Gram-positive bacteria, such as the Enterococcus species, S. aureus and Clostridium difficile can be treated with vancomycin and similar glycopeptides, which are medications of last resort. Vancomycin's bactericidal effect was long hypothesized to be impervious to resistance as it binds to the bacterial cell envelope rather than to a protein target, as is the case for other antibiotics. Vancomycin's therapeutic efficacy is threatened by two types of complex resistance mechanisms, each involving a multi-enzyme route, that have arisen and are becoming extensively more common in pathogenic species. Precursor degradation and substitution with DAla-D lac or D-Ala-D-Ser alternatives, which vancomycin has a poor affinity for, constitute the mechanisms of resistance. Vancomycin resistance has been studied extensively for >30 years, and significant progress has been made in the understanding of the molecular biology of the enzyme cascades involved (1,2). The large size and high molecular weights of glycopeptides make them ineffective against gram-negative bacteria as they cannot penetrate past the outer membrane. Vancomycin is effective against all gram-negative bacteria, but nearly all gram-positive bacteria are vulnerable to glycopeptides (3). Inhibition of the bacterial cell wall by glycopeptides results in exposure of the fragile cytoplasmic membranes surrounding the bacterial cells, resulting in their bactericidal activity. There is an increase in osmotic pressure inside bacterium cells, causing them to quickly expand until they burst open (4). This review summarizes the mechanisms of gram-positive vancomycin resistance, which may be useful in the cure of illnesses caused by these pathogens.

There has been evidence of both vancomycin resistance and dependency in some Enterococci strains of the vanA and vanB vancomycin resistance classes. The vancomycin-dependent Enterococci have been isolated by plating them on vancomycin-containing agar, such as that used for the separation of Campylobacter or Gonococci, from clinical samples that seemed to be culture negative (36). If these organisms stop producing D-Ala-D-Ala, one hypothesis is that they will be unable to continue grow without a new structure to replace it. It is only when vancomycin is present that most vanA and vanB types of resistance occur, as vancomycin induces the creation of vanH dehydrogenase and of D-ala-D-lac-ligase (30). As long as vancomycin is present, bacteria may be unable to produce D-Ala-D-Ala, and even vanX destroy bacteria. This may explain why bacteria are unable to synthesize cell walls whilst D-Ala-D-Ala is present. As soon as the vancomycin is removed, the cell stops growing and replicating as it needs to either produce D-Ala or D-Ala D-Lac in order to continue growing and replicating (36,30). If vancomycin independence is restored, it may be explained by a mutation leading to constitutive D-Ala-D-Lac production or via (re-)activation of the standard mechanism for synthesizing D-Ala-DAla (36,37).

S. aureus strains, particularly methicillin-resistant strains, have been found to exhibit tolerance to vancomycin, according to early findings (38). Tolerance has been proposed as the reason for the failure of vancomycin treatment of infections caused by these bacteria despite the presence of MIC-detected susceptibility. Studies have also indicated that these resistant S. aureus strains increase mortality in individuals with bacteremia or endocarditis, despite vancomycin therapy (38,39). Vancomycin-resistant S. aureus (VRSA) strains have been identified, and these bacteria have in-vitro MIC values >4 µg/ml. Vancomycin resistance has been observed in both S. aureus and coagulase-negative staphylococci (40). VRSA is more prevalent in Africa and Asia than in Europe and the United States, and vanA and SCCmec II were the most common genetic factors associated with VRSA (41).

The emergence of VRSA was particularly noticeable in institutions with a high prevalence of methicillin-resistant Staphylococci and a policy of indiscriminate glycopeptide administration. The extensive use of glycopeptides (vancomycin or teicoplanin) has permitted the selection of resistant strains from both S. aureus and coagulase negative Staphylococci, primarily from S. epidermidis, followed by S. hemolyticus, S. hominis and S. warneri (42-44). Additionally, glycopeptide-resistant mutants of S. aureus have been selected experimentally by gradually increasing the quantity of vancomycin present during in vitro development, supporting this theory (9). Furthermore, the discovery that vancomycin heteroresistance is a widespread occurrence among Staphylococci also supports this theory (45,46). When vancomycin is used to treat bacterial infections, the presence of heteroresistance may allow for the selection of resistant strains in the body.

