In this document we include the nomenclature from 2009 for ESBL (ESBL_A, ESBL_M-C and ESBL_CARBA) (1). ESBL_A is used as a synonym to classic acquired, extended-spectrum class A beta-lactamases and ESBL_M-C as a synonym to plasmid-mediated AmpC. For ESBL_CARBA please see document “Enterobacterales: Detection of carbapenemases”.

 

Background 

Enterobacterales can acquire resistance mechanisms, which alone or in combinations, confer varying levels of resistance to extended-spectrum cephalosporins (1, 2). These mechanisms include acquired extended-spectrum beta-lactamases (ESBLs), reduced permeability (porin loss), increased expression of chromosomal beta-lactamases (AmpC), and acquisition of plasmid-mediated AmpC-beta-lactamases (pAmpC).

 

Table 1. Overview of ESBL_A and ESBL_M-C in Enterobacterales

	Ambler Class A	Ambler Class B	Ambler Class C	Ambler Class D1
ESBL group	Classical ESBLs (ESBLA): CTX-M, SHV2, TEM2	-	Plasmid-mediated AmpC (ESBLM-C)3 CMY, DHA	-
Main features	Usually hydrolyse 1-4. gen. cephalosporins and aztreonam, but not carbapenems and cephamycins (cefoxitin, cefotetan)4		Hydrolyse cephamycins (cefoxitin and cefotetan) and 1-3. gen. cephalosporins, usually not 4. gen cephalosporins
Common species	E. coli, K. pneumoniae complex5, P. mirabilis, Salmonella spp., Shigella spp.		K.pneumoniae complex5, P. mirabilis Salmonella spp. E. coli and Shigella spp.6
Inhibited by (used in phenotypical ESBL tests)	Clavulanic acid		Cloxacillin and boronic acid

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

¹ Class D ESBLs (OXA-variants) exist, but they cannot be detected by phenotypical methods.

² All SHV and TEM enzymes are not ESBLs.

³ AmpC is chromosomal in many Enterobacterales species, but only plasmid-mediated AmpC is included in ESBL_M-C

⁴ The enzymes are diverse and have variable substrate profiles.

⁵The most important species in Klebsiella pneumoniae complex are K. pneumoniae sensu stricto, K. variicola and K. quasipneumoniae 

⁶ In E. coli and Shigella spp. phenotypical tests cannot discriminate between chromosomal AmpC and ESBL_M-C

⁷When several enzymes co-exist in one isolate, the synergy with various inhibitors can be masked.

 

 

Resistance mechanisms and phenotypical detection

ESBL (classical ESBL-enzymes; ESBL_A) include acquired plasmid-mediated CTX-M, ESBL-variants of TEM and SHV, as well as less prevalent variants (Table 1). ESBL_A producing Enterobacterales (usually Escherichia coli or Klebsiella pneumoniae complex) are by definition resistant (R) to one or more 3rd-4th generation cephalosporins.

The susceptibility to cephalosporins is restored in vitro with beta-lactamase inhibitors, such as clavulanic acid and tazobactam. Synergy between different 3rd-4th generation cephalosporins and clavulanate can be detected by several methods: ESBL gradient tests, combination disks and disk approximation test (2) (Figure 1). Some ESBL_A only hydrolyse certain 3rd and 4th generation cephalosporins. For example the CTX-M group 9 hydrolyzes cefotaxime effectively, while affecting ceftazidime susceptibility to a limited degree. ESBL_A producing Enterobacterales are therefore not considered resistant to all 3rd-4th generation cephalosporins.

 

Some Enterobacterales harbour chromosomal beta-lactamases which may interfere with phenotypical detection of acquired resistance mechanisms. At the same time, nomenclature of Enterobacterales is continuously changing as new (sub)species closely related to major human pathogens are designated new species. NordicAST Species List contains species names a clinical laboratory may encounter and classes of their chromosomal beta-lactamases in relation to the flow charts presented below. 

 

 

Figure 1.

