JAC Antimicrob Resist doi:10.1093/jacamr/dlab092 JAC- Antimicrobial Resistance Extended-spectrum f-lactamases: an update on their characteristics, epidemiology and detection Mariana Castanheira’, Patricia J. Simner? and Patricia A. Bradford @ ** 4 JMI Laboratories, North Liberty, A, USA; "School of Medicine, Johns Hopkins University, Baltimore, MD, USA; "Antimicrobial Development Specialists LLC, Nyack, NY, USA “Corresponding author. &-moil: poracford@antimicroboldev com Extended-spectrum B-loctamase (ESBL)-producing Gram-negative pathogens are a major cause of resistance to expanded-spectrum f-lactam antibiatis, Since their discovery in the early 1980s, they have spread worldwide and an are now endemic in Enterobacterales isolated from both hospital associated and community-acquired infections. As a result, they ore a global public health concern. In the past, TEM= tand SHV-type ESBLs were the predominant families of ESBLs. Today CTX-M-type enzymes are the most commonly found ESBL type with the CTX-M-15 variant dominating worldwide, followed in prevalence by CIXGMe14, and CTDX%GM-27 is emerging in certain parts of the world. The genes encoding ESBLs ore often found on plasmids and harboured within transposons ar insertion sequences, which has enabled their spread. In addition, the population of ESBL-producing Escherichia coli is dominated globally by a highly virulent and successful clone belonging to ST131. Today, there are many diagnostic tools available to the Clinical microbiology laboratory and include both phenotypic and genotypic tests to detect B-lactamases Unfortunately, when ESBLs are not identified in « timely manner, appropriate antimicrabial therapy is fre quently delayed, resulting in poor clinical outcomes. Several analyses of clinical trials have shown mixed results with regards to whether a carbaperiem must be used to treat serious infections caused by ESBLs or whether some of the older B-lactam-f-lactamiase combinations such as piperacillintazobactam are appro: priate, Some of the newer combinations such as ceftazidimefavibactam have demonstrated efficacy in patients, ES2L-praducing Gram-negative pathagens will continue to be major contributor to antimicrabial resistance worldwide. Its essential that we remain vigilant about identifying them both in patient isolates ‘and through surveillance studies. 1. Introduction Although naturally occurring in some species of bacteria f-lactamnases have become moblized on plasmids and have be- come widespread in response to the use and overuse of facta antibiotics. In Gram-negative bacteri, broad-spectrum enzymes such os TEM-1 and SHV-1 arose following the introduction of first- and second-generation cephalosporins.” Subsequently, expanded-spectrum B-lactam antibiotics were intraduced the were refractory to hydrolysis by these enaymes, In particular, the oxyimino- cephalosporins such as ceftazidime and cefotoxime be came widely used. This led to evolution of new B-lactamases that hydrolysed these new drugs." The most epidemiologically import- tant group of such enzymes is the extended-spactrum f-lacta- ‘mases, which have become endemic worldwide, ESBLS are serine Blacternases, belonging to Ambler molecular and structural classification as class A. They are biochemically characterized by their abilty to hydrolyse expanded spectrum p-lactam antibiotics, and inhibition by B-lactamase inhibitors, specifically clavulanate.’ ESBLs have been found in many genera of frobacterales as well os in Pseudomonas aeruginosa They confer resistance to most lactam antibiotics, including expanded-spectrum cephalosporins and monobactams, but not to carbapenems and cephamycins. The original ESBL enzymes were variants of TEM and SHV variants that had rine acid substitutions leading to « change in, thei substrate profile to include the expanded-spectrum cephalo: sporins. With the widespread usage of gene sequencing to identify fFiactamase genes in clinical isolates, multiple vavionts of the common TEM and SHV enzymes have been identified. AS ofthis, writing, 243 variants of TEM and 228 variants of SHV have been, identified, eithough nat al ofthese possess the ESBL phenetype {https:ivon.ncbi nim. govlpathogensisolatestretgene!TEM nttpsifwmwcb nlm.rih govlpathagensisolatestrefgeneiSHV) ESBL-producing Gram-negative pathogens ore now corman- place in both the hospital and community settings.° The impact ©The Author(s) 2021, Published by Oxford University Press on behalf ofthe British Society fr Anti irobial Chemotherapy. This is an Open Access article distriouled under the (erms of the Crealive Commons Aliibulian Non-Commercial License (hiipicreativecom ‘mons orglicenses/by-nel4,0N, which permits non-commercial re-use, dstnbution, and reproduction in any medium, provided the original work s propery cited. For commercial re-use please contac jour nals permissions@oup.com oy papeojuned quapenej sc a sero veyaensenuenelw: jsen6 Aa yeazeeoreo0 L202 fine £4 uoReview of ESEL:postive Enterobacterales on the choice of empiical and definitive antimicrobial therapy has been substantial, resulting in the increased use of carbapenems in many institutions, which led the increase of carbapenem resistance in these organisms." This review wil focus on the phenotypic and genetic characterza- tion of ESBLs, their epidemiology, the state ofthe art of detection ofthese enzymes, and therapeutic options. 2. ESBL placement in f-lactamase classification scheme The fist clasifcation scheme for lactamases that recognized ESBLs was established in 1989 by Karen Bush," in which group 26 was defined as felactamase enzymes that can hydrolyse oxy mino-Srlactams such as cefotaxime, ceftazidime and aztreonam trates ot least 10% that of benzylpenicilin and that are strongly inhibited by clovlanate (Fgure 1. Subsequently, these enzymes were designated os group 2be in the functional ciassiiation scheme of developed by Bush, Jacoby and Mederios® In this scheme, FSBLs retained the strict defition of cass A facta mases that could hydrolyse these expanded-spectrum 6-lactam antibiotics and are also susceptible to inhibition by the original frlactemase inhibitors clavulancte, sulbactam and tazobactam. The original classification scheme also included only plasmide mediated enzymes, however the updated scheme now recognizes, 1 fluidity of genes expressing ESBLS between plasmids and chromosomes® Traditional ESBLS are inhibited by all of the frlactamase inhibitors including the older inhibitors clavulonat, sulbactam and tazobactar cs well os by the newer inhibitors such 2s avinactam, relebactam and vaborbactam. Although the notion, of including aay enzyme that can hydrolyse the oxyirino- lac: tams in the cassfcaton as an ESBL has been proposed, the strict defiition ofan ESBL remains thatthe inhibition by ciovuianate is @ requirement for designation inthis group.” 