Central Florida , K. oxytoca demonstrates the highest rate of non-CTX-M enzymes, although its overall ESBL rate remains below 6%. Additionally, among SPiCE-M organisms, is E. cloacae complex the most frequent carrier of CTX-M.

ESBL Testing

Traditionally, ESBL testing relies on the inhibitory effect of clavulanic acid against beta-lactamases. The double-disk diffusion test measures the capability of clavulanic acid to restore susceptibility to ceftazidime or cefotaxime. ESBL presence is confirmed when the combination with clavulanic acid produces an inhibition zone at least 5 mm larger than the zone observed with the antibiotic alone.

Although certain automated panels include ESBL testing, traditionally, the confirmatory test has been performed reflexively based on observed resistance to third- or fourth-generation cephalosporins, typically extending the turnaround time by approximately 24 hours.

The lowering of cephalosporin breakpoints provided the basis for discontinuing routine ESBL testing except when epidemiological or infection prevention considerations necessitated confirmation. Nevertheless, this confirmatory testing remains labor-intensive, time-consuming, and typically delays results by approximately 24 hours.

Ceftriaxone has consistently been a critical and routinely used indicator for ESBL detection. As outlined in our abstract at ASM Microbe, our team at AdventHealth demonstrated that ceftriaxone non-susceptibility in E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis yielded a sensitivity of 98% for identifying ESBL. Independently, ceftazidime and cefepime showed sensitivities of 59% and 36%, respectively. However, combining ceftriaxone with ceftazidime and/or cefepime improved overall sensitivity, achieving 99.8%.

Validating MIC Distribution for ESBL Confirmation

Several years ago, as we prepared to update our Vitek2 Gram-negative cards, the introduction of separate cards for Enterobacterales and non-fermenters emerged as our optimal solution.

We streamlined processes and improved turnaround times by optimizing card space, focusing on agents with clear clinical utility for these groups. For instance, is it necessary to allocate wells for cefazolin or ertapenem when testing non-fermenters?

The validation and implementation of the Vitek2 Advanced Expert System (AES) were crucial steps. Utilizing AES enabled us to remove the ESBL test from the Vitek2 card, freeing up to six wells for additional antimicrobials. AES subsequently became our standardized approach for directly and after validation, confirming the presence of ESBL-producing organisms.

The underlying principle is straightforward: beta-lactam's MIC values for various beta-lactamases are analyzed using a machine learning algorithm to establish common resistance patterns. For ESBL detection specifically, our evaluation identified ceftriaxone, ceftazidime, and cefepime as the most critical indicators, although other than cephalosporins were initially included in the analysis.

The Vitek2 AES relies on extensive global datasets, but as our experience revealed, these generalized data points may not always align perfectly with local epidemiological patterns, highlighting the importance of regional validation.

During our comprehensive validation of AES ESBL calls using the double-disk diffusion test as the confirmatory method, we verified that ceftriaxone, ceftazidime, and cefepime were the most effective agents for ESBL detection in E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis. Our validation demonstrated that for any of these organisms, reaching the minimum threshold with at least one of these agents confirmed ESBL production via the double-disk diffusion test.

The only noted exception was P. mirabilis when ceftazidime alone met the threshold. According to the AES database, a ceftazidime MIC of ≥4 served as the criterion for ESBL detection. However, our local data revealed a critical discrepancy. Specifically, every P. mirabilis isolate with a ceftazidime MIC of 4 that remained susceptible to ceftriaxone and cefepime tested negative for ESBL by double-disk diffusion, despite being flagged as positive by AES. Conversely, isolates with ceftazidime MICs of ≥8 consistently tested positive.

Setting the Rules in Epic

Although implementing these rules and algorithms in Vitek2 was straightforward, transferring and adapting the data into our Laboratory Information System (LIS) required additional steps. Furthermore, the specific ceftazidime MIC threshold adjustment needed for P. mirabilis could not be customized directly within Vitek2.

Consequently, the final rules established that any isolate of E. coli, K. pneumoniae, K. oxytoca, or P. mirabilis from any clinical source meeting or exceeding one of the specified MIC thresholds would be flagged accordingly.

●       Ceftriaxone: MIC ≥2

●       Ceftazidime: MIC ≥8

●       Cefepime: MIC ≥4

Implementing these rules results in multiple changes to culture reporting:

●       The organism's identification is updated to reflect ESBL production (e.g., E. coli becomes E. coli ESBL, or K. pneumoniae becomes K. pneumoniae ESBL, etc.).

●       Susceptibility results against penicillins, penicillins combined with beta-lactamase inhibitors, aztreonam, cefazolin, ceftriaxone, ceftazidime, and cefepime are reported as resistant, regardless of initial susceptibility. The MIC are reported as tested.

●       An automatic electronic alert is generated for infection prevention protocols, prompting patient isolation measures.

While ceftriaxone serves as a highly effective indicator for ESBL detection, incorporating ceftazidime and cefepime significantly enhances the accuracy and reliability of screening and confirmation processes.

Based on our internal data, relying exclusively on ceftazidime or cefepime without including ceftriaxone for ESBL screening is not recommended. Both ceftazidime and cefepime demonstrate lower sensitivity compared to ceftriaxone, due to the high prevalence of CTX-M enzymes, which exhibit greater affinity for ceftriaxone/cefotaxime than for ceftazidime or cefepime.

We are currently developing an expanded set of rules to further enhance our screening and diagnostic capabilities. One of these rules specifically aims to detect ceftazidime/avibactam-resistant KPC variants, such as KPC-31 and KPC-44, carbapenem-susceptible KPC variants that phenotypically behave as ESBLs. KPC-31, for example, is a carbapenem-susceptible variant that, from a phenotypic perspective using common Tier 1 and 2 agent panels, would flag as an ESBL. Since the criteria for Carbapenem-Resistant Enterobacterales (CRE) and for suspecting a carbapenemase-producing CRE (CP-CRE) is carbapenem resistance, these variants create an epidemiological concern. Without routine use of ceftazidime/avibactam (to which these variants are resistant), KPC-31 may be easily missed during routine testing.

This detection will be facilitated by leveraging the ceftazidime/avibactam and meropenem/vaborbactam testing capabilities included in the new Vitek2 GN815 card.

Another rule aims to identify plasmid-mediated AmpC enzymes more accurately in non-SPiCE-M organisms, optimizing treatment with cefepime and reducing unnecessary carbapenem use.

Identifying and tracking ESBL-carrying organisms is critical not only for optimizing patient care and guiding appropriate antimicrobial stewardship but also as a vital epidemiological and infection prevention intervention. To be most effective, this process should be centralized and standardized at the laboratory level, moving away from a reactive, case-by-case protocol triggered by individual requests. Such a proactive and systematic approach ensures timely and accurate information, empowering healthcare professionals to make informed decisions that benefit patient outcomes and decrease the spread of these resistant pathogens.

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