Why are gram positive bacteria more susceptible to antibiotics

A GRAM-POSITIVE BACTERIAL CELL WALL COMPONENTS

Gram-positive bacteria do not contain LPS, yet they trigger a toxic shock syndrome similar to that induced by LPS. This response is caused by cell wall components of Gram-positive bacteria, such as peptidoglycan (PGN) and LTA (Fig. 4). PGN is an alternating β(1, 4) linked N-acetylmuramyl and N-acetylglucosaminyl glycan whose residues are crosslinked by a short peptide. LTA is a macroam-phiphile, equivalent to LPS in Gram-negative bacteria, containing a substituted polyglycerophosphate backbone attached to a glycolipid, and is anchored in the membrane by the glycolipid. Recently it has been demonstrated that TLR2 may act as a receptor for PGN and LTA from Gram-positive bacteria. Whole Gram- positive bacteria, soluble PGN, and LTA induced the activation of NF-κB in 293 cell expressing TLR2 but not in cells expressing TLR1 or TLR4 (Schwander et al., 1999). Similarly, CHO fibroblast cells expressing TLR2 were activated by heat-killed Staphylococcus aureus and Streptococcus pneumonia and peptioglycan from S. aureus, whereas CHO expressing TLR4 did not respond to these stimuli (Yoshimura et al., 1999). TLR-mediated activation by these Gram-positive cell wall components was further assessed using TLR2 and TLR4 KO mice (Takeuchi et al., 1999a). These results demonstrated that the response to Gram-positive bacterial peptidoglycan is mediated by TLR2, but not TLR4. In contrast to the results from in vitro overexpression studies, the LTA response was found to be mediated by TLR4, but not TLR2. This finding shows that both TLR2 and TLR4 are responsible for the recognition of Gram-positive bacteria.

Gram-positive bacteria

Gram-positive bacteria have a thick PG layer approximately 10–20 layers thick that retains the crystal violet stain in a Gram stain giving the bacteria a purple color (Fig. 6C; Scheffers and Pinho, 2005). Most Gram-positive bacteria cell walls are also composed of teichoic acids (TAs) or related glycopolymers, capsular polysaccharides and cell wall associated proteins, and collectively they make up a polyanionic mesh-like matrix that surrounds the cytoplasmic membrane giving the Gram-positive bacteria cell wall elasticity, porosity and tensile strength (Xia et al., 2010; Neuhaus and Baddiley, 2003). TAs are composed of wall teichoic acids (WTA) and lipoteichoic acids (LTA). WTAs are water-soluble anionic polymers of glycerol phosphate or ribitol phosphate linked by phosphodiester bonds covalently bound to MurNAc residues and known WTA structures have a great diversity between bacterial species and even between clonal groups. While LTAs are a macroamphiphile with a glycolipid anchored covalently to the cytoplasmic membrane (Neuhaus and Baddiley, 2003). Together WTAs and LTAs may contribute to the net negative charge of Gram-positive bacteria cell walls (Xia et al., 2010), but their overall function remains unclear, although it has been suggested that they have a role in adhesion, cell wall maintenance, and host-mediated immune responses (Neuhaus and Baddiley, 2003; Xia et al., 2010).

Pre-Clinical Research

Gram-positive bacteria, including staphylococci (Staphylococcus aureus, S. epidermidis,) streptococci (Streptococcus pyogenes, S.pneumoniae, etc.), enterococci, and many anaerobic gram-positive bacteria (e.g., Clostridium difficile, C.perfringens, Listeria monocytogenes), are susceptible to teicoplanin in vitro. Beta-Lactamase-producing or methicillin resistant strains of staphylococci are generally equally susceptible to teicoplanin, as are nonresistant strains. In general,MIC90 values for teicoplanin are the same, or a 2-fold dilution lower, than those of vancomycin against most susceptible species. However, teicoplanin is several times more potent as an inhibitor of enterococci than is vancomycin. Gram-negative bacteria are not susceptible to teicoplanin Campoli-Richards et al (1990).