3. ESBL families Although ESBLs have common biochemical properties with regards to the hydrolysis of expanded-spectrum B-actam antbi tics ond inhibition by clevulanate, the genes encoding these tenzymes are diverse in nature and can be grouped into several families (Table 2).' Some of these families such os the TEM- and SHV-type ESBLs are highly related, with variants differing by only a few amino acid substitutions. Other families such as the CTX-M- type ESBLs are much mare genetically diverse. Each of the ESBL families have some unique characteristic. 3.1 TEM TEM-type €58LS ore variants of the original plasmid mediated - lactamnase, TEM-1, which was described in the early 19605." This enzyme was sonamed because t was orginlly found in anisolate of Escherichia coli isolate that came from a blood culture from a Greek patient named Temaneira”’ The fist derwvative of TEM, TEM-2, has a single amino acid substitution of Gin39Lys from the original TEM-1 Brlactamose.** This change did not alter the sub- strate profile from TEM-1, however TEM-2 served os the progenitor for many of the TEM-type ESBLS. The first TEM-type variant that showed the ES@L phenotype was TEM-3, which was reported in 1983."° As of this writing, 243 different TEM variants have been described, although not all are ESBLs {hitps:iiuww.ncbinlm in gouipathogensirefgener#TEM) The amino acid substitutions that occur within the TEM enzyme occur at a limited number of positions’ The amino acid residues (Ambler numbering) are most frequently involved in conferring the ESBL phenotype to TEM-type enzymes are Giy238 and Glu240 located on the b3 pplected sheet; Argi64 located on the neck of the 2 loop; and Glu20é located directly across from iy238 Glu240 at the opening of the active-site cavity (Figure 2). OF these, the substitutions Giy238Ser and Glu240Lys appear to be have the most impact on producing the ESBL phenotype.’ Some of the newer TEM variants have subtle changes inthe substrate pro- file. For example, TEM-18% (amino acid substitutions at GSK, aK, 112TV, RL64S ancl M1827) hyerelysed ctreonam mare ef ficiently than ceftazidime or cefotoxime. Although so many new variants are being discovered by WGS, few of these are being henatypically characterized to determine if they have properties of an ESBL. However, computer modeling and network analysis, has enabled the prediction of whether aparticular sequences ike ly to belong to functional groups 2b (original broad spectrum), 2be (€SB1) or 2b inhibitor resistant)? {At the height f prominence for TEM-type ESBL, the prevalence cof some of the variants were regional in nature. For example, TEM- 3 was very commonin France, but rarely seen in the USA” In con- trast, TEM-10 was the most prevalent TEM type ESBL in the USA? Interestingly, TEM-26 wos detected in isolates from across the globe." As the CTX-M-type flactamases became the most prevalent ESBL worldwide, TEM-type enzymes became more ifte quent. In a recent survey of European isolates, TEM-type ESBLs were detected in less than 1% of FSSl-producing F. cali and ‘ebsielia preumonize.” 3.2 SHV The SHV-type Blactomases (so named for sulfrydyt reagent vari- able) originated as chromosomally encoded enzymes in K pneumoniae The fs ESBL described in 1985 was SHV-2 and was found in a single strain of Kiebsiela azaenae isolated in Germany that difered from S#V-1 by a single amino acid substitu ‘on of Gly to Ser at poston 238.” Sila to what is seen in TEM ype ESBLs, the majority of SHV-type ESBLs also have mutations at Ambler positions 238 (Gly to Ser) and 240 (Lys to Gu) (igure 2). ‘Te substitution of serine at position 238 appears tobe crtcal for the efficient hydrolysis of ceftazidime, whereos the substitution of Lys at residue 240 is ertical for the efficient hydrolysis of cefatax- ime.” The relevance of the vorious amino ac substitutions with regards to phenotypic changes in substrate profile has recently been investigated using a mathernatical model To date, 228 sequence variants of SHV have been detected, although not all have been functionally characterized to determine if they possess ‘the ESBL phenotype thitps//aw.nebinim.ningovlpathogensiso latestiretgenetSiN). Worldwide, SHV-5 ond SHV-12 have been ne mast cornmon ESBL variants found in Enterobacterals.”?"° SiW-type ESBLS are most often found in clinical isolates of pneumoniae, however, these enzymes have also been found in, ther genera of Enterebactercies and P. aeruginasa as wall?" 2of a1 mwapenejcsdy wow pepeoKuMcd a 1202 fn £1 uo 8808 Aq Leszzesrzsorepseiemmenwesetwe:Review JAR Penicillins oO rtyix oy Hl ° oom Benzyipeni Ovacitin Cephalosporins and Monobactam ory Cefalotin Cefoperazone Cefepime Carbapenems eee yy apes 4 os oo Meropenem imipenem Traditional Inhibitors Yo paw BYo Fe EX om om Newer Inhibitors is. orvttyg,, Avibactam Vaborbactam Figure 1. structures of f-actam ontiiotics ond flactamase inhibitors, In recent European survellance, SHV-type ESBLS were found in 3.19%-17 0% of clinical isolates of K. pneumoniae, depending on region.”” However, in @ clinical trial that targeted ceftazidime- resistant pathogens from complicated intra-abdominal infections ony J mati Cefiderocol ‘aitnesiat {C1AD) and complicsted urinary tract infections (CUT), SHV-type SBLs were rarely encountered nd were only found in strains that dso produced @ plasmid-mediated AmpC 0: carbapenemase Although TEM- and SHV-type ESBLS are stil encountered, it 3 of 21 pepeotunod 1202 An £1 uo sen8 Ae Leazzeerecogeprerepmenuerelwuos dre suepene/ stReview Chavacteristies Point mutotion voiants of TEM-1 or TEM-2 Point mutation voriants of SHV-1 TEM votints that ae resistant to inition by lovulanate ond sulbactom, but do not hove ESBL phenotype ‘TeMvotiants that ee resistant to nition by clavulanete end sulbectem and iso have ES8L phenotype Derived from the chromosomal f-Lactamase from Klayvera spp. Preferentilly hydroiyses cefotaxime More prevalent in? aeruginase than Enterobacterales Some veiants also hydrolyse carbapenems More prevalent inP. ceruginosa and A. baurmanoi then Enterobactercles Inhibition by newer flactamase inhibitors's variable Preferentially hydroiyses cetaz.dime and aztreonam compared with cefotaxime: Inhibitonby newer lactamase inhibits variable Preferentially hydroiyses ceftozidime and eztreonom compared with cefotaxime Preferentilly hydroiyses cetozidime and aztreonam compared with cefotaxime Teble 4. £581 families Family Nomenciature Te ‘Temoneiva, the potient infected with the frst isolate expressing TEM 1 sav Sulfhyary reagent yarable iT Inhibtorsistane JEM om Complex mutant derved from JEM-1 rem Cefozoxime-hydralysing g-octomase isolatecin Munich ces ‘Suiane extended spectrum Per Pseudomenos extended resistant ves Vietnam extended: spectrum Plactarase eet Belgium extended prlactamase TA Nomed after the Rahuica Indians (Mexico, fram whom the frst ‘solate wos obtained so From Sevrati fonticola oxy From klebsele onvtoce Incucble ‘chromosomally encoded Adopted from Jacoby." ‘appears that the impact of their presence among clinical isolates isminimal 3.3 Inhibitor-resistant p-lactamases Inhibitor-resistant PHlactamases are derivatives of TEM and SHV enzymes that have amino acid substitutions that confer resistance to inhibition by the p-lactamase inhibitors clowulanate cand. sulbactam, In the functional classification scheme, they belong t functional group 2br."