Which of the following will be resulted from an antimicrobial that inhibits bacterial cell wall synthesis?

1 Introduction

β-Lactam antibiotics have been in use for several decades following the discovery of penicillin (derived from Penicillium chrysogenum) in 1928 by Sir Alexander Fleming at the St. Mary's Hospital, London, and the subsequent commercial production of the antibiotic in large quantities due to the efforts of Howard Florey, Ernst Chain, and their associates. Since their initial discovery, many β-lactam antibiotics have become available for clinical use. Much has been learnt about their mechanism of action, clinical applications, pharmacokinetic (PK) profile, and toxic effects. In addition, the medical community has become aware of the various mechanisms of resistance developed by bacteria against the β-lactam antibiotics and the impact this may have on the appropriate management of critically ill patients in particular.

Mechanism of Action

β-Lactam antibiotics act on enzymes called penicillin-binding proteins (PBPs) responsible for building the bacterial cell wall.18 Therefore they are active only against rapidly multiplying organisms in which the binding of penicillin within the cell wall interferes with production of cell wall peptidoglycans and results in cell lysis in a hypo-osmotic or iso-osmotic environment. There may be anywhere from two to eight PBPs in a bacterium. When β-lactam antibiotics bind covalently and irreversibly to the PBPs, the bacterial cell wall is disrupted and lysis occurs. Differences in the spectrum and activity of β-lactam antibiotics are due to their relative affinity for different PBPs. To bind to the PBPs, the β-lactam antibiotic must first diffuse through the bacterial cell wall. Gram-negative organisms have an additional lipopolysaccharide layer that decreases antibiotic penetration. Therefore gram-positive bacteria are usually more susceptible to the action of β-lactams than gram-negative bacteria. Because the penicillins poorly penetrate mammalian cells, they are ineffective in the treatment of intracellular pathogens.

β-Lactams

β-Lactam antibiotics inhibit bacteria by binding covalently to PBPs in the cytoplasmic membrane. These target proteins catalyze the synthesis of the peptidoglycan that forms the cell wall of bacteria.172 Alterations of PBPs can lead to β-lactam antibiotic resistance.173

In gram-positive bacteria, resistance to β-lactam antibiotics may be associated either with a decrease in the affinity of the PBP for the antibiotic174 or with a change in the amount of PBP produced by the bacterium.175 Multiple mechanisms seem to be present in some clinical isolates. Penicillin-resistant strains of S. pneumoniae isolated in South Africa have shown several changes in PBPs (i.e., decreased affinity of some PBPs, loss of others, and appearance of PBPs not present in the more susceptible cells).176 The genes that encode these PBPs are mosaics, composed of segments from susceptible pneumococci and segments from resistant commensal streptococci.177 In S. aureus178-180 and E. faecium,181 additional PBPs may be inducible (i.e., their production is stimulated by exposure of the microorganism to the β-lactam antibiotic). These inducible PBPs have a lower affinity for β-lactam antibiotics, making them less susceptible to inhibition by low concentrations of the drug. Changes in the types of PBPs observed in susceptible and resistant strains also have been seen with the viridans streptococcal species Streptococcus mitis.182

Impermeability resistance

β-Lactam antibiotics cross the outer membrane to reach PBPs in Gram-negative bacteria through porin proteins (p. 27; see also Figure 2.1, p.11). Alterations in porin production can lead to decreased permeability and concomitant β-lactam resistance. Porin deficiencies coupled with high β-lactamase production have been reported in clinical isolates of Enterobacteriaceae such as Enterobacter cloacae and Serratia marcescens that produce cephalosporinases. Resistance due to decreased permeability is also important among Ps. aeruginosa strains, which possess a less permeable membrane and contain efflux pump mechanisms for β-lactam antibiotics, coupled with production of chromosomally mediated β-lactamase activity. Because Gram-positive bacteria lack an outer membrane, this mechanism of resistance does not apply.

β-Lactams

All β-lactam antibiotics consist of a 4-membered β-lactam ring system as carbon-skeletal backbone and bind to penicillin-binding proteins. Clinically, five classes of β-lactams including penams, cephems, carbapenems, clavams, monobactams are important (Fig. 3A). The electrophilic β-lactam antibiotics mimic the D-Ala-d-Ala termini of the pentapeptide part of peptidoglycan layer (Fig. 3B). Penams, cephems, and carbapenems covalently bind to penicillin binding proteins, preventing the enzyme transpeptidase (Tipper and Strominger, 1965).

