The race to out-smart bacteria, as they evolve to become resistant to antimicrobials, is not new. In fact, the first beta-lactamase enzyme that can break down drugs like penicillin was identified in Escherichia coli (E. coli)—even before penicillin was ever used in patients. Although organisms are naturally successful at developing methods for protecting themselves from the danger of antibiotics, exposure to antibiotic therapy has increased the diversity and complexity of these resistance mechanisms over time.

To complicate matters, identifying resistance mechanisms in gram-negative rods is complex, and the laboratory methods available to detect beta-lactamase expression are limited and imperfect. Correct identification and reporting of bacterial resistance mechanisms directly impact patient care but require consideration of many caveats, including variability in the way resistance mechanisms are mediated in gram-negative organisms and the variety of species that acquire and display extended-spectrum beta-lactamase (ESBL) genes. Nonetheless, antibiotic resistance continues to be a growing problem that kills millions of people worldwide each year. Understanding and detecting these mechanisms is imperative for proper treatment and to prevent spread.

AmpC Beta-Lactamases: Bugs Born to be Bad

Some gram-negative organisms have chromosomally-mediated beta-lactamases, which means that the resistance mechanism is a feature of virtually all members of the species. These enzymes are called AmpC beta-lactamases, and their expression is typically low but can be induced by exposure to beta-lactam antibiotics. An easy way to think of organisms with AmpC beta-lactamases is that they are “bugs that were born to be bad,” and taunting them with antibiotic (beta-lactam) exposure may trigger them to turn up the expression of this resistance mechanism. Some bacteria in the Enterobacterales order can do this so effectively that they can cause an antibiotic treatment to fail.

Enterobacterales considered at significant risk of clinically meaningful AmpC production (sometimes abbreviated ‘HECK-Yes’) include Enterobacter cloacae, Citrobacter freundii and Klebsiella aerogenes. These organisms can cause infections in the urinary tract, soft tissue, gastrointestinal tract and lower respiratory tract. Therefore, it is essential to be aware of these organisms in the clinical setting, including how they may respond to common empiric treatment. Although they are initially susceptible to cephalosporins like ceftazidime or ceftriaxone, treatment may encourage overexpression of the resistance gene and lead to treatment failure. In many cases, clinical laboratories will not report some antibiotics (cephalosporins) for the HECK-Yes organisms.

The Extended-Spectrum Beta-Lactamases (ESBLs) 

Sometimes, the genes that confer resistance to beta-lactam antibiotics are plasmid-mediated, meaning they are transferred on mobile genetic elements called plasmids. These bacteria were not “born to be bad” like organisms with AmpC beta-lactamases, but rather they acquired antibiotic resistance genes through conjugation with other bacteria. The enzymes that result from this process are called extended-spectrum beta-lactamases, and they cause resistance to most penicillins, cephalosporins and aztreonam. ESBLs are particularly worrisome in terms of spread and outbreaks because, unlike chromosomally-mediated resistance mechanisms, ESBLs can be spread within species and between other organism types, like a baton handed off during a relay race.

Clinical Significance and Diagnostic Methods for Extended-Spectrum Beta-Lactamases (ESBLs) 

To date, ESBLs have become a significant clinical and epidemiologic concern. They are associated with adverse patient outcomes, like longer hospital stays, treatment failure, and mortality. In 2017, there were over 197,000 cases of ESBL infection in the United States alone. Broad-spectrum antibiotics such as carbapenems are typically required to treat these infections, but carbapenem resistance is also rising. Identifying ESBLs in the clinical microbiology laboratory is imperative for successful treatment and infection prevention. However, the diagnostic techniques are still developing, and identification can be complex and challenging. There are several molecular and phenotypic methods available to identify ESBLs; however, these may be limited by a lack of laboratory resources, local clinical preference and the absence of formal guidelines. Confirmatory testing for ESBLs is a controversial topic that is actively discussed in the microbiology and infectious disease communities.

The Historical Context of ESBLs 

Overall, ESBL genes can occur in nearly any gram-negative rod but are most commonly found in E. coli, Klebsiella pneumoniae, Klebsiella oxytoca and P. mirabilis. Over the years, hundreds of these enzymes have been identified. The first plasmid-mediated beta-lactamase ever described was from an E. coli isolated from the blood culture of a Greek patient named Temoniera in the early 1960s and was subsequently named the TEM-1 enzyme. Due to the ease of spread associated with plasmid-mediated mechanisms, TEM-1 quickly spread across the globe and has been identified in various other gram-negative organisms.

SHV-1, a chromosomally-mediated beta-lactamase first identified in Klebsiella, was identified after TEM-1. While these original beta-lactamases were considered “narrow spectrum” (meaning limited in action to only certain antibiotics, like ampicillin), they gave rise to broad-spectrum beta-lactamase mutants that began causing significant clinical problems in the 1980s. ESBLs can hydrolyze 3rd and 4th generation cephalosporins and monobactams but cannot break down cephamycins, such as cefoxitin, or the carbapenems (meropenem, imipenem, ertapenem, doripenem). Furthermore, the hydrolytic activity of ESBLs is inhibited by beta-lactamase inhibiting drugs like clavulanic acid.

To date, the most dominant ESBL is CTX-M. This enzyme originated from a species of Kluyvera, also an Enterobacterales, which is  typically found in the human gastrointestinal tract and considered an opportunistic pathogen that rarely causes infection. It is ubiquitous in human and animal flora and is readily found in the environment. In 1986, this beta-lactamase was identified in an E. coli isolated from the fecal flora of laboratory dogs being used for pharmacokinetic studies in Japan, and subsequently from an E. coli causing a middle-ear infection in a four-month-old child in Munich. The enzyme ultimately received its name from the case of the infected child in Munich; it conferred resistance to ceftriaxone (CTX) and was isolated in Munich (M), CTX-M.

Dissemination of the CTX-M enzyme exploded globally throughout the 1990s and 2000s and did so with increasing gene sequence diversity. These enzymes have been identified in numerous members of the Enterobacterales, as well as in Pseudomonas aeruginosa and Acinetobacter species. They are present in hospital and community settings, the environment, the food supply and livestock.
 

A consensus for the way naturally-occurring beta-lactamase genes should be named.​

Final Conclusions 

One of the most significant challenges we face in the fight against antimicrobial resistance is detecting resistance and managing patients accordingly. Currently, CLSI does not recommend one particular confirmatory phenotypic test. Whether to use ESBL testing, or which test to choose, rests with each microbiology laboratory and its associated medical center. The challenges associated with identifying these resistance mechanisms are further deepened by the complexities surrounding breakpoint updates. New guidance regarding the treatment of ESBLs is available. However, the clinical microbiology laboratory must determine if and how to test for and report organisms harboring these mechanisms in conjunction with PharmD and infectious disease clinician colleagues. Every effort should be made to ensure that reliable susceptibility information is released from the clinical microbiology laboratory and that these results are communicated in ways that optimize patient care and prevent the spread of drug-resistant organisms.
 

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