-lattamasiPenicillinasi-cefalosporinasi-carbapenemasi

-lactamase

Clinically isolated distinct -lactamase now number > 1400.

The simplest classification is by protein sequence, four classes, A, B, C, and D,

Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl

enzyme through an active site serine, whereas class B -lactamases are metalloenzymes that

utilize at least one active-site zinc ion to facilitate -lactam hydrolysis.

Two major classification schemes exist for categorizing -lactamase enzymes: Ambler classes A through D, based onamino acid sequence homology, and Bush-Jacoby-Medeiros groups 1 through 4, based on substrate and inhibitor profile. A “family portrait” reveals the structural similarity of class A, C, and D serine -lactamases . Class B -lactamases (“a class apart”) are metallo—lactamases (MBLs) .

Carbapenemases are β-lactamases that hydrolyze penicillins, in most cases cephalosporins, and to various degrees carbapenems and monobactams (the latter are not hydrolyzed by metallo-β-lactamases).

-lactamaseClassA enzymes

ClassA enzymes are very prevalent among bacteria, especially in the Enterobacteriaceae. TEM-1 and TEM-2 were some of the first beta-lactamases described in the 1960s. SHV-1 was discovered later. These enzymes can be encoded on conjugative plasmids,

which have served as genetic vehicles for wide dispersal of these resistance genes. SHV-1 is also found on the chromosome

of Klebsiella pneumoniae. TEM-1, TEM-2, and SHV-1 confer resistance to commonly used antibiotics, such as ampicillin and amoxicillin. Other important substrates for these three enzymes include carbenicillin, pipercillin, cefazolin, and cefuroxime. The extent of hydrolysis for these drugs can vary depending upon

the quantity of the -lactamase that is produced. Class A enzymes are inhibited by clavulanic acid (sulbactam,

tazobactam). In addition, clavulanic acid has proved useful in the laboratory for detection of some classA enzymes.

-lactamaseClassA ESBL enzymes

ESBLs hydrolyze penicillins, narrow- and extended-spectrum cephalosporins (including the anti-methicillin-

resistant S. aureus [MRSA] cephalosporin), and the monobactam aztreonam. In contrast, ESBLs cannot

efficiently degrade cephamycins, carbapenems, and –lactamase inhibitors. More than 200 different ESBLs have been identified, posing a significant risk to public health

and to hospitalized patients in intensive care units, where infection with an ESBL may lead to significant morbidity and mortality The majority of ESBLs are from the SHV,

TEM, and CTX-M families; less frequently they are derived from BES, GES-1, VEB, and PER enzymes, and sometimes

these enzymes do not belong to any defined family.

-lactamaseClass A serine carbapenemases

Class A serine carbapenemases include the nonmetallocarbapenamase of class A (NMC-A), IMI, SME, and KPC. Members of this group of -lactamases can hydrolyze

carbapenems as well as cephalosporins, penicillins, and aztreonam. These carbapenemhydrolyzing enzymes have been identified primarily in Enterobacter cloacae, Serratia

marcescens, and K. pneumoniae, bacteria which often harbor multiple resistance determinants, narrowing the range of

treatment options. The bla gene for the former two organisms is typically found on the chromosome, while the K.

pneumoniae carbapenemase blaKPC gene is carried on plasmids containing Tn4401. MICs of carbapenems in

carbapenemase-expressing strains can vary from moderately increased (2 to 4 g/ml) to resistant ( 32g/ml)

-lactamaseClass B metallo--lactamases

Class B enzymes are Zn- dependent -lactamases that demonstrate a hydrolytic mechanism different from that of the

serine -lactamases of classes A, C, and D. Organisms producing these enzymes usually exhibit resistance to penicillins,

cephalosporins, carbapenems, and the clinically available -lactamase inhibitors . Interestingly, the hydrolytic profile of MBLs

does not typically include aztreonam. MBLs likely evolved separately from the other Ambler classes, which have serine at

their active site. The blaMBL genes are located on the chromosome, plasmid, and integrons . P. aeruginosa, K.

pneumoniae, and A. baumannii produce class B enzymes encoded by mobile genetic elements. In contrast, Bacillus spp.,

Chryseobacterium spp., and Stenotrophomonas maltophilia possess chromosomally encoded MBLs, but the majority of these pathogens are generally not responsible for serious infections.