Penicillins are naturally occurring compounds belonging to a special class of compounds, the β-lactam antibiotics, of great clinically, pharmacologically, and economically importance. β-lactam antibiotics are typically used to treat a broad array of Gram-positive and Gram-negative bacteria. Natural penicillins are derived from the Penicillium fungi/mold and produced from the fermentation of the fungus Penicillium chrysogenum. The cephalosporins are a class of cephem-based β-lactam antibiotics originally derived from the fungus Cephalosporium (Wilke et al., 2005; Sharma et al. 2007).

Beta-Lactam Antibiotics

β-Lactam antibiotics have a β-lactam ring in their molecular structure (Figure 8-1). They include penicillins, cephalosporins, monobactams, and carbapenems. They are bactericidal antibiotics (see Chapter 6) that bind covalently to and inhibit penicillin binding proteins (PBPs). These PBPs are needed to catalyze the cross-linking (or transpeptidation) of the peptidoglycan layer of bacterial cell walls, which is continuously remodeled by bacteria (Figure 8-2). When PBPs are inactivated by β-lactam antibiotics, bacterial enzymes that hydrolyze the peptidoglycan cross-links during cell wall remodeling continue to function, which breaks down the cell wall further. The accumulation of peptidoglycan precursors also triggers activation of cell wall hydrolases, with further digestion of intact peptidoglycan. The end result is bacterial rupture.

The peptidoglycan layer of gram-positive bacteria is 50 to 100 times thicker than that of gram-negative bacteria and is highly cross-linked, which maintains the structural integrity of gram-positive bacteria.1 Therefore, compared to gram-negative bacteria, gram-positive bacteria are more easily inactivated by β-lactam antibiotics. Bacteria possess multiple PBPs, which are assigned numbers based on their molecular weight. PBPs vary in their affinities for different β-lactam antibiotics, which in part explains the difference in spectrum of activity and bactericidal action of β-lactam antibiotics. For example, inhibition of PBP1a and PBP1b leads to cell lysis, whereas inhibition of PBP2 results in rounded cells called spheroblasts. Drugs that produce rapid lysis (e.g., carbapenems) are the most bactericidal and have highest affinity for PBP1.

Resistance to β-lactam antibiotics is now widespread and results primarily from β-lactamase production.2 It can also result from production of altered PBPs (such as PBP2a) and, among gram-negative bacteria, exclusion of drugs that normally diffuse through porins to their site of action. Gram-negative β-lactamases are strategically located just beneath the outer lipopolysaccharide layer, which acts as the barrier to drug penetration. Gram-positive bacteria secrete β-lactamases into their immediate surroundings. There are many different β-lactamase enzymes that vary in their specificity for β-lactam drugs.3

β-Lactam antibiotics have a short half-life and exhibit time-dependent pharmacodynamics (see Chapter 6). Drug concentrations should be maintained above the minimum inhibitory concentration (T > MIC) for at least 50% of the dosing interval. For penicillins and carbapenems, T > MIC can be less than 50%, but aiming for a target of 50% ensures that most patients will have adequate exposure. To maintain this target, some β-lactam antibiotics with short half-lives require frequent administration or slow infusion. For other drugs, a long half-life prolongs the T > MIC to allow for infrequent administration. For example, cefpodoxime proxetil has a half-life longer than those of other oral cephalosporins, so it can be administered only once per day. Cefovecin has an extremely long half-life in dogs and cats and can be effective for some infections when administered at 14-day intervals. For other cephalosporins (e.g., injectable drugs with short half-lives such as cefotaxime), three- to four-times-daily dosing may be required for treatment of gram-negative bacterial infections.

F Antimicrobial Agents

β-lactam antibiotics, including penicillins and cephalosporins, inhibit platelet aggregation responses, and some can induce a bleeding diathesis when given in high doses. These include carbenicillin, penicillin G, ticarcillin, ampicillin, nafcillin, cloxacillin, mezlocillin, oxacillin, and piperacillin.293–303 The effects on platelet function appear to be dose-dependent, taking approximately 2–3 days to manifest and 3–10 days to abate after discontinuation of the drug.293,298,299,302,303 Penicillins lacking the α-carboxy group (mezlocillin, piperacillin, apalcillin) appear to adversely affect platelet function less often than carboxypenicillins or moxalactam.302 Cephalosporins may also impair platelet function.302,304,305 Moxalactam has been reported to induce platelet dysfunction associated with prolonged bleeding times and clinical hemorrhage.302,305 However, other third-generation cephalosporins appear to show little effect on platelet function.302 Some β-lactam antibiotics (such as moxalactam, cefamandole, cefoperazone, cefotetan) may induce hypoprothrombinemia and thereby contribute to hemorrhage.302,305 These compounds contain an N-methyl-thiotetrazole (NMTT) side chain, which inhibits vitamin K epoxide reductase, thus interfering with the carboxylation of the vitamin-K dependent coagulation factors.