 

 

Plasmid-mediated AmpC (ESBL_M-C)

AmpC production in Enterobacterales may be due either to increased production of intrinsic chromosomal (derepressed) AmpC or acquired plasmid-mediated AmpC (pAmpC) (3). Only pAmpC is included in ESBL_M-C. Most Enterobacterales have a chromosomal AmpC. The exceptions are Proteus mirabilis, Klebsiella pneumoniae complex and Salmonella spp. Phenotypic detection using cloxacillin is only conclusive in these species. In E. coli and Shigella spp., an AmpC phenotype may be due to either overproduction of chromosomal AmpC or acquisition of pAmpC. A molecular test is needed to confirm pAmpC in these species (Figure 2).

The incidence of pAmpC (ESBL_M-C) in clinical Enterobacterales isolates varies in different parts of the world, but is most commonly observed in E. coli and usually confers clinical resistance to cefotaxime and ceftazidime. Overproduction of chromosomal AmpC can be detected in approx. 1% of clinical E. coli isolates in major population studies, but is typically associated with I-group susceptibility to cefotaxime and R to ceftazidime (4, 5). pAmpC (ESBL_M-C) in E. coli has been associated with nosocomial outbreaks. Therefore, there may be reason to examine E. coli with an AmpC phenotype for ESBL_M-C. However, the laboratories should refer to national guidelines on screening and confirmation of pAmpC (ESBL_M-C).

 

Figure 2. 

 

 

Recommendations

 

1. E. coli, Klebsiella pneumoniae species complex, Proteus mirabilis, Salmonella spp. and Shigella spp.

Comprehensive studies of substrate profiles for clinically relevant ESBL types show that either cefpodoxime alone or cefotaxime and ceftazidime in combination as test antibiotics can be used to demonstrate reduced susceptibility to 3rd-4th generation cephalosporins in Enterobacterales (2). To avoid delaying reporting of susceptibility testing results of the most relevant 3rd-4th generation cephalosporins, NordicAST recommends the use of both cefotaxime and ceftazidime in the primary susceptibility testing of clinically relevant Enterobacterales isolated from hospital patients (Figure 1 and 2).

 

General recommendations: 

When S for cefpodoxime alone or S for both cefotaxime and ceftazidime in primary susceptibility testing, the isolate can be reported S for 3rd-4th generation cephalosporins without further investigation.

When resistance to one or more 3rd-4th generation cephalosporins is detected, the susceptibility categorization of the individual extended-spectrum cephalosporins and aztreonam is interpreted directly from inhibition zones using EUCAST clinical breakpoints for disk diffusion or MIC values, ​and reported without delay. A warning on uncertain therapeutic outcome for infections other than urinary tract infections may be enclosed (6). Further testing for ESBL_A / ESBL_M-C detection is recommended for epidemiological and infection control purposes. In suspected or detected ESBL_A / ESBL_M-C producing strains with susceptibility to amoxicillin-clavulanate, ampicillin-sulbactam or piperacillin-tazobactam, the susceptibility of beta-lactamase inhibitor combinations can be reported as tested, with a note on appropriate dosing (6). Mecillinam can be reported as tested for lower urinary tract infections without warnings. 

When detecting ESBL_A and/or ESBL_M-C, the following comment is recommended in addition to the susceptibility categorization: "Production of extended spectrum beta-lactamases (ESBL) detected which may involve infection control measures”. Infection control measures should be adapted to local guidelines.

 

2. Enterobacterales with chromosomal class A beta-lactamase

Hyperproduction of chromosomal class A beta-lactamase in, for example, Citrobacter koseri and phylogenetically closely related Citrobacter spp., Klebsiella oxytoca, Proteus vulgaris, Raoultella spp. and Serratia fonticola provides a phenotypic profile corresponding to that of ESBL_A with clavulanic acid synergy (4, 7). Thus, in principle classical ESBL_A tests cannot distinguish between hyperproduction of chromosomal class A beta-lactamases and acquired ESBL_A in these species. However, K. oxytoca hyperproducing its chromosomal K1 typically has a higher MIC to cefotaxime than to ceftatzidime. Correct species identification is therefore important to interpret such phenotypic findings. Susceptibility categorization is interpreted as for other resistance mechanisms directly from inhibition zones using standardized disk diffusion methods or MIC values.