-lactamaseClass C serine cephalosporinases

Class C AmpC –lactamases include CMY-2, P99, ACT-1, and DHA-1, which are usually encoded by chromosomal bla genes, although

plasmid-borne AmpC enzymes are becoming more prevalent. Organisms expressing the AmpC –lactamase are resistant to penicillins, -lactam–-lactamase inhibitor combinations, and

cephalosporins, including cefoxitin, cefotetan, ceftriaxone, and cefotaxime. AmpC enzymes poorly hydrolyze cefepime and are

inhibited by cloxacillin, oxacillin, and aztreonam. Members of the Enterobacteriaceae family, such as Enterobacter spp. and

Citrobacter spp., are AmpC -lactamase producers that resist inhibition by clavulanate and sulbactam, although Klebsiella spp.,

Salmonella spp., and Proteus spp. normally do not harbor chromosomal blaAmpC genes. Production of chromosomal AmpCs

in Gram-negative bacteria is at a low level (“repressed”) but can be “derepressed” by induction with certain -lactams, particularly

cefoxitin.

-lactamaseClass D serine oxacillinases

Class D -lactamases were initially categorized as “oxacillinases” because of their ability to hydrolyze oxacillin at a rate of at least 50% of that of benzylpenicillin, in contrast to the

relatively slow hydrolysis of oxacillin by classes A and C. In bacteria, OXA –lactamases can also confer resistance to

penicillins, cephalosporins, extended-spectrum cephalosporins (OXA-type ESBLs), and carbapenems (OXA-type

carbapenemases). OXA enzymes are resistant to inhibition by clavulanate, sulbactam, and tazobactam, (with some exceptions;

e.g., OXA-2 and OXA-32 are inhibited by tazobactam but not sulbactam and clavulanate, and OXA-53 is inhibited by

clavulanate). Examples of OXA enzymes include those rapidly emerging in A. baumannii (e.g., OXA-23 and OXA-24/40) and

constitutively expressed in P. aeruginosa (e.g., OXA-50).-

Inibitori delle β-lattamasi

Alcuni β-lattamici, derivati dalla ricerca di nuovi antimicrobici, sono stati inizialmente scartati a

causa della loro modesta potenza antibatterica nei confronti dei vari patogeni. Un numero ristretto di

queste molecole è stato in seguito identificato come capace di legarsi in modo covalente alle β-

lattamasi. Attraverso tale meccanismo l’enzima viene catturato e reso indisponibile per inattivare

l’altro eventuale farmaco presente.

Inibitori delle β-lattamasi

Gli inibitori suicidi oggi in uso sono: acido clavulanico, trovato in colture di

Streptomyces clavurigerus, tazobactam che è un sulfone dell’acido penicillanico e sulbactam che è un 6-desaminopenicillino sulfone. Questi

composti sono abbinati a penicilline o cefalosporine garantendo loro

immunità dalle più diffuse β-lattamasi.

Currently available lactamase inhibitors are effectively limited to many class A -lactamases, excluding the KPC carbapenemases.

Ceftolozane--tazobactamCXA-101 (oxyimino-aminothiazolyl cephalosporin) + tazobactam (beta-lactamase inhibitor) (2:1 ratio)

Microbiology Coverage •P. aeruginosa (MIC90 = 1-8 μg/ml)

•CXA-101 is not a substrate of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps nor the carbapenem-specific porin OprD in P. aeruginosa

Spectrum Gaps •Bacteria producing metallo-lactamases and certain ESBLs (OXA-15 and OXA-11, -14 and 16) confer losses of CXA-201 activity (MIC >32 μg/ml); derepression of ampC in P. aeruginosa can increase MIC up to 8-fold; MIC90 vs. MDR P. aeruginosa of >64 μg/ml

•Ceftazidime-R E. coli, K. pneumoniae ceftazidime-R or KPC producers, Enterobacter, Citrobacter, ESBL+ Proteus spp. (MIC90= 16/>16/>16/>16/>16 μg/ml)

•No activity against MRSA and other key gram-positives

IV only

The addition of tazobactam at 8 μg/ml resulted in restoring the susceptibility of 93% of the ESBL producers and 95% of the AmpC overproducers. Tazobactam was unable to lower MICs, however, for Enterobacteriaceae producing KPC carbapenemases

Molecole in evoluzione

Tra gli inibitori suicidi che non includono i beta-lattamici vi sono acido boronico, fosfonati (poco

stabili e sensibili alle fosfodiesterasi) e diazabiciclo

octanoniAvibactam inibisce B, C e D beta-

lactamasi, ma non le metallo beta-lattamasi.

E’ in fase di sperimentazione clinica

Avibactam DBO diazabiciclo octanoni

Avibactam has an extremely broad spectrum of activity against classes A and C serine -lactamases, including ESBLs and class A carbapenemases This molecule inhibits selected class D -lactamases including OXA-48, but apparently not other class D carbapenemases, as judged by the absence of synergy with imipenem against resistant strains of A. baumannii producing OXA-51 and OXA-58 and does not inhibit class B MBLs.