3.5 Response of Gram-positive and Gram-negative bacteria to plant extracts

Gram-positive bacteria are more susceptible to EOs and various plant extractions. Plant EOs appeared more active with respect to Gram reaction, thereby exerting a greater inhibitory effect against Gram-positive bacteria than Gram-negative bacteria, which are more resistant to the EOs (Deans, Noble, Hiltunen, Wuryani, & Penzes, 1995). Yoda, Hu, Zhao, and Shimamura (2004) investigated the antibacterial activity of tea catechin (epigallocatechin gallate) against various strains of Staphylococcus (Gram-positive cocci) and E. coli, K. pneumonia, and Salmonella (Gram-negative rods). The results of Yoda et al. (2004) showed that 50–100 μg/ml is required to inhibit the growth of Staphylococcus, whereas concentrations higher than 800 μg/ml are required to inhibit Gram-negative rods. In similar work, S. aureus was the most sensitive and E. coli was the most resistant against various extracts of dietary spices (100 mg/ml) when these bacteria were tested using the agar well diffusion method (Shan, Cai, Brooks, & Corke, 2007). Belguith et al. (2009) also reported the impact of aqueous garlic extract mainly on Gram-negative bacteria, and their outer membrane, which is absent in Gram-positive bacteria. It was found from in vitro experiments that citrus oils (lemon, orange, and bergamot) are more effective on Gram-positive bacteria (Listeria monocytogenes, S. aureus, Bacillus cereus) than on Gram-negative (E. coli O157 and Campylobacter jejuni) (Burt, 2004; Delaquis, Stanich, Girard, & Mazza, 2002; Fisher & Phillips, 2006). Earlier, Shelef, Naglik, and Bogen (1980) and Tassou, Drosinos, and Nychas (1995) reported that Gram-positive bacteria are more sensitive to EOs than Gram-negative bacteria. Thongson, Davidson, Mahakarnchanakul, and Weiss (2004) reported higher MIC values for Salmonella Typhimurium than L. monocytogenes, indicating the greater sensitivity of Gram-positive listeriae to spice extracts than the Gram-negative salmonellae. Commonly, Gram-negative bacteria are more resistant to plant oils due to the presence of their hydrophilic cell wall structure. Gram-negative bacteria contain a lipopolysaccharide that blocks the penetration of hydrophobic oil and avoids the accumulation of EOs in target cell membranes (Bezic, Skočibušić, Dunkić, & Radonić, 2003; Zaika, 1988). Davidson and Branen (2005) also suggested that the outer membrane surrounding the cell walls of Gram-negative bacteria may restrict the diffusion of hydrophobic compounds through the bacterium's lipopolysaccharide cell wall coverings. This could explain why Gram-positive bacteria are more sensitive to plant extracts than Gram-negative bacteria (Rahman & Kang, 2009).

Abstract

Gram-positive bacteria coordinate social behavior by sensing the extracellular level of peptide signals. These signals are biosynthesized through divergent pathways and some possess unusual functional chemistry as a result of posttranslational modifications. In this chapter, the biosynthetic pathways of Bacillus intracellular signaling peptides, Enterococcus pheromones, Bacillus subtilis competence pheromones, and cyclic peptide signals from Staphylococcus and other bacteria are covered. With the increasing prevalence of the cyclic peptide signals in diverse Gram-positive bacteria, a focus on this biosynthetic mechanism and variations on the theme are discussed. Due to the importance of peptide systems in pathogenesis, there is emerging interest in quorum-quenching approaches for therapeutic intervention. The quenching strategies that have successfully blocked signal biosynthesis are also covered. As peptide signaling systems continue to be discovered, there is a growing need to understand the details of these communication mechanisms. This information will provide insight on how Gram-positives coordinate cellular events and aid strategies to target these pathways for infection treatments.

3.9.1 Type of microorganisms and their initial population in the food

Gram positive bacteria offer higher resistance to the PEF procedure relative to gram negative strains, as they are covered in a thick multilayered peptidoglycan layer (Pillet et al., 2016). Typically, yeast cells have higher sensitivity to electric fields in comparison with bacteria based on their large sizes, though in low-magnitude electric fields they seemingly offer higher resistance than gram negative cells (Kethireddy et al., 2016). The initial population of microorganisms is important for any PEF treatment, since it is the effective parameter for measuring the efficacy of the process (González-Arenzana et al., 2019).

Other components of gram-positive cell envelopes

Gram-positive bacteria lack an outer membrane but proteins, lipoproteins and other macromolecules are associated with the cytoplasmic membrane and peptidoglycan. Many wall proteins are anchored directly to the peptidoglycan, and contain a conserved amino acid motif – LPXTG – which is involved in transporting these proteins across the cytoplasmic membrane. Lipoproteins, which may be highly immunogenic, are anchored to the cytoplasmic membrane by the N-terminus which is lipid modified. Expression of both wall and cytoplasmic membrane proteins may be influenced by environmental factors such as iron availability. Also associated with the surface of some gram-positive bacteria are immunglobulin-binding proteins such as staphylococcal protein A and streptococcal protein G, which bind the Fc region of antibody molecules. Proteins with analogous function are now being detected in some gram-negative bacteria. Release of such proteins may protect the organism by binding to antibody and preventing its deposition on the bacterial surface. Other proteins such as the M protein of Streptococcus pyogenes have an antiphagocytic function.