The following generalizations can be made regarding the impact of β-lactam antibiotics on platelets. First, the effects on platelet function and hemostasis are dose-dependent and time-dependent with effects becoming discernible over several days.293,298,299,302,303 Second, the patients at particular risk of bleeding appear to be those with concurrent illnesses including sepsis, malnourishment, thrombocytopenia, and malignancy; intensive care units are the typical setting. Third, the platelet inhibitory effect of β-lactam antibiotics is influenced by plasma albumin. Both the platelet binding of the antibiotic and the impact on platelet responses are inversely related to albumin concentration.306 Lastly, with some of the β-lactam antibiotics, the bleeding is related to a concomitant inhibition of synthesis of vitamin K-dependent coagulation factors.302,304,305

A number of mechanisms have been invoked to explain β-lactam antibiotic-induced platelet inhibition. Some of these drugs inhibit platelet aggregation and secretion as well as platelet adherence to subendothelial structures and collagen-coated surfaces.297 Many studies have evaluated the effects on platelets only in vitro and often at concentrations above those attained in vivo. Short-term in vitro exposure of platelets to penicillin has been reported to result in impaired aggregation responses and decreased binding of agonists to specific receptors (ADP, VWF, α2-adrenergic) on platelets.307,308 Although the latter effect of penicillins was found to be rapidly reversible,307,308 the platelet inhibitory effect of β-lactam antibiotics appears to persist for several days after discontinuation, indicating that the sustained effect is related to other mechanisms. Other reported effects of β-lactam antibiotics include inhibition of intracellular signaling events, calcium mobilization,309 TXA2 synthesis,308,309 and alterations in activation-induced changes in membrane integrin αIIbβ3 and GPIb-IX.310

The context in which the bleeding events are encountered in patients on antibiotics precludes clear definition of the role played by the antimicrobials, because of the simultaneous presence of thrombocytopenia, disseminated intravascular coagulation, infection, and/or vitamin K deficiency. Discontinuation of a specifically indicated antibiotic may not be possible or necessary. Supportive measures using blood products and other interventions (e.g., correcting metabolic abnormalities and vitamin K deficiency) are indicated in the overall management.

Other antimicrobials shown to inhibit platelet function include nitrofurantoin,311 hydroxychloroquine,312 and miconazole.313

7.17.2.2 Mode of Action of β-Lactam Antibiotics

β-Lactam antibiotics mimic the terminal d-Ala-d-Ala moiety of the pentapeptide. The reactive β-lactam ring is able to acylate the active serine residue of the transpeptidase, leading to a stable acyl–enzyme intermediate that is still appended with a bulky substituent (the second ring of the β-lactam antibiotics), thus preventing the access of an incoming amino group, required to achieve cross-linking (Figure 5). Before the resolution of x-ray structures of β-lactamases and transpeptidases, the mode of action of β-lactam antibiotics was solely explained by the acylating power of the β-lactam ring, as measured by the typical high infrared frequency of the β-lactam carbonyl band (>1780 cm−1).12 However, this model did not take into account the enzyme environment. Thus, another model was introduced, based on the distance between the β-lactam carbonyl oxygen atom and the carbon atom of the carboxylate function (3.0–3.9 Å for active versus >4.1 Å for inactive derivatives; Figure 6).13

The molecular rationalization of the mode of action of β-lactam antibiotics requires the amidic character of the carbonyl group to be reduced, to enable acylation of the active serine residue of the transpeptidases. This is achieved by preventing the overlap of the β-lactam nitrogen π orbital with those of the β-lactam carbonyl group by (1) geometric constraints resulting from the presence of a fused five-membered ring (penams), (2) an inductive effect (monobactams), (3) delocalization of the β-lactam nitrogen electron lone pair into an adjacent unsaturated system (cephems), or (4) by a combination of two effects (penems and carbapenems). In addition, the β-lactam carbonyl group must be located in the oxyanion hole, to provide the correct activation and orientation of the carbonyl group for attack during the acylation and deacylation steps, respectively (see Figure 8).

The unique feature of β-lactam antibiotics is that they target a family of related enzymes (transpeptidases). However, due to the high structural variability of these enzymes in their active site region, it is not possible to obtain high affinity for all of them. Therefore, inhibition relies mainly on the irreversible selective acylation of the active serine residue by the β-lactam ring. Selectivity with regard to other host targets arises from the fact that transpeptidases process an ‘unnatural’ d-configured substrate. Furthermore, β-lactam antibiotics must remain stable with respect to the action of β-lactamases. These enzymes are closely related to transpeptidases but are able to open the β-lactam ring of the antibiotics.

