What is β- lactams and β-lactamases ?

β- lactam antibiotics are a broad class of antibiotics, consisting of all antibiotic agents that contains a β- lactam ring in their molecular structures which include penicillin derivatives, cephalosporins, monobactams and carbapenems (Holten et al 2010). They are the most commonly used antibiotics that kill bacteria by inhibiting the carboxy / transpeptidase or penicillin binding proteins (PBPs) involved in the late stage of peptidoglycan biosynthesis. Although introduced nearly 60 years ago, they still represent the most widely used class of agents in the clinic today (Hugo and Russel 2004). β-lactam antibiotics are used in a wide variety of both systemic and localized infections including Respiratory tract infections, GI tract infections, CNS infections, Skin and soft tissue infections, Urinary tract infections etc.

These days β-lactams are clinically relevant due to resistance arising via modification of the normal penicillin-binding proteins (PBPs), bypassing of the normal PBPs, impermeability of the Gram-negative organism outer membrane and production of β- lactamases (Livermore 1998).

β-Lactamases are a heterogenous group of proteins with structural similarities, composed of α-helices and β-pleated sheets and are the members of a superfamily of active site serine proteases (Knox et al 1998). β lactamases  are the major cause of bacterial resistance to β-lactam antibiotics and can be either chromosomal or  plasmid, or transposon encoded and produced in a constitutive or inducible manner. They provide antibiotic resistance by breaking the antibiotic structure which have a common element in their molecular stucture which is a four atom ring β-lactam. the lactamase enzyme hydrolyses the β-lactam ring and deactivate the antimicrobial properties.

There is rapid dissemination and emergence of β-lactamases of various types; extended spectrum β-lactamases, AmpC β-lactamases and Metallo β-lactamases that can easily hydrolyse these antibiotics by breaking down the β-lactam ring and rendering the antibiotic harmless. Carbapenems are the most powerful penicillin-related antibiotics, often used against difficult bacterial infections, such as imipenem and meropenem, are antibacterial agents with activity against many multi drug resistant Gram-negative, Gram-positive, and anaerobic microorganisms (Franklin et al 2006).

Two schemes are currently used to classify β-Lactamases: the Ambler classification scheme and the Bush-Jacoby-Medeiros classification system. The Molecular classification scheme of Ambler was proposed in 1980 and functional classification scheme was proposed by Bush in 1989 and was later modified in 1995 by Bush et al. The Ambler classification scheme separates β-lactamases into four distinct classes A through D based on similarities in amino acid sequence. Classes A, C and D are serine β-lactamases, whereas class b are metallo β-lactamases that require zinc for activity (Ambler, 1980). Bush-Jacoby-Medeiros classification system classifies β-lactamases according to functional similarities i.e. substrate-inhibitor profiles. There are four categories and multiple subgroups in this classification scheme (Group 1, 2, 3 and 2a, 2c, 3a etc) (Bush et al 1995). The Groups 1, 2 and 3 of Bush-Jacoby-Medeiros classification system fall respectively on the Ambler Molecular class C, A/D and B.

β-lactamases catalytically disrupt the β-lactam (amide) bond to form an acyl enzyme complex. A conserved serine in the active site acts as the reactive nucleophile in the acylation reaction. A critically positioned water then acts as the attacking nucleophile in the deacylation process resulting in the release of penicilloyl and cephalosporyl moiety. Penicillin-binding proteins (PBPs) have the similar mode of action, however, their structure don’t allow easy acess of water such that β-lactamases have hydolysis rate for β-lactams upto 2-3000 times higher than PBPs.

Extended Spectrum β lactamases (ESBLs)

ESBLs are β-lactamases capable of conferring bacterial resistance to the penicillins, first, second and third-generation cephalosporins and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and these enzymes are inhibited by β-lactamase inhibitors such as clavulanic acid (Paterson and Bonomo 2005). They can be found in a variety of Enterobacteriaceae species; however, majority of the ESBL producing strains are K. pneumoniae, K. oxytoca and E. coli. They have also been found in P. aeruginosa and other Enterobacteriaceae strains like Enterobacter spp, Citrobacter spp, Proteus spp, Morganella morganii, S. marsescens, Burkholderia cepacia and Capnocytophaga ochracea (Bradford et al 2001; Thomson et al 2001).

Reported risks factors include an increased length of stay in the hospital (Mangely et al 2000), an increased length of stay in intensive care unit, increased severity of illness , the use of a venous or arterial catheter , the use of a urinary catheter, ventilator assistance hemodyalisis, emergency abdominal surgery, the use of a gastrostomy or jejunostomy tube, gut colonization, prior administration of an oxyimino-beta-lactam antibiotic and prior administration of any antibiotics (Lautenbach et al 2001;  Kim et al 2002).

Various β- lactamases studied till date include TEM, SHV, CTX-M, OXA, PER-1, VEB, TLA, SFO etc. TEM-1 is the most commonly encountered β-lactamase in Gram-negative bacteria. Up to 90% of ampicillin resistance in E. coli is due to the production of TEM-1 (Livermore 1995). This enzyme is also responsible for the ampicillin and penicillin resistance that is seen in H. influenzae and N. gonorrhoeae in increasing numbers. TEM-1 is able to hydrolyze penicillins and early cephalosporins such as cephalothin and cephaloridine. TEM-2, the first derivative of TEM-1, had a single amino acid substitution from the original β -lactamase.The SHV-1 β -lactamase is most commonly found in K. pneumoniae and is responsible for up to 20% of the plasmid-mediated ampicillin resistance in this species (Tzouvelekis and Bonomo 1999). The majority of SHV variants possessing an ESBL phenotype are characterized by the substitution of a serine for glycine at position 238. The serine residue at position 238 is critical for the efficient hydrolysis of ceftazidime. The majority of SHV-type ESBLs are found in strains of K. pneumoniae  (Bradford 2001).

CTX-M is a new family of plasmid-mediated ESBLs that preferentially hydrolyze cefotaxime. They have mainly been found in strains of S. enterica serovar Typhimurium and E. coli, but have also been described in other species of Enterobacteriaceae. Kinetic studies have shown that the CTX-M-type β-lactamases hydrolyze cephalothin or cephaloridine better than benzylpenicillin and they preferentially hydrolyze cefotaxime over ceftazidime. It has been suggested that the serine residue at position 237, which is present in all of the CTX-M enzymes, plays an important role in the extended-spectrum activity of the CTX-M-type β-lactamases (Bradford 2001).

The OXA-type enzymes are another growing family of ESBLs which belong to molecular class D and functional group 2d. The OXA-type β-lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly inhibited by clavulanic acid (Paterson and Bonomo 2005). These have been found mainly in P. aeruginosa.

Besides these, several other types of enzymes have been reported from around the globe. Some of them include: PER-1 in strains of P. aeruginosa, PER-2 in S. enterica serovar Typhimurium strains, VEB-1 of E. coli and P. aeruginosa, TLA-1 in E. coli, SFO-1 from S.  fonticola  (Bradford 2001).