Ceftazidime-avibactam

Microbiology •Covers ESBL-producing Enterobacteriaceae and P. aeruginosa isolates producing class A and C β-lactamases, including KPC producers as well as AmpC-overexpressing strains

Spectrum Gaps •Not active against bacteria producing metallo-lactamases, OXA or VEB ESBLs, OXA carbapenemases in A. baumannii, NDM-1 producers, and efflux-mediated ceftazidime resistance in P. aeruginosa

IV only

Ceftaroline - avibactam

•Gram-positives: MRSA, VRSA, MRSE, VRE ( E. faecalis, amp-sensitive), S. pneumoniae, S. pyogenes

•Gram-negatives: H. influenzae, M. catarrhalis, ESBL-producing Enterobacteriaceae, wild type Acinetobacter, KPC-producers

•Atypicals: No coverage •Anaerobes: No, requires combination with metronidazole Spectrum

Gaps: Acinetobacter producing OXA β-lactamases , Enterobacteriaceae producing metallo-β-lactamases, P. aeruginosa producing AmpC or with reduced outer membrane permeability, amp-resistant E. faecalis

Mutant Selection: Frequencies for stable mutants from 25 enterobacteria with ESBL, AmpC or KPC β-lactamases were mostly < 10-9

IV only

Aztreonam-Avibactam

Monobactams are hydrolyzed by ESBLs

but are inherently stable to hydrolysis

by MBLs, an avibactam-aztreonam combination resulted

inhibitory to MBL-producing pathogens.

MK 7655

MK-7655, a new member of the DBO series. A combination of imipenem and MK-7655 has excellent activity against a KPC-2-producing isolate of K. pneumoniae and displays moderate improvements in the imipenem efficacy against most AmpC-overexpressing isolates of P. aeruginosa.

Imipenem-MK7655

Spectrum with Imipenem

•Gram-positives: streptococci, MSSA, MSSE, non-VRE

•Gram-negatives: At 4-8 μg/ml MK-7655, imipenem MICs of all strains were below the resistant breakpoint of IPM for Pseudomonas and Klebsiella containing KPCs

•Atypicals: No coverage

•Anaerobes: Coverage of B. fragilis and others Spectrum Gaps: VRE, MRSA, Stenotrophomonas spp., Burkholderia spp. strains containing class D metalloproteases; high levels of AmpC or KPC; certain class A β-lactamases Mutant Selection: 10-8 - 10-9 for 2 isolates of P. aeruginosa; 2 x10-7 to <3 x 10-8 for KPC+ K. pneumoniae for imipenem + MK-7655

IV only

Preclinical Findings

•Restored activity of imipenem to kill rapidly

•Low protein binding (20%)

RPX7009RPX7009, a boronic acid-containing -lactamase inactivator with inhibitory activity against class A and class C

serine -lactamases, particularly highlighting both in vitro and in vivo

activity against KPC-producing K. pneumoniae. Boronic acid inhibitors of PBPs and of classes A, C, and D -

lactamases had previously been identified, but none has achieved success as a clinical candidate. Preclinical testing of RPX7009

indicated that the inhibitor had no off-target effects, and it was well tolerated at high doses, with no

safety signals that would preclude future development

Biapenem-RPX7009

Biapenem (“RPX- 2003”) is a broad-spectrum carbapenem with in vitro activity against Gram-negative and Gram-positive bacteria similar to that of meropenem. Like other carbapenems, biapenem is not affected by the presence of

ESBLs but is labile to hydrolysis by both serine and metallo-carbapenemases. Although

carbapenem resistance is increasing, 75% of recent Japanese pseudomonal isolates were susceptible to biapenem and meropenem.

Pharmacologically, biapenem is notable for its low proconvulsive activity compared to that of

imipenem

Carbapenem + carbapenem

A combination of ertapenem and doripenem in both an in vitro chemostat and an in vivo murine thigh infection model. Overall, the combination of doripenem plus ertapenem

demonstrated enhanced efficacy over either agent alone (2011)

Double-Carbapenem Therapy Not Proven To Be More Active than Carbapenem Monotherapy against KPC-Positive Klebsiella

pneumoniae (2012)

Ertapenem plus doripenem or meropenem were given in three patients suffering from pandrug-resistant, KPC-2-positive

Klebsiella pneumoniae bacteremia (2 patients) and urinary tract infection (1 patient), respectively. All responded

successfully, without relapse at follow-up. The results obtained should probably be attributed to ertapenem’s increased affinity

for the carbapenemases hindering doripenem/meropenem degradation in the environment of the microorganism (2013)

Enzimi autolitici

La lisi cellulare ottenuta con le penicilline non è dovuta alla semplice inibizione della sintesi del peptidoglicano ma all’induzione di enzimi: un’amilasi che scinde i legami tra i tetrapeptidi e, una glucosilasi che scinde il glucano.

Quando questi enzimi sono inattivati o per mutazione o per crescita dei batteri a basso pH, la penicillina ha effetti batteriostatici.