* Most ofthese remain susceptibie to inhibition by tazobactam and avibactom.”>"* The majority of inhibitor-esistantf-lactamases ate derivatives of TEM-1 and were formerly called IRF (for inhibitor resistant TEM), but are now given sequential TEM numbering.”® Common substitutions in the TEM variants hove been characterized at amino acid postions Met69, Ser130, Arg244, Arg275 and Asn276 (Figure 2). It appears that the cost of the mutations resulting in resistance to clavulanate and sulbactam a reduction in the efficiency of hycrelysing some penicilins and cephalosporins such as cefalotin.”” though these ‘mutants are rarely detected, «strain of K. pneumoniae expressing the inhibitorresistant TEM-30 was identified in several KPC- producing isolates from an outbreak of carbapenem-resistant Enterobacteroles (CRE) in New York City.”? Several SHV-type loc- tamases have been characterized as inhibitor resistant, including SHV-69, -56 ond -107, which were identified in K. pneumoniae Clinical isolates from patientsin Europe.’* “° A few complex TEM mutant (CMI) Brlactamases have been described that are mutants of TEM -lactamises that have both the ESBL phenotype and inhibitor resistance. © These CMT variants will not be detected with any of the screening methods used ta detect ESBLs because those tests rely on inhibition with clavulon- ate, One such complex mutant, TEM-152, was found in an isolate of € callin a patient hospitalized in'Fronce.”* This mutont harboured amino acid substitutions Arglé4His and Gui26dLys, previousy observed in ESBLs, plus Met69Val and Asn276Asp, previ ously observed in the inhibitor-resistant enzyme TEM-36, which resulted in eficient hydrolys's of ceftazidime and a 50% reduction in inhibition by clavulenate. Because these complex mutants Gre not resistant to avibactam, ceftaziime/avibactarn or one of the other new p-lactamase inhibitor combinations maybe thera peutic option to treat infections caused by organisms expressing cone ofthese enzymes.” Itisikely that the prevalence of TEM or SHV-type inhibitor-esistant Blactamases is underestimated be- cause thereisnot a phenotypic test that laboratories can routinely Useto identify these strains * 34 CXM, CTX-MHtype Blactomase enzymes were initially reported in the late 1980s, emerging concomitantly in several ocotions. The no- ‘menclature CTXM (cefoteximase from Munich) wos inital used ina report from Germany However, CiX-M-type enzymes identi fied in other regions received cifferent names, including FEC-1 {Uopan),Toho-1 (opan) and MEN (Franceinon talon patient * ‘These intial reports were followed by outbreaks in several coun- ties. The worldwide expansion of isolates canying these ESELS later would be referred as the ‘CTX-M pandemic. Since the early 2000s, CTX-N-type enzymes have been recognized as the most common ESL group, replacing TEM and SHV as the dominant ESBL type. CTX'M variants have been reported among several 4of21 (spquepueaetuice'dno-aqapeneyrsdny way papeajuMoG 1202 fine £1 uo 8808 Aq yeazzeoresone1pirReview JAR Amino Aoid Reside TEN-1 suv-1 omen "MS IQHFRVALI ~~~ -PF FAAFCLPVFAH~PETLVKVKDAEDQLGARVGYIELDINSG -MRYIRLCIT-~-~SLLATLPLAVHAS-POPLEQIKLSESOLSGRVGMIEMDLASG .MUxCXSLRQPPLMATATVPLLLGS--VPLYAQTADVQOXLAELERQSGGRUGVALINTAD~ Amino Acid Residue ae 3 eM-1 KILESFROEERFI \VLSRVDAGOEOLGRRIHY SONDLVEYSPVTEXHLE suv RILITAWRADERE Pe sSRERWLCCAVLARVDAGDBQLERKTHYRQOULVDYSPVSERHLA ora NOQILYRADERE? VARVLKASESEPNLLNQRVEIKSULVNEMPIAEKEVD Amino Reid Residue TEM-1 sHV-1 oem Amino Acid Residue TEM-1 suv-1 emem-1 8 3 DGATVRELCSAATIMSDNTAANLLLT [GG PRELTAF LENMGDHVIRLDFWEPELNEAIP GMT VGELCAAATIMSDNSAANTLLATVGGPAGLTAFLRQIGDNVTRLDRWETELNEALP (GIMSLAELSAAALQY SDNVAMNIC.ISHVOGPASVTAPARQLGDETFRLDRTEPTLNTAIP. 'NDERD?TMPAAMATTLRKLL-?GELLTLASRQQI IDHMEADIVAGPLLRSAL PAGWPTADK. (GDARD??PTPASMAATLRKLLTSQRLSARSQRQLLQWMVDDEVAGPLIRSVLPAGMPTADK. (GDPRDT?PSPRAMAQTLENLTLGXALGD SQRAQLVTWMKGNTTGRASIQAGL PASHVVGDK. £2 'SGAGERGSRGITAALGPOGKPSRIVVIYPTGSQATMDERNRQIAEIGASLIXIW: "TGAGERGARG IVALLLGPNNKAERIVVI YERDTPASMAERNOQTAGIGAALTEHWO "70S GUY GTTNDIAVIWPKDRAPLILVTYPTQPQPKAE SRRIVLASAAKE ENGL eM-1 sHv-1 ome Figure 2. Amino acid alignrvents of TEM-1, $HV-1 and CTX-M-1. The omino ocd sequences WP_000027057.1 (TEM-1), W_001620085.1 (4V-1) and \WP_013188473.1 (CTX-N-1) were obtcined from NCBI and aligned using Clustal Omega."*"* Numbering accarding to Ambler.” Asterisk (1) nd ates positions that have o single, fully conserved residue. Coion() indicates conservation between groups of stronaly similar properties. Period {) indicates conservation between groups of weokYysilar properties. The yellow highlights show the active site Ser70-X-X-1ys active ste common to all serine f-tactamases. Rec emino acids denote residues where substitutions provide ES8L phenotype (TEM ond SHY). Blue amino acids denote where substitutions provide inhitr resistance phenotype. Green incicates postion 240 in CTX-M-1, which has been identified as being assocated with increased hydrolys's of cefotaxime, members of the order Enterobacterales and in aeruginosa and Acinetobacter spp." Isolates canying CTX-M-encading genes have been detected in nosocomial and community settings as well as in companion animals, the environment, food products, andiivestock Most CTX-M enzymes can be clustered into five groups based fon sequence homologies: CTX-M-1, CTX‘M-2, CTXM-8, CTKM-9 and CTX-M-25. By for the most common CTX-M-1 group is CTX-M- 15, followed by CTDM-3 and CTXM-L" Tn the CTKM-8 group, CIXM-9 ond CDM-14 were the most common enzymes, ut more recently CTX-M-27 has often been reported"? CIX-M-2, CTXN-8, and CTX-M-25 are the most frequent variants within thei ‘in groups. The ahaiysis ofthe upstream sequences flanking the genes encoding CTX-M-2 groups belonged to Kluyvera spp. Further analysis demonstrated that this group derived from KLUA-1, an enzyme from Kiuyvera ascorbate.” Siilay, CTX-M-134 (CD&-M-1 group) is derived from KLUC-1 from Kluyvera cryocres- cens and the CTK-M-9 group have similarity with KLUG-1 from Kiuyvera georgiana °°" Notably, CTX-M enzymes also exhibit structural similarity and hydrolytic profiles with other class A B-1oc- tamases from environmental organisms, such as Erwinia persicina ‘ond Rahneita aquatii°*°° The ecrly CTXM variants efficiently hydrolysed cefotaxime cond ceftriaxone, hence the name cefotaximase.”* Contrary to the TEM- and SHV-type ESBLs reported to that point, the early CTX-M enzymes had limited activity against ceftazidime, however CM, Variants with enhanced ceftazidime hydroiytic activity were later described. Important examples of CTX-M enzymes displaying ceftazidime hydrolysis are CTX-M-15 and CTX-M-27, which ore from the CIX-M-1 and CTX-M-9 groups, respectively CTGNE5 is derived from CT&M-3 and displays a single amino ‘cid change in aosition 240 (Asp to Giy) when compared with its ancestor (Figure 2).