Due to the discovery of the vancomycin heteroresistance phenomena in Staphylococci, the procedures employed in the clinical microbiology laboratories to distinguish vancomycin resistance may need updating/adapting. The inoculum tested must be large enough for justification of the occurrence of the phenotype. Staphylococci vancomycin resistance has been shown to be reversible in the lab. S. aureus isolates with vancomycin resistance were repeatedly cultured on non-selective media in an effort to demonstrate this phenotypic reversal. Moreover, the vancomycin-resistant subpopulations were eliminated from several obtained strains. Reversion of vancomycin resistance phenotype may account for the difficulty in obtaining vancomycin-resistant clinical Staphylococci isolates from patients who do not respond to vancomycin therapy, as well as some of the challenges in identifying these isolates within the clinical microbiology laboratory (47). Primary testing of a vancomycin-resistant S. epidermidis strain was performed in a case study. A second test was accomplished on the isolate cultured to validate the first result, and the outcome was that the strain remained sensitive to vancomycin even after multiple tests. As it was unclear whether the initial primary test was flawed, researchers performed a second round of testing on the same patient (48). According to the data, there was no error in the primary susceptibility test, but there was a problem with an obscure phenotype (49). These findings may lay the groundwork for a novel approach to the clinical laboratory cultivation of Staphylococci. Vancomycin-resistant isolates can be maintained on the normal media as well as a medium containing low levels of the antibiotic, which may be preferable. The basic results of vancomycin susceptibility testing, not the results from subcultures, should be taken into account as well. The method by which Staphylococci develop resistance to vancomycin remains unknown at present, to the best of our knowledge. However, in vitro experiments and mouse skin transfers of the vanA gene cluster from Enterococci to S. aureus have been demonstrated in the laboratory. Clinical isolates of Staphylococci, which frequently co-colonize wound infection sites with Enterococci, may exhibit this route of genetic transmission (50). However, no investigation has shown the presence of any van gene clusters in clinical Staphylococci isolates, and hence a mechanism of this nature has not been observed (51).

Vancomycin-resistant Staphylococci in a liquid media form bacterial cell aggregates when exposed to vancomycin. Following this, the drug is no longer detected in the medium, and the bacterial aggregates break down into single cells with the same appearance as cells grown in an antibiotic-free environment. Electron microscopy examination of the bacterial aggregates revealed the development of substantial amounts of extracellular material with staining qualities comparable to those of the cell wall. Bioactive vancomycin may be extracted from cell wall material. These findings suggest that bacteria may sequester antibiotic molecules in a bound form on an additional cell wall substance, making them resistant to the activity of vancomycin (52-54).

A total of 16 vancomycin-resistant S. aureus clinical isolates from 7 countries were tested in a drug-free medium. Vancomycin-susceptible strains (MIC 4 µg/ml) were obtained after 10-84 days of these passages, but reversion to vancomycin resistance was achieved by a single step of vancomycin selection. All vancomycin-resistant strains had thicker cell walls that became thinner when vancomycin resistance was lost and thick again when vancomycin resistance was regained in a laboratory setting. S. aureus vancomycin resistance can develop under the selective pressure of long-term vancomycin use, but the resistance can be overcome by discontinuing the drug's use. It is possible, according to this research, that vancomycin resistance is caused by thickening of the bacterial cell wall, or some property associated with this (55).

Even though vancomycin-resistant Staphylococci thickening has been linked to complex reorganization of cell wall metabolism (10), with additional wall material displaying lower peptidoglycan cross-linking of the side chains of D-Ala (56). The complex reorganization of cell metabolism and lower peptidoglycan cross-linking prevents vancomycin from reaching the intracellular target molecules as it binds to these free termini outside the cell wall (54). In these bacteria, the thickening and metabolic reorganization of the cell wall are likely acquired through a genetic mutation (38,39). In order to better understand how these strains develop vancomycin resistance, additional research is needed to improve our understanding of vancomycin resistance in these strains and highlight potential therapeutic options.

The lactic acid bacteria Lactobacillus, Leuconostoc and Pediococcus are more likely to exhibit/attain vancomycin resistance. Human commensal lactic acid bacteria have been isolated from the digestive and genitourinary tracts of individuals, as well as from dairy products. Vancomycin is ineffective against lactic acid bacteria as these bacteria are genetically resistant to it, due to the weak affinity of vancomycin binding to their cell walls (32,33). This weak binding was shown to be caused by the establishment of peptidoglycan precursors terminating in the depsipeptide D-Ala-D-Lac observed in Enterococci's vanA and vanB resistance classes to vancomycin, highlighting a potential direction for analysis of the peptidoglycans present in their cell walls. DNA probes for the vanA or vanB genes failed to hybridize with glycopeptide-resistant lactic acid bacteria. These findings suggested that these natively glycopeptide-resistant bacteria included a natural ligase that catalyzes the production of D-Ala-D-Lac instead of D-Ala-D-Ala, to which vancomycin binds with high affinities (24,43).

There have been significant breakthroughs in our understanding of the molecular processes and genetics of vancomycin resistance in gram-positive bacteria. Despite this, it remains unknown where the resistance genes have originated from. A potential source of resistance for glycopeptide-producing organisms is the presence of genes encoding homologues of vanA, vanH, vanR, vanS and vanX in these species' secondary metabolic products. For infections with multi-resistant strains of Staphylococci, Streptococci and Enterococci, glycopeptides, alone or in combination with another antibacterial, are typically the sole mode of treatment. Vancomycin-resistant gram-positive bacteria can become resistant to all antibiotics if they develop and spread significant levels of resistance to the drug. Therefore, knowledge of the molecular and structural vancomycin resistance pathways may highlight potential avenues for addressing this serious issue.