We May Be Missing Important Carbapenemases. Carbapenem-Susceptible KPC Variants.

When discussing carbapenemases, resistance to carbapenems typically comes to mind first. These enzymes represent one of the most clinically significant and widely recognized mechanisms of resistance within the beta-lactam antibiotic group, especially in Gram-negative bacteria.

Among the most prevalent carbapenemases are KPC, NDM, and OXA-48. Within the serine-beta-lactamase group, KPC unquestionably stands out due to its broad spectrum and significant clinical impact.

As a versatile carbapenemase, KPC's extensive antibiotic resistance spectrum wreaked havoc in clinical settings prior to the availability of dedicated anti-KPC agents. Unfortunately, despite these advances, routine laboratory screening and confirmatory procedures remain inadequate. Simply reporting Enterobacterales as carbapenem-resistant (CRE) is insufficient; laboratories should further identify specific carbapenemases for accurately guiding therapeutic decisions.

According to current practices, laboratory screening for CRE primarily depends on detecting resistance to carbapenems and related beta-lactam antibiotics. However, this approach potentially overlooks an important subgroup of KPC enzymes, specifically carbapenem-susceptible KPC variants. These variants, despite their susceptibility to carbapenems, pose a significant challenge due to their resistance to novel beta-lactamase inhibitors, underscoring the need for broader screening methodologies that can accurately detect these unusual resistance mechanisms.

The Near Missed (A Surely Prior Missed) KPC-31.

In May 2024, our laboratory identified a Klebsiella pneumoniae isolate using the GenMark BCID assay from a patient previously diagnosed with urinary tract infections involving KPC- and ESBL-producing strains. Following our rapid response protocol, we promptly initiated treatment with meropenem/vaborbactam. It along with ceftazidime/avibactam are part of our escalated anti-KPC management strategy, reflecting our comprehensive approach to resistant infections.

Sixteen hours later, our rapid direct blood culture panel revealed an antimicrobial profile consistent with our ESBL criteria, displaying resistance to all beta-lactams except meropenem and ertapenem. Recognizing the unusual resistance pattern for our local KPC epidemiology, we immediately initiated repeated susceptibility testing from the growing colonies and for meropenem/vaborbactam, ceftazidime/avibactam, and imipenem/relebactam. Initially suspecting a potential false-positive KPC result, subsequent verification using the Cepheid Carba-R assay conclusively confirmed the presence of the KPC enzyme in the isolate.

Confirmatory colony antimicrobial susceptibility testing consistently demonstrated susceptibility to meropenem, ertapenem, meropenem/vaborbactam, and imipenem/relebactam. However, resistance to ceftazidime/avibactam was notably present, a distinctive phenotype highly suggestive of specific carbapenem-susceptible KPC variants, such as KPC-31 or KPC-44.

Although the immediate association with KPC is typically resistance to carbapenems, novel variants such as KPC-31 and KPC-44 illustrate an important deviation: carbapenem-susceptible yet inhibitor-resistant KPC enzymes. KPC-31 arises from a D179Y mutation in the Ω-loop of the KPC-3 enzyme, leading to resistance against ceftazidime/avibactam while paradoxically becoming susceptible to carbapenems. First identified clinically in patients previously treated with ceftazidime/avibactam, its emergence underscores the selective pressure exerted by such antimicrobial therapies.

Similarly, KPC-44 is characterized by a duplication of 15 amino acids (Ala262-Glu276) in the KPC-2 enzyme, altering its structural and functional profile. Unlike KPC-31, KPC-44 retains some carbapenemase activity, although diminished compared to wild-type enzymes. This variant exhibit notable resistance to ceftazidime/avibactam and moderate resistance to carbapenems, predominantly emerging in patients with prolonged exposure to ceftazidime/avibactam therapy.

Thanks to the rapid coordination and intervention by our Pharmacy and Infection Prevention teams, optimal treatment escalation and targeted infection control measures were promptly executed, driven by our Microbiology laboratory's rapid response capability. This collaborative approach facilitated effective containment of the infectious agent and ensured a successful patient outcome. 