 

3. Enterobacterales with chromosomal class C beta-lactamase (AmpC) 

In species with chromosomal AmpC, resistance to 3^rd generation cephalosporins is usually due to elevated expression of an intrinsic chromosomal AmpC beta-lactamase. This is particularly relevant for Enterobacter cloacae complex, Klebsiella aerogenes, Citrobacter freundii complex, Hafnia alvei, Providencia spp., Serratia spp., and Morganella morganii.

 

The expression of the chromosomal AmpC beta-lactamase is normally low and mediates resistance only to ampicillin/amoxicillin and first generation cephalosporins. However, mutants with stable high expression (derepressed) of chromosomal AmpC can be selected and will display resistance to all penicillins (including penicillins plus beta-lactamase inhibitors), 1st-3rd generation cephalosporins, and aztreonam.  There is robust evidence to suggest that the risk of selection of mutants with derepressed chromosomal AmpC must be stratified according to the bacterial species, the severity of the infection, the site of infection and the expected inoculum of infection (8, 9). In a prospective single centre study of patients with infections caused by Enterobacterales with chromosomal AmpC, the overall incidence of the emergence of resistance during extended spectrum cephalosporins therapy was 5% (11/218), represented by E. cloacae species complex (12.3%, 8/65), K. aerogenes (2.9%, 2/51), and C. freundii (2.6%, 1/39) (10).

 

NordicAST recommends that comments about the risk for selection of resistance are communicated to the treating clinicians (in writing and sometimes also verbally), particularly in severe infections, challenging foci of infection or in immunosuppressed individuals. The comment should convey that there is a risk of selecting resistance during treatment with piperacillin-tazobactam, cephalosporins or aztreonam. The risk is likely to be below 15% in most species and in some species below 5%. The wording of the comment should be adapted to national antimicrobial policies and treatment traditions.

 

When R for cefotaxime and ceftazidime without clavulanate synergy combined with multidrug-resistance (resistance to aminoglycosides, fluoroquinolones and/or trimethoprim-sulfamethoxazole), the strain should also be investigated for the occurrence of ESBL_A. This can be done by examining susceptibility for 4th generation cephalosporins (cefepime or cefpirome) with and without clavulanic acid, possibly examining susceptibility to 3rd generation cephalosporins in the presence of both clavulanic acid and cloxacillin. Reduced susceptibility to 3rd and 4th generation cephalosporins with significant clavulanate synergy is interpreted as ESBL_A expression. Alternatively, molecular methods may be used.

 

References

1.       Giske C et al. J Antimicrob Chemother. 2009;63:1-4.

2.       EUCAST Detection of resistance mechanism version 2, 2017

3.       Jacoby GA. Clin Microbiol Rev 2009;22:161-82.

4.       Tofteland S et al. Scandinavian Journal of Infectious Diseases, 2012; 44: 927–933.

5.       Haldorsen B et al. Journal of Antimicrobial Chemotherapy (2008) 62, 694–702

6.       EUCAST Expert Rules v. 3.2 on Enterobacterales (June, 2019, corrections May, 2020 and January, 2023)

7.       Naas et al. Antimicrob Agents Chemother 2007 Apr;51(4):1365-72.

8.       Kohlmann et al. J Antimicrob Chemother. 2018; 73(6):1530-1536.

9.       Mizrahi et al. J Antimicrob Agents 2020 Feb;55(2):105834.

10.   Choi et al. Antimicrobial Agents Chemother 2008;52:3:995-1000

 

 

Document responsible

NordicAST representatives, subgroup for gram-negative bacteria, see http://www.nordicast.org

 

Changes

Version	Changes
2024-02-7	Title changed from “Detection of classical ESBL (ESBLA) and plasmid-mediated AmpC (ESBLM-C)” to “Detection of classical extended spectrum beta-lactamases (ESBLA) and plasmid-mediated AmpC (ESBLM-C), with consideration of species harbouring chromosomal beta-lactamases”. Addition of a paragraph on species harbouring chromosomal beta-lactamases and a link to species list with class A and C chromosomal betalactamases. Change in Recommendations regarding species with chromosomal class C betalactamases (AmpC) Minor revisions in other paragraphs. Addition of references.
2021-02-16	New document