°° The Asp240 residues located inthe terminal part of the B3 fistrand and is responsible for the flexibility of this structure and the accommodation of ceftazidime, which is a bulkier molecule than cefotaxime.“ Despite the modest increase Sof 21 wo papeotmed eopeysunen(nioe dnersqepeceyrey 1202 fine 41 uo 8808 Aa yeazzearcsone1penReview inhydrolytic activity observed against ceftazidime, this change sig- nificantly increased ceftazidime MIC values of constructs canrying (CTX-M-15.°°°? CTX-M-27 has the same resicue in position 240 that is present in CTX-M-15, This residue confers elevated ceftazidime MIC values, despite the overall poor activity of CTX-M-27 against cther substrates compared with its ancestor CTXM-14."" Data from clinica isolates collected from the SENTRY Antimierobia, Surveillance Program showed that the ceftazidime MIC for CTX-M- producing isolates varies, with MIC values ranging from 0.25 mg. to>32meil.(M.Castanheita, JMI Laboratories, unpublished data) In 2019, Poitel et a.“ described a new CTX-M variant, CTX-M- 3, that had an alteration in posttion 109 (Asp to Ser) compared with CIX-M-15, This enzyme displayed decreased ceftazidime hydrolysis, but significant meropenem hydrolysis, elthough this transiated inte only a modest increase in meropenem MIC in an isogenic pair. However, the clinical isolate of K. pneumoniae isolate carying this new variant also had impaired permeability resulting ina meropenem MIC of mg/l. CTX-M-type P-lactamases are widespread enzymes. Although there are stil treatment options for isolates carrying these enzymes alone, the combination of these enzymes in isolates with other resistonce mechanisms and the exponsion of hydrolytic profiles with single amino acid muta~ tions could limit the activity of meropenem and newer agents. 3.5 ESBL phenotype OXA-type f-lactamases ‘The OXA-type fr-lactamases hydrolyse oxacilin and are grouped 1s Ambler ciass D and Bush-Jacoby-Medeiros functionol group 2d enzymes” In general, OXA-type enzymes are a broad group that displays variably in substrate profiles and omino acid sequences. However, several OXA-type variants have been noted to hydrolyse cephalosporins, cephems, ond/or maonobactoms. These OXA enzymes with an ESBL ‘phenotype are categorized in Bush functional subgroup 2de.* Whether er not these oxacilinases with activity against expanded-spectrum cephalosporins are defined as ESBLs is debatable.” Many researchers do not apply the ESBL terminology to oxacilinases because these enzymes are not Classified in the 2be group and are refractory to inhibition by clavu- lancte or otherinhibitorsin the some manners the true ESBLs. According to arecent review, there are 27 oxacilinase enzymes described as extended spectrum. These enzymes’ substrates in- clude third- and/or fourth-generation cephalosporins in addition to penicilins and early cephalosporins.** Most extended-spectrum ‘oxaclinases derive from OXA-10 (also named PSE-2) and OXA-2 ‘The OXA-10 dervatives include OXA-11, OXA-13, OXA-14, OXA-16, OXA-17, OXA-19 and OXA-28." In addition, OXA-16 has only a partiol sequence submitted as its first_descrition (GenBank ¥WAE043100), Among the OXA-2 derivatives, OXA-15, OXA-32, (OXA 34, OXA 36 (partial sequence), OXA.53, OXA-141, OXA 161, (OXA-210 and OXA-226 have been described." Many OXA-2 and, OXA-10 derivatives are detected in isolates of. aeruginosa. Despite not being considered os extended-spectrum oxacili- rnases, OXA-1 and OXA-30 hove been named for their ability to hydrolyse cefepime.°”*? OXA-1 and OXA-30 were intially reported to differ by one amino acid; however, it was corrected later that these enzymes were identical.” OXA-1 combined with loss of porins has been implicated in flse-ESBL phenotypes among E.coli isolates and resistance to frlactamase inhibitor combinations.” Contrary to most oxacilinases, which have a dimeric described structure, OXA-1 was reported to be a monomer.”® An OXA-31 that was detected in an isolate of P. aeruginosa had three amino ‘acid substitutions compared with OXA-1, including the amino acid differences from OXA-4 and also displayed activity against cefepime.’* OXA-48 derivatives, namely OXA-163 and OXA-405, have been described to display cetivity against extended spectrum Blactams with or without many of the OXA-“8-lhke enzyme's characteristic carbapenemase activty.””” 3.6 Other ESBL families The GES (Guiana extended-spectrum f-lactamase) family is the ‘most prevalent group ofthe ess common FBI's, The gene encod- ing GES-1 is not ciosely related to any other plasmid-mediated Brioctamase but does show 36% homology to a carbenicllin- hydrolysing enzyme from Proteus mirabilis ~ Despite intially being reported among species of Enterobacterales, GES enzymes are ‘more common among isolates of P. aeruginosa and A, baurmonnil isolates."-*? GES enzymes ore notable for ther ability to acquire single or double amino acid substitutions and expand their spec- ‘umtocorbapenems The ESBL GES-1 was frst described in 1998 in a K. pneumoniae isolate collected in France from a patient who had recently been hospitalized in French Guianc." At the same time, another group described a similar enzyme, named IBC, from an €, cloacae isolate from Greece.®® Subsequent enzymes GES-? and IBC-? were both found in isolates of P. aeruginosa isolates "°** I8C-1 was later renamed GES-7 and IBC-2, GES-8. Interestingly, GES-2 had a ‘single amino acid substitution {Gly170Asp) compared with GES-1 and displayed some hydrolytic coctivity against carbopenems.” Later described GES f-lactamases fell into two categories: enzymes that were ESBLs ond those that showed some modest carbapenemase activity, The original GES enzymes were ESBLs that hydrolyse pencils and cephalosporins wel, but not aztreonam. These enzymes ore inhibited by clavu lanate, tazcbactarn, and the newer lactamase inhibitors such as cvibactam, relebactam and vaborbactam.**** This means that isolates expressing GES enzymes are often susceptible to ceftaz dimelavibactam, but not ceftolozanetazobactom*? GES-1 hydrolyses ceftazidime better than cefotaxime. Amino acid substi- tutions of Glu1O4Lys or Gly243Ala/Ser that were detected in GES variants described later have been shown to confer greater tesist- ‘ance to cephalosporins ond aztreonam.” The PER-1 frloctamase (Pseudomonas extended resistant) was intialy desebed from an folate of P. aeruginesa displaying resistance to cephalosporins and inhibition to clavulanate.”** This enzyrne hydrolysed most penicilins well and cephalosporins including cefalotin, cefoperazone, cefuroxime, ceftriaxone and ceftazidime. PER-T' did not hydrolyse axaclin, cephamycins or imipenem, Only o few years later, PER-2 was described in a P. aeruginosa isolate from Argentina, which wos 86.4% homolo- (gous with PER-1."