Whole-Genome Sequencing. A Guiding Tool for Improving Phenotypic Screening.

Next-generation sequencing (NGS) plays a pivotal role in resolving complex microbiological challenges, especially when discrepancies arise between identified resistance markers and observed phenotypic profiles. This advanced technique significantly enhances our ability to precisely characterize resistance mechanisms and guide appropriate therapeutic strategies. 

At AdventHealth Central Florida, our implementation of next-generation sequencing (NGS) extends beyond organism identification, genotyping, epidemiological surveillance, and direct sample metagenomics. Access to this technology in-house enhances our approach capability for full organism characterization, especially in cases involving complex or unusual resistance profiles.

Genomic DNA extraction was performed using the ZymoBIOMICS kit, followed by whole-genome sequencing utilizing the Oxford Nanopore Flongle flow-cell, achieving a genome coverage of 69X. Subsequent analysis using the OneCodex database confirmed the organism's identity as Klebsiella pneumoniae spp. pneumoniae.

Running the FastA file through the Pasteur Institute's MLST database and analyzing the seven housekeeping genes for this specie revealed an unexpected and concerning profile: the isolate was identified as Klebsiella pneumoniae sequence type (ST) 307, recognized as a hypervirulent strain. ST307 has garnered international attention due to its rapid global spread and frequent association with multidrug resistance. Epidemiologically, ST307 poses a substantial public health threat because of its efficiency in spreading within healthcare settings, often carrying numerous resistance determinants. Clinically, infections caused by ST307 have severe implications, including bloodstream infections and pneumonia, particularly in vulnerable populations. The emergence of ST307 carrying variants such as KPC-31 further complicates therapeutic approaches, underscoring the urgent need for vigilant surveillance and advanced diagnostic techniques to effectively monitor and control this challenging strain.

Resistome analysis was performed utilizing BioMérieux's EpiSeq software, which identified two distinct plasmids. The first plasmid, IncFIB(K), harbored multiple resistance determinants, including genes encoding KPC, ESBL (TEM-1), tetracycline resistance, and aminoglycoside resistance. Notably, the KPC gene identified was specifically the blaKPC-31 variant, co-existing alongside additional beta-lactamase genes such as blaOXA-1 and blaTEM-1.

The second plasmid, the pKPC-CAV1193, carried two ESBL genes, blaCTX-M-15, and blaTEM-6. An additional ESBL gene, blaSHV-28, along with quinolone and fosfomycin resistance genes were found although unable to map in any specific plasmid.  

Besides these genes, only an ompk37 porin was present along with nine point mutations across its gene. OmpK37, an outer membrane porin, plays a critical role in antibiotic permeability and resistance in K. pneumoniae. Unlike its counterparts OmpK35 and OmpK36, OmpK37 is less frequently described but significantly contributes to resistance profiles, particularly in strains where OmpK35 and OmpK36 are deficient or absent. Its importance arises from its potential to mediate resistance against beta-lactams, highlighting the need for its consideration in phenotypic and genotypic resistance screening strategies.

The identification of Klebsiella pneumoniae ST307, carrying the KPC-31 variant, represents a clinically significant finding. ST307 is recognized as a hypervirulent strain with notable epidemiological importance due to its rapid global spread and multidrug-resistant characteristics. Clinically, this strain poses severe risks, especially as it displays a common and deceiving phenotype: carbapenem susceptibility resembling an ESBL-producing organism. This highlights the urgent need for laboratories to adopt screening strategies capable of accurately identifying such atypical resistance profiles, ensuring the correct prioritization of treatments such as meropenem/vaborbactam. 

Change of Practice and A New Approach.

The identification of KPC-31 presents a considerable epidemiological challenge, particularly for microbiology laboratories relying on traditional CRE screening methods. Given its distinctive phenotypic profile, a revised approach is necessary to effectively detect and manage this variant, thereby ensuring accurate surveillance and appropriate clinical intervention.

Although CLSI guidelines categorize antimicrobials into reporting tiers, it is important to clarify that this classification does not preclude routine testing of Tier-2 or Tier-3 antimicrobials, including anti-KPC agents, from initial susceptibility testing. However, current limitations from AST panel manufacturers have prevented the availability of comprehensive and expanded testing panels at the point of initial susceptibility testing.