Pharmacology and mechanism of action

Beta-lactam antibiotic of the acylureidopenicillin class. Like other beta-lactams, piperacillin binds PBPs that weaken or interfere with cell wall formation. After binding to PBPs, the cell wall weakens or undergoes lysis. Like other beta-lactams, this drug acts in a time-dependent manner (i.e., it is more effective when drug concentrations are maintained above the MIC values during the dose interval). Compared to other beta-lactam antibiotics, piperacillin has good activity against Pseudomonas aeruginosa. It also has good activity against streptococci, but is not active against methicillin-resistant Staphylococcus. Piperacillin has a short half-life in animals and must be given by injection (usually IV), which limits its usefulness. Tazobactam is a beta-lactamase inhibitor. When administered in combination with piperacillin, it increases the spectrum to include beta-lactamase-producing strains of gram-negative and gram-positive bacteria.

7.6 Resistance to Other Antimicrobials (β-Lactam Antibiotics and Chloramphenicol)

β-Lactam antibiotics act by binding to penicillin-binding proteins (PBPs) and disrupting peptidoglycan cross-linking during cell wall synthesis, resulting in bacterial lysis and cell death (Iovine, 2013). With exception of imipenem, the majority of Campylobacter strains show resistance to a large number of β-lactam antibiotics (Aarestrup & Engberg, 2001). In general, Campylobacter are intrinsically resistant to penicillin G and narrow-spectrum cephalosporins due to their limited ability to bind to PBPs and their low permeability (Aarestrup & Engberg, 2001; Tajada, Gomez-Graces, Alos, Balas, & Cogollos, 1996). However, resistance to other β-lactam antibiotics, such as amoxicillin, ampicillin, and ticarcillin, is mainly due to the production of β-lactamase, which inactivates β-lactam antibiotics by hydrolyzing the structural lactam ring (Aarestrup & Engberg, 2001; Griggs et al., 2009). The β-lactamase, blaOXA-61, has spread widely in C. jejuni and C. coli, and the prevalence of the blaOXA-61 gene in ampicillin-resistant Campylobacter can reach up to 91% (Griggs et al., 2009). A study showed that inactivation of the blaOXA-61 gene increased susceptibility to ampicillin, penicillin, carbenicillin, oxacillin, and piperacillin, in both ampicillin-sensitive and -resistant strains, but had no effect on cephalosporin or imipenem (Griggs et al., 2009). In another study examining the in vitro susceptibilities of 100 thermophilic Campylobacter strains to various β-lactam agents, 77%, 88%, and 100% of the isolates were susceptible to amoxicillin–clavulanic acid, cefepime, and imipenem, respectively (Tajada et al., 1996).

Resistance to β-lactam antibiotics is also associated with alterations of outer membrane proteins and efflux (Iovine, 2013). Several studies showed a significant increase in susceptibility to ampicillin in CmeABC-inactivated C. jejuni mutants and a decrease in susceptibility in CmeABC-overexpressing mutants (Lin et al., 2002; Pumbwe, Randall, Woodward, & Piddock, 2004). β-Lactam antibiotics are generally not the drugs of choice to treat Campylobacter infections, and the prevalence of resistance to β-lactam antibiotics in Campylobacter has not been monitored in the NARMS program.

Chloramphenicol inhibits bacterial protein biosynthesis by binding to the 50S ribosomal subunit and preventing peptide-chain elongation. Chloramphenicol resistance is mainly due to the acquisition of a plasmid carrying the cat gene encoding the chloramphenicol acetyltransferases (CAT), which enzymatically inactivates the drug and prevents it from binding to ribosomes (Wieczorek & Osek, 2013). Prevalence of chloramphenicol resistance in Campylobacter has been very low, but the cat gene has been identified in C. coli (Wang & Taylor, 1990; Zhao, Tyson, et al., 2015). Recently, a plasmid-borne MDR cfr-like gene [cfr(C)] was detected in 10% of C. coli strains isolated from cattle farms in the United States (Tang, Dai, et al., 2017). The cfr gene encodes an rRNA methyltransferase, confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A, and was first detected in Staphylococcus sciuri in 2000 (Long, Poehlsgaard, Kehrenberg, Schwarz, & Vester, 2006). The amino acid identity between Cfr(C) and Cfr, and between Cfr(C) and Cfr(B) was 55.1% and 54.9%, respectively. Cloning cfr(C) in C. jejuni NCTC 11168 and conjugative transfer of the cfr(C)-carrying plasmid confirmed its role in conferring resistance to chloramphenicol, florfenicol, and other unrelated antimicrobials, including linezolid, tiamulin, and clindamycin (Tang, Dai, et al., 2017).