Teichoic and lipoteichoic acids are acidic polymers of glycerol or ribitol and are bound to the peptidoglycan and cytoplasmic membrane respectively. They may be important antigens in staphylococci and streptococci, stimulating both humoral and cell-mediated immune responses.

4.3.2 QS in Gram-positive bacteria

Gram-positive bacteria are a common feature of two component membrane systems using signals of modifiable oligopeptides and histidine kinase sensor receptors. The phosphorylation cascade mediates cell signaling, which in its turn regulates reaction regulator activity, in particular the transcriptional factors that are binding on DNA. Since Gram-negative bacteria (GNB) use Lux IR quorum sensing systems, Gram-positive bacteria are equally sensitive to signal structures by using cognitive receptors. Therefore, intra species communication confers peptide quorum-sensing circuits as in LuxIR systems. Although, the peptide signals do not spread across the membrane, henceforth to mediate cell signaling by committed oligopeptide exporters. Signals of peptide QS are reported to be derived from larger precursor peptides that are later adapted to contain lactone and thiolactone rings, lanthionines, and isoprenyl groups, though biochemical processes leading to these events are not clearly comprehended [43, 46–48]. Moreover, in combination with other types of QS signals, Gram-positive bacteria communicate with multiple peptides, e.g., S. aureus is an enthusing example for the sensing of peptide quorum. It is normally a benign human commensal, but it becomes a deadly pathogen [49] by open penetration in host tissues. S. aureus is used for biphasic strategy for the transmission of the disease. When cell counts are small, however, as cell density increases, the bacterium suppresses these features to produce proteins that facilitate attachment viz-à-viz colonization and then induce toxin and protease production, which eventually results in dissemination [50]. This switch to gene expression programs is regulated in Agr QS system. S. aureus are classified according to the AIP thiolactone sequence [41]. Every AIP leads remarkably to the activation of the AgrC receiver and blocks the expression by competitive binding of all other noncognized receivers [51]. Thus, in all three other groups, S. aureus inhibits virulence cascade activation. Competition of intra-species exists when two different S. aureus are co-infected [43]. Hence QS in S. aureus inhibits the spread of nonkin progeny while allowing the spread of closely related offspring. This means that the communication between cells was instrumental in setting up a specific niche for each strain [52]. Signal-receptor pair divergence in these bacteria could be part of the molecular mechanisms underlying the development of new bacterial species. Streptomycetes has an important clinical importance as it is a reservoir of secondary metabolites, many of which are used as antibiotics, of the diverse family of Gram-positive soil dwelling bacteria [53]. Streptomycetes control morphological differentiation with quorum sensing and secondary production of metabolites. They utilize γ-butyrolactones as auto inducers. These signals are interesting because they are structurally linked to autoinducers from AHL. No reports of cross-communication between Streptomycetes and other Gram-positive bacteria that communicate with AHLs are yet available.

Polysaccharides

Gram-positive bacteria often contain polysaccharides within their cell wall. These polysaccharides are classified in different categories according to their function. Capsular polysaccharides (CPS) that are covalently attached to PG and establishing a thick layer called capsule. The CPS structure and composition are species- or even strain-dependent and depend on growth conditions for a given strain. The capsule is a highly hydrated structure since it is composed of up to 99% water. On the other hand, exopolysaccharides (EPS) are not covalently bound to the CW and are either loosely adsorbed via weak interactions or secreted in the environment. The major EPS components are hexose, pentose, hexosamine and ketose. They are able to increase water absorption ability and, thus, influence the bacterial surface hydrophilicity. These EPS are of great interest for the dairy industry due to their physico-chemical properties that can modulate the texture, the stability and the flavor of fermented dairy products (Chapot-Chartier, 2014).

Envelope of the Vegetative Cell

Gram-positive bacteria and, in general, monoderms have complex envelopes comprising one bilayer lipid membrane separating the cytoplasm from the exterior of the cell. The membrane is part of a very complex structure that comprises many layers (up to 40 in the case of B. subtilis) of murein, or peptidoglycan, a complex of peptides containing d-amino acids (in particular mesodiaminopimelic acid), and amino sugars. The cell envelope also has several layers of teichoic acid (Figure 1).

The possible existence of a periplasm in B. subtilis in a distinct cell compartment surrounded by the cytoplasm membrane and the cell wall is a controversial issue. Cytoplasm, membrane, and protoplast supernatant fractions were prepared from protoplasts generated from phosphate-limited cells. The protoplast supernatant fractions was found to include cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from a fraction of protoplasts. By this operational definition, 10% of the proteins of B. subtilis can be considered periplasmic.