° PER enzymes have since been described from A, baumannii and Aeromonas spp, and in various species of Enterobacterales PER-1 and PER-2 are the most common members of the PER farnily. These enzymes have been reported to be inhibited by avi-, bactam to a lesser extent than other clas A frlactamases, with signficantefferences in MIC values for avibactam and relebactam when tested in combination with other frlactams””? More Gof 21 (spquepueaetuce dno-aqapeneyrsdhy wey papeaKuMoG 1202 fine £1 uo 8808 Aq yeazzeorceone1perReview JAR detailed studies are warranted due to the difference in activity of these two inhibitors of the same class, Recent analysis demon: strated that A. baumonni isolates harbouring PER enzymes can display elevated MIC values against cefiderocol, a siderophore cephalosporin.” PER enzymes are most commonly found in iso- lates from Turkey and Mediterranean countries.” VEB-1 (Vietnamese extended: spectrum f-lactamase) was first detected from an E, col isolate recovered from a Vietnamese in fant.” VEB-1 conferred high MIC values for ceftazidime and o7 treonom, but only modest elevation of MIC values for cefotaxime when expressed inan €. coli background. A -fold increase in cofe- ime MIC values and no activity against imipenem was observed. This enzyme was wel inhibited by clavulanate, but avbactam was initially reported to not reduce the ceftazidime MIC values for P. aeruginosa isolates harbouring these enzymes.” Further studies demonstrated that when various VEB enzymes were expressed in, «nF, colisogenic background, the ceftazidime/avibactam MIC va tues were reduced in a concentration-dependent manner, lowering 1 ceftazidime MIC values ~8-fold when mg/L of inhibitor was Used.”” VEB-1 and other VEB variants have been described among various Gram-negative pathogens of including multiple species of Enterobacterles, Vibrio spp, Achromobacter xyiosoxidons cand more clinically relevant species such as P. aeruginoso and A, baumannii!" Less common FS@Ls have been described, but their occurrence is limited, These less common ESBLs include SFO-1 from Serratia fonticola, TLA-1 from the Mexican indigenous people group Tlahuicas, TLA-2 from Germany that displays only $1% homology TLA-1,BES-1 from Brazi, ond BEL-1 from Belgium.* Ina review that addresses these rare ESBLs, Naas eta” summarized the MIC values for solotes corying these enzymes egainst various flac tams. The cefotaxime MIC value for a Blass transconjugant was mail and the result for ceftarcime was + mall. Fora bloga. s-har- bouring recombinant strain, the cefotaxime MIC resuits was 1 mg/L, but ceftazidime was mai. Higher ceftazidime MIC values were noted for clinical isolates carying blages (L6mg/L} or blonas (2256mgiL ‘several other ESBLs have been detected in the chromosome of Enterobacterales and non-fermentative species, Among those, the OXY f-actamases in Klebsiella oxytaca are probably the most common cause of resistance in clinicalisolates °°" 4, Molecular characterization of ESBL-producing isolates 4.1 Genetic environment of ESBL genes Mobile genetic elements (MGES) such as plasmids, transposons, in sertion Sequences, integrons and bacteriophages contribute to the dissemination of various ESBL-encoding genes. MGES can move themselves and/or genes from one location to another within the cell or be transfered fiom cel to cell orizantally by conjugation, nsformation er, in the case of bacteriophages, by transduc- n° More often than not, MGES carry multiple resistance genes that confer an MDR phenotype to their hosts. Some of the main elements of MGEs tha corr efferent ESEL types are highightedin the section below Genes encoding TEM-1, TEM-2 and ther ESBL derivatives are usually carried by Th-, Tn2-, or Tn3-Lke transposons (Figure 3).:°° These structures were intially named TnA and display 99% nu cleotide homology, with most nucleotide differences identified close to their resolvase (res) site.°” A limited number of studies specifically report on the MGE-carying, blarayrencoding ESBL enzymes. Inn early study, blozew-12 wos reported to be port of Tn841, which exhibits homology to Tn3.* The gene encoding TEM-3' was located on an interrupted copy on Tht. Tn2 was reported to camry blarey.1o whereas blorew-n, Was associated with Tni.!°"°” in all cases, these structures were embedded in plasmids." A study by Marcadé et a.” that evaluated replicon types of conjugative plasmids carrying ESBL genes revealed that 667% of the plasmids harbouring TEM-type ESBL genes belonged to the InoMC type. Most of these plasmids caried biaray 2s, but the plasmids also carried blorey.s, blorew0 and blcre.21. Others Confirmed the occurrence of blarkurencoding FSBLS in IncA/C plasmids °°" Notably, the blorews: reported by Marcodé etal" was embedded in incl plosmids. The presence of 1526 flanking blas. was inticly described in the eorly 1990s. Inthe frst report of bie, S26 was identified as, ‘the mobilizing element for multiple resistance genes and provided ‘a promoter forthe expression of blasy, (Figure 3)."* Intact copies of 1526 have been reported inthe plasmids or the chromosome of various bacterial species flanking blasi, portions ofits 5" proximal termini or defective 1526 elements.'"™"'® Genes encoding SHV- type ESBLs can be found either in plasmids or the chromosome. Seven plasmid replicon types that predominantly carry Blasi encoding ES8L enzymes Inc Ine; IncHI, Incl, IncLM, Ine cand IncX3—have been identified?” Different bios variants have been detected in each of these plasmid types, with the exception of Inck3, which has only been detected carrying blosiv-12® Additionally, Billard-Pomares et al reported a bios. 3-canying E.coli where this gene was embedded in a P1 bacteriophage structure. ISEcpI has been identified upstream of several lacrx m types (Figure 2) Lattigue et cl. *? observed an ISEcp1 upstream of the genes belonging to the CTKM groups 1,2, and 9, In another study, ert et al” analysed the genetic environment of 28 isolates carrying 7 unique blacrm types and observed ISEcp1 in 23 of them. Analysis ofthe sequences surrounding ISEcp1 and bldcre-w ‘ypes revealed signature sequences indicating that transposition events were respansibe fo the mobilization of blacrx m'”” Beyond promoting the dissemination of these genes, ISEcp! provided a strong promoter for the expression of blacrxs.'* ISEcp1 also has been detected flanking other frlactamase genes, including KLU enzymes in the Kluywera spp." Inaddition to ISFept, blac, have been detected in the 3" end of complex class 1 integrons between two qackD1/sull ele- ments." The ESL gene was not part of a gene cassette like the genes upstream of gacEDI/suli, but rather in all cases the ES2L gene was flanked upstream by o/f513. This structure has been named ISCR1 and was postulated to mobilize genes by rolling cr- cle. Notably, of513 might function as @ transposase that dsploys similarities to 1591-Uke transposases.”” Structures harbouring biecre including combinations involving 1526, can be observed in several combinations, most likely due to the development of multiple recombination exchanges overtime.” Elements harbouring blacrs ware usually carried by conjugative plasmids. In a study evaluating CTX-M-15-producing isolates, from seven countries located on four continents, Coque et al "?