Laboratories often implement reflex protocols for anti-KPC agents, either upon provider request or as part of routine internal testing, particularly when the prevalence of KPC-producing organisms is low. However, from a surveillance standpoint, this practice may be counterproductive, especially in regions where carbapenem-susceptible KPC variants have been identified. Such protocols may inadvertently miss these atypical variants, highlighting the need for enhanced detection strategies.

By leveraging phenotypic profiles and pattern recognition, incorporating ceftazidime/avibactam and meropenem/vaborbactam into the initial Enterobacterales susceptibility panel can serve as an effective early detection strategy for KPC variants that might otherwise go unnoticed with traditional screening methods.

In the described case, the detection of this KPC variant occurred specifically because routine KPC screening via BCID is systematically performed for all Gram-negative isolates from blood cultures. Had this variant originated from urine or respiratory cultures, sources known to have a higher prevalence of carbapenemases, it likely would have gone undetected. Given this scenario, it is plausible that similar variants may already exist undetected within our patient populations.

Our laboratory currently employs a validated ESBL and CRE screening algorithm integrated within Epic, designed to automatically assess antimicrobial susceptibility profiles for all Enterobacterales isolates. The algorithm identifies potential ESBL phenotypes based on MIC patterns for ceftriaxone, cefotaxime, or ceftazidime, and when meropenem is non-susceptible (CRE) it triggers confirmatory PCR testing. To further enhance detection capabilities, particularly for carbapenem-susceptible but inhibitor-resistant KPC variants, we propose incorporating an additional screening criterion: isolates displaying an ESBL pattern (susceptible to meropenem) and intermediate or resistant MIC to ceftazidime/avibactam would also trigger confirmatory PCR testing. This proposed approach adds a critical new screening layer against CRE.

Additionally, including aztreonam in combination with the anti-KPC agents in the initial susceptibility panel, provides the data for a phenotypical screening strategy aimed to preliminary distinguish between serine-beta-lactamases from metallo-beta-lactamases. By incorporating these antimicrobials into the initial panel, we ensure rapid availability, enabling prompt and appropriate therapeutic decisions immediately upon PCR confirmation.

Epidemiological and Clinical Impact.

The emergence of carbapenem-susceptible yet inhibitor-resistant KPC variants poses significant epidemiological and clinical challenges, particularly for microbiology laboratories relying on traditional CRE screening methods. These variants, although phenotypically resembling ESBL-producing organisms, require optimized detection strategies due to their unique resistance profiles. Without enhanced diagnostic methods such as the routine use of rapid molecular assays (e.g., BCID for blood cultures), these ESBL-like KPC variants may go unnoticed, potentially resulting in misdiagnosis and suboptimal treatment. An optimized and targeted screening approach thus represents a rapid, practical, and cost-effective solution for accurate identification.

Although these variants are susceptible to carbapenems, treatment without an inhibitor is not advisable. Optimal therapeutic choices include agents such as meropenem/vaborbactam and imipenem/relebactam, which should be promptly initiated once the expanded CRE screening protocol is activated.

Although preliminary, our observations indicated susceptibility of aztreonam/avibactam against this KPC-31 strain. Additional in vitro testing suggested that KPC-31 exhibits an antimicrobial-dependent inhibitor-resistance profile. Avibactam effectively inhibited the enzyme when paired with aztreonam but failed to do so with ceftazidime. Moreover, the inhibitory capacity of vaborbactam appeared diminished when combined with ceftazidime. Further in vitro and clinical investigations are necessary to fully characterize this unique resistance phenotype and guide therapeutic strategies.

We emphasize the importance of implementing a comprehensive initial antimicrobial susceptibility testing panel that integrates Tier-1, Tier-2, and Tier-3 antimicrobials, including proven anti-MDR agents available in the hospital formulary. By adopting such a comprehensive initial screening approach, laboratories can rapidly provide accurate susceptibility results, significantly improving clinical management decisions and enabling timely identification and treatment of challenging resistance variants.

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