* 7of 21 wo papeotmed 1202 fn £1 uo 8808 Ae LeazzeerceoqePrerepmenueselwos dno wepene/c sthReview @ 0A mp Bag © 1826 Blagyy © See! bese nt ee ee Gene casetes a4 == a 305 7s BTIISCRT — Wiz 18 Baa w int eg, = ————— ——____ ra roan ©) iti Ma poms qacEAl sul! ofS —_ oa Figure 3. Genetic structures harbouring genes encoding ESBLs, Genetic structures most commonly reported to hotbour {a} blo (6) Blas, (4 leery fd blany oF () close 1 integrons that ean carry uncommon ESBL genes. Schematic representations were adapted from Rossolin: et $5 Potel et ai and Destaet al? ‘observed that blacryw-15 was embedded in the narrow host range plasmid IncF with replicon types FIT alone or in association with FIA or FIB, Subsequent anaiysis demonstrated that this was true for various other isolates that harboured blac;xqs.3s. The dissemin- lion of blocrcw group 9 genes seems to be associated with cn IncHiI2-type plasmid, but there have also been reports of IncFll- types.) Other blacrs y carried various incorpatbity-type plas- ‘mids, including norraw and broad range conjugative plasmids that ‘may also cany additional resistance genes. 4.2 Common strain types for ESBL-producing isolates MLSThas been used extensively to track and monitor the spread of. resistance determinants in bacterial pathogens. Several widely disseminated sequence types have been found in epidemics and outbreaks due to resistant clones that are highly associated with specific resistance mechanisms. Unt the mid-2000s, it appeored that CDGM enzymes spread in an seemingly random pattern, with ‘no major clones responsible for theircissernination. However, in the last two decades, the dissemination of CTX-M-producing enzymes has been mainly associated with spread of E, coibetong- ing to. anew clonal group $1131.""° E.coli ST131 derives from the phylogenetic group B2 and sero- type 025b: Hé and exhibits multiple virulence factors such as tachesins,siderophores, toxins and a group 2 capsule. ST131 iso- lates differ from most other MDR E, colby being quite pathogen- ic!" E calibelonging to ST131 causes a wide variety of infections, but is most commonly found in urinary tract infections including cystitis, pyelonephritis ond urosepsis = E.coli ST131 isolates have been reported to cary a varity of BHloctamases and several CIXM types, most commonly, CTX M-15.*" Five groups (A through E) have been described according Bof 21 (spquepueaetuce dno-aqapeneyrsdhy wey papeaKuMoG 1202 fine £1 uo 8808 Aq yeazzeorceone1perReview JAR to the virulence factors identified among E. coli ST131 isolates. These clones vary according to geographic region. Interestingly, Virotypes A, 8 and over haif of virotype C cory blacrx-wss, whereas isolates from virotype O carry other B-lactamase genes, including blac. group 9 genes and blasiy 12!" In addition to blacre 1, other characterises of virotypes A, B and C include resistance {0 fluoroquinolones and the Type 1 fimbria gene fimH/30." This group also corres a ISL3-ike transposase within its fimit gene. ‘Typing fm highlighted thatthe subgroups H30, H30-R, and H30- Rare associated with MDR clones of €,col/ST131.."° These groups seem to have evolved in a stepwise manner, frst by acauiing, fluoroquinolone resistance for H30-R and then incorporating blacre1u 35 forthe H30-Rx group.""° ‘The occurrence of ST131E.colisolates carrying blaceavs hove been well documented globally. In an early survey, Coque etal 7? reported that ST131 €. coli solates producing CTX-M-15 and belonging to $1131 were detected in all seven countries for which isolates were analysed. Among E.coli clinica isolates collected as part of the SENTRY and MYSTIC programmes in 2007, it was found {hol 56/127 (47.19%) isolates belonged to ST131."" These isolates, were estimated to correspond to 17% of the overall isolates. Almost 70% of the ST131 isolates were resistant to fluoroquine- lones or broad-spectrum cephalosporins that was mediated mainly by CTX-M-15,!"° Peirano et al? reported that 46% of the ESBL-producing E. col isolates collected in 11 Canadian hospitals belonged to ST131. Most of these isolates harboured bloc. 145 but other lacy types were also observed. More recentiy, Mendes et ot” reported that $3.6% of the bloodstream and 58.2% of the urinary tract infection isolates collected in US hospitals as part of the SENTRY programme belonged to ST131 oF to clonal complex {CC} 131. These solates were collected during 2016in 36 USstates and were screened using WGS after displaying elevated MIC values of ceftazidime, ceftriaxone, aztreonam or the carbapenems. A recent study from Colombia showed that E.coli solates from patients with urinary tract infections that expressed CTGM-15 al belonged to ST131 and the epidemic sub- clone 025b: H4-B2-H30-Rx."" The emergence of other E.coli and & pneumoniae St disseminating blac.» genes has been recertly documented, with Bldcr:y13 Being the most prevalent"? ‘Among these, $T1193 F coll appears to have rapidly emerged worldwide." ‘Among the less common ESBLs, GES- and VEB-encoding genes are usually gene cassettes within cass |integron structures.”"?"° These structures can be mobilized os single genes and often are carried olongside other resistance genes that confer resistance to aminoglycosides, quinolones andior trimethoprim/sulfameth- oxazole, The genes encoding PER are a part of a composite transposons such as T1213, Tn4176 and ISCR. TLA-1 and PME-1 cre cartied by ISCR structures."™*""°"*" Lastly, 1526 has been detected flanking both ends of loses and las!” 5. Epidemiology In the early 2000s, reports suggested that CTX-M-producing iso lates were becoming widespread in Europe, Latin America andthe Asia-Pecific region." '"* Previously, TEM- ond SHV-type enzymes had been the most predominant ESBLs worldwide.” Later, this shit in the ESBL nopuiotion toward higher numbers of CDeNeprecucing isolates was observed in the USA with two studies: fist CTX-M-producing isolates were found ina single hos- pital and then these isolates were found in 80% ofthe hospitals participating in the MYSTIC survelance programme, with CTX-M- 15 and CTXM-14 being the most prevalent types identified. “°""! Since the year 2000, the incidence of ESBL infections has risen in the USA with an increase of 53% between 2012 and 2017, largely due to an increase in community-onset cases." Evaluating the epidemiology of EBL from aliterature review is challenging. As studies use varying isolate selection criteria and a range of methodologies to detect genes, remarkable disparities in outcomes are generated. Unpublished data (M. Castanheira, IMI LLobaratores) from the SENTRY Antimicrobial Surveilance Program demonstrated that among 22548 non-carbapenem-resistant E.coli cnd K. pneumonige cinial isolates consecutively collected in US hospitals, 3363 isolates exhibited on MIC value >? malt. for ‘wo of the following agents. ceftazidime, ceftriaxone or aztreo- nam. These isolates were screened for f-loctamases using previ ously described methods. “7° An ESBL gene was detected in 2059 (13.3%) €, coli and 836 (11.8%) K. pneumoniae, OF these, 92.5% carried CTX-M-encoding genes belonging to the CTXM group 1 (70.0%) or CTX group 9 (22.8%). SHV genes encoding ESBL enzymes were noted among 8.6% of sequenced isolates, mostly in K. pneumonice (6.5%). TEM ESBLs were only detected ‘among 20 isolates, including 16 F. coli isolates. The prevalence of €S8Ls can vary with geographical location, even within one country. A recent study of Grom-negative biood culture isolates token across the USA showed on overall prevalence of 11% for blacrx.m, however the percentages ranged from 54% (Michigan) 10 26% (Washington, DC). ‘Among 15449 non-CRE €. coli and K. pneumonige clinical isolates collected as a part of the SENTRY programme in Europe, Asic-Peciticand Latin America, an ESBL gene was detected among 82%, 15.4% ond 303% of the isolates, respectively {(M.Castonheira, JMI Laboratories, unpublished data). These rates Varied among individual countries (Figure 4), Simitar tothe scenario inthe USA, most isolates carrying cn ESBL gene from the Europe, the Asia-Pacific region and Latin America harboured a CTX-M gene (95.1%, 85.2%, ond 98.1%, respectively). Genes belonging to CTX M group 1 and CIM group 9 were the most common. Canton and Coque’”” reported endemicity of CTX-M-producing isolates in vorious geographic areas. The authors highlighted a drastic increase among E. clisolates producing CTX-Min the early 2000s. In their analysis, CTXM-3 and CTX-M-15 were the most ‘common genes detected among the CTX group 1 and CTXM-9. Additionally, CTX-M-14 was most frequent gene observed among group 9. Livermore ot ot reported similar observations when evaluating several European countries. A literature search by Bevan et of.“ revealed a significant increase in ESBLs in all of the \WHO regions analysed, Ths increase in the prevalence of ESBLS, ‘was mainly caused by the dissemination of TM genes. blacresus5 was the most common gene in all regions except Latin America, where Blacr: 2 Was the most common gene observed. Among ther groups, Blacrcats Bldcrew ANA Blocrxw7 have also spread globally ‘study evaluating the epidemiology of ESBL-producing E.colfin Spain demonstrated o decrease of TEM-producing isolates from >19% in 2000 to 1.2% in 2006." These investigators aiso highlighted the dominance of CTX-M-producing €. coli, ana noted that CTXCM-14 was the most common type of ES@L. Subsequent of 21 wo papeotmed eoqueysunen(pioe dhersqepeceyredy 1202 fine 41 uo 8808 Aa yeazzearcsone1penReview eet ame Eo aoe chet eatin xa a — cen i is) B a 3 3 i al] [ER 4 2 : Figure 4, vistibution of 7 studies by the same group highlighted on increase of CTX-M-15- producing isolates tht appeared to replace the CIX-M-14- producing population."**"°’ Rodriguez-Vilalobos et al! high- lighted an increase in ESBL production and differences in ESBL _ TEM and SHV- producing isolates in the USA, Asia-Pacific, Furope and Latin America types when comparing clinical isolates from 2008 to 2006. These outhors evaluated Enterobacterales isolates collected in 118 Belgium clinical aboratories and noted that ESBL rates and CTXM production increased in E. cali, K. preumonige and €. cloacae 100f 21 1202 fine 11 uo ant Aa yeazzcoresoReIpeie/eonvenwereinuco-cho oqwapeneicedhy WOM PapEOIWMERReview JAR isolates. Simitar trends were not observed among K. aerogenes Similaty, Peano et a.” reported CTK-M enzymes replacing SHV- type ESSLs among K. pneumoniae when surveying Canadian isolates, which has aiso been observed in other regions 2° ESBLS are less common in P. aeruginosa than in isolates of Enterobacterales. Croughs et al" evaluated 15282. aeruginosa isolates from referral hosptalsin the Netherlands: 113 isolates ds- playing ceftazidime MIC values >8mag/L were screened for ESBL genes and only 6 isolates (0.4% overall 5.3% among ceftazidime: resistant isolates) possessed ESBLs. These Dutch ESBL-carnying P. aeruginosa isolates harboured blarew-12 (2 isolates), blows.» (2), biases (1) and oxacilinase genes (2) Laudy et ol." reported th ‘among 300. aeruginosa isolates recovered during 2010-14 from four hospitals in Poland, 99 carried (11.0%) ESBL genes. Among the ES8Ls, 69 isolates had VEB-9 and 14 isolates had GES (6 with GES-1, 1 with GES-5, 5 with GES-13 and 2 with GES-15). In Braz CTXM-2 was detected among 19.6% of carbapenem-resista P. ceruginosa screened for ESBLs.** In this study, 2/56 isolates carried GFS-encoding genes. In Greece, PER-L-producing, P. aeruginosa isolates belonging to the international high-risk clo- nal complex 11 were dented among the isolates that displayed a ceftazidime MIC >8 mg/L and a postive ESBL phenotypic Additionally, outbrecks of lay producing P. aeruginosa were described in France ond Tunsia"**"* Data regarding P. aeruginosa producing of ESBLs in US isolates is scarce. the anc- lysis of 155. aeruginosa isolates reported as part of « previous study evaluating resistance mechanisms against vavious ant pseudomanal frlactams revealed that only 3 (1.9%) isolates horboured Sats." ESBL-producing A. boumanni isolates have been described in specific locations andlor as part of outbreaks, ESBL genes that have been reported in A. baumannilinclude GES, VEB, PER, TEM and CTXNF415, among others." & study by Endimian’ et al screened 407 A. boumannilisolates collected in an Itaian hospital luting a 7 year period for resistance to ceftazidime. OF the 119 thot hed MIC values >8 mg/L, 31 isolotes were found to possess blarey.s I @ study from Celenza et al,"® 150, baumannii iso- lates from a Bolivian hospital were screened for ESBLs and found that 106 carried blacreyy2, 32 corfied blacress ond 12 carried Dlares 2. Mary other studies highlight single occurrences or groups of isolates harbouring PER, VEB ond GES in A. baumanni isolates suggesting these are the most common ESBLS in this species.” The dissemination of ESBL-producing bacteriol pathogens is likely due to many factors such as geagraphical location, popula tion density, hygiene and usage of antibitics. For example, the prevalence of ES8LS in E col is low in Europe but is very high in Southeast Asi, Attica, and Central Ameria," There even coun: tty to country variation with regions. For exemple, the prevolence of Enterobacterales expressing ESBLs is higher in Meciterranean countries but is very low in the Netherlands and Scandinavia, Today, our globol society is quite mobile, whether it be os vacationers, medical tourists or refugees. Al ofthese factors heve Contributed to outbreaks and the overall global dissernnation of SBL-mediated resistance. The epidemiology of ESBL-mediated resistonce primarily fol- lows the type of infections where the pathogen encountered often require heavy usage of exponded-spectrum f-lactam antibiotics. For ESBL-producing Enterobacterales, the main source of these pethogens is the genitourinary tract of patients, with infections most often caused by strains with which the patient is, already colonized.” The transmission of ESBL-producing Enterobacterales can occur betiveen patients with or without the involvement ofa healthcare worker as an intermediate vector. The rate of transmission is also Ikely to vary based on differences, in various species due to differences in virulence factors. The ransmission rate from patients colonized with ESBL-producing K. pneumoniae wos shown to be 2-fold higher than from those colonized with ESBL producing Ecol." Initaly, E5@L-producing clinical isolates were found onlyin the hospital setting, however, they quickly spread into nursing homes cand then into the community" Although most outbrecks of ESBL-producing Fnterabacterales accur in the ICU or in immuno: compromised patients, other patient populations can also be affected. In Japan, there was an outbreak of ESBL-producing E.coli in neonates that was traced to shared breast mik from donor mothers.” A 2017 survey of US hospital infections caused by -BL-producingE,coliand K. pneumoniae showed thatthe rates of nese infections were increasing."”* Both TEM- and SHV-type ESBLs were detected throughout the USA and Europe inthe Ite 1980s and 1990s with specific variants, noted to have variations in regional prevalence."**17""*° For ex ‘ample, TEM-10 was identified in several unrelated autbrecks of ESBL-producing Enterabacterales in the USA, but was rarely seen, in Europe.” The prevalence of both TEM- and SHV-type ESBLs has now diminished at the same time as the worldwide dominance of solates producing CTX-M-type frlactamases has occurred.** ESBLS have also been reported fram many environmental, food cand veterinary samples. 6. Detection of ESBL-producing Gram-negative organisms in clinical microbiology laboratories Detection of ESBL-producing Enterobacterales has traditionally relied on phenatypic methods for detection in cinical microbiology laboratories. These methods exploit the fact that £SBLs are inhibited by traditional f-lactamase inhibitors such as clavulanate. Both CLSI and EUCAST have endorsed screening ond confirmatory (ests for detection of ESBL producers and guidance on use of these {ests vaties bosec on the cephalosporin breakpoints applied by the laboratory.'**"* although the methods described by these stand ‘rds setting organizations are similar, differences exist in the rec ‘ommended organisms to test, screening and confirmatory test methods ond interpretations (Table 2). These ESBL methods re- ue overnight incubation and have known limitations that affect oth sensitivity (e.g, false negatives due ta the co-production of an AmpC frlactamase) and specificity (e.g. false positivty due to hyperproduction of narrower-spectrum f-lactamases combined with altered permecbilty). Commercially available automated “ntimicrobie susceptibilty testing systems have adopted compar- ‘able built-in €SBL screening and canfrmation tests on thelr ponels However, these systems are known to report false positive ESAL results and some lack US FDA clearance for P. mirabilis due to poor performance." Over a decade ago, both CLSI and EUCAST lowered the cephalosporin breckpoints to increase the sensitivity of identifying 1iof ai wo papeotmed 1202 fn £1 uo 8808 Ae LeazzeerceoqePrerepmenueselwos dno wepene/c sthReview Tal 2, ESBL screen and confirmatory tests as recommenced by CLST and FUCAST Citeria rgerisms Screening test methods Screening agents and cutoffs Positive serening results Confirmatory test methods Test Positive interpretation asi F col K axytoce, K. preumonige and P, mirabils Disc diffusion ond BMD methods Aatreonam, cefotaxime, ceftazidime and cefticxone MIC of >2 mg/l. Cefpodaxime MIC af 22 mail for P. mirabilis MIC 28mg for, col K.preumonige ond K axytoco Ether ()cefpodoxime alone Or (i extrecnam (excluding P.mirbils, cfetox- ime, ceftezid'me or ce/tnaxone screen positive Disc diffusion ond BMD methods Ceftezidime and cefotaxime + ciovulenate Disc diffusion: 25 mm increase in zane diameter for either agent tested in combinelion with lawulonte versus the zone diameter af the gent tested alone BMD: 23 2old concentration decreases in an MIC for ether agent tested in coronation with ciaws- lanate versus the MIC of the agent tested clone Reporting cephalosporin results fr FSBL-producing isolates Report all pencilins, cephalospotins ond aztreonam asresistent Use of obsolete cephalosporin breakpoints Use of current cephalosporin breakpoints Report the MICs and interpretations as tested eucast ‘Group 1-F. col Kebsiella spp. (not including Klebselio formerly Enterobacter aerogenes) P. mirbils, ooutelle spp, Saimoneta sap. and Shigeo sp. ‘Group 2 (Enterabacterales with inducible chrome- somal AmpC): Enterobacter spa, Ctrabacter freundi, Morgana morgani, rovidencia stuart Serratia spp, of ave! Broth elution, agar dilution a ise aiffusion Cefpadoxime, cefotaxime, ceftaridime and cefleaxane MICof>2mght Either (i cefpodoxime clone {i cefotaxime or ceftriaxone AND ceftazidime screen postive CDT, DDST, ESBL gradient test ond BMD test Group 1: Ceftazidime and cefotaxime = cowlenate, ‘ode cefepime + clavulanate if cafoxtin as been tested and hos an MIC of 236 mg/l ‘Group 2: Cefepime + ciovulanate DI: Same interpretation es the CLS se difusion test DST: Zones of inhibition oround cepholosperin discs are cugmented ot there ia keyhole inthe direction ofthe dsc containing covalanete MD: >8-foldrecucion is oaserved inthe MIC ofthe cephalosporin combined with clavulanate comn- pared with the MIC of the cephalosporin alone ‘Gragient diffusion: Ine same es above fr BMD or if ‘9 phantom zone or deformed elipseis resent BMD, brath microdution; COT, combination dsc test; DDS, double-disc syneray test Adopted fram CLSI™" and EUCAST."" ESBL-producing organisms, to decrease the burden confirmatory testing placed on microbiology laboratories and because of Updated. pharmacokinetics (Pkl/pharmacodynamics (PD) data, MIC dlstrbutions ond limited cinical outcome dota suggesting improved patient outcomes with lower breakpoints."*""* With the lowering of the breakpoints, they revised the recommenda- tions to perform ESBL confirmatory tests for epidemiological or in- fection control purposes only."®"®? Based on this guidance, many laboratories updated their cephalosporin breakpoints and stopped performing routine ESBL confirmatory testing. As such, the MICs and interpretations are reported as tested for the penicilins, cephalosporins and aztrecnam without identifying the mechan- ismleading to third-generation cephalosporin resistance ‘The MERINO trial was the fst randomized clinical tral compar- ing the outcomes of patients receiving piperacilin/tazobacta and meropenem for the treatment of presumed ESBL-producing bloodstreaminfections.** The original analyses found inferior out- comes for patients treated with piperacilin/tazobactam (although they were later medified due to inaccurate susceptiblity testing) This has led to a renewed interest in understanding the role of £SBL testsin clinical practice. In the absence of ESBL confrrmation testing, some clinicians and more recently the IDSA antimicrobial, 120f21 (spquepueaetuce dno-aqapeneyrsdhy wey papeaKuMoG 1202 fine £1 uo 8808 Aq yeazzeorceone1per