STUDY OF GENETIC POLYMORPHISMS AMONG STAPHYLOCOCCUS AUREUS RESPONDING VARIOUSLY TOWARDS VANCOMYCIN

VRSA Proposal

STUDY OF GENETIC POLYMORPHISMS AMONG STAPHYLOCOCCUS AUREUS RESPONDING VARIOUSLY TOWARDS VANCOMYCIN

By: Kiran Babu Tiwari
Research Scientist
Research Laboratory for Agricultural Biotechnology and Biochemistry
email: kiranbabu.babukiran@gmail.com


1) Rationale:
a) Vancomycin is the terminal antibiotic for MRSA
Staphylococcus aureus is one of the most common causes of nosocomial infections, especially pneumonia, surgical site infections, and bloodstream infections. This bacterium has the ability to rapidly acquire antimicrobial resistance. Most S. aureus strains (>90%) are resistant to penicillin, and since the 1980s, methicillin-resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide . One decade later during the 1990s, S. aureus isolates with diminished susceptibility to vancomycin (vancomycin-intermediate S. aureus [VISA]) were reported (Bozdogan et al., 2004; Avison et al., 2002; Bhateja et al., 2005). Glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant Staphylococcus aureus (MRSA). Incidences of vancomycin resistance in S. aureus (VRSA) have been increasing worldwide for the last 5 years. (Avison et al., 2002) The first clinical VRSA, Mu50, was isolated in Japan in 1997. Glycopeptides, such as vancomycin, are often the therapeutic drugs of choice for serious MRSA infections. However, failures of vancomycin therapy against S. aureus, due to the emergence of strains that are significantly less susceptible to vancomycin [vancomycinresistant S. aureus (VRSA)], are now well established (Avison et al., 2002). Recent reports of three S. aureus clinical strains with the vanA gene open a new era in staphylococcal antibacterial resistance (2-4). This latter development limits further potentially therapeutic options against these strains. (Bozdogan et al., 2004).

b) Mechanism of vancomycin resistance is simple mechano-biochemical reorganization of peptidoglycan biosynthesis
A thickened cell wall is responsible for the vancomycin resistance of VRSA strain Mu50, however, the mechanism of vancomycin resistance in other VRSA strains remained unclear (Cui et al., 2003). Thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for Vancomycin resistance in S. aureus. (Cui et al., 2003). VRSA strain Mu50 produces excessive amounts of peptidoglycan to make the thickened cell wall which become thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains (Cui et al., 2003). The differences in the cell wall thickness between VRSA and passage-derived strains, vancomycin-resistant mutant strains and passage-derived strains, and VRSA and control strains were all statistically significant (P < 0.001)(Cui L et al., 2003). Vancomycin binds to the stem peptide of the membrane-anchored murein monomer (lipid II) at its Lys-d-Ala-d-Ala residue and inhibits the transglycosylation and transpeptidation reactions, preventing incorporation of the precursors into the bacterial cell wall (Périchon et al., 2004). The amount of glutamine-non-amidated muropeptide subunits (i.e. those containing d-glutamate rather than d-glutamine) in the Mu50 cell wall increases, and non-amidated muropeptides are poorer substrates for transpeptidases than amidated ones, although this is yet to be confirmed. Cell wall thickness and cross-linking result in vancomycin resistance is that the modified cell wall binds more vancomycin, due to the increased amount of terminal d-alanyl-d-alanine dipeptide (Avison et al., 2002). And thus the thickened cell wall not only traps a greater number of vancomycin molecules but also significantly reduces the time that vancomycin completely inhibits peptidoglycan synthesis (Cui L et al., 2003). The fine structure of the Mu50 cell wall is similar to that of an MRSA strain such as N315, except that the Mu50 peptidoglycan chains show significantly less cross-linking, and an increased content of pentapeptide chains (Avison et al., 2002). However, the resistance in enterococci (VRE) is due to synthesis of VanA-type glycopeptide, a modified precursors, ending in d-Ala-d-lactate (d-Ala-d-Lac) in place of d-Ala-d-Ala that result in 1,000-fold-lower affinity for glycopeptides (Périchon et al., 2004).

c) Development of resistance in the hospital ecological environment is, hence, likely to be faster
Isolates of vancomycin resistant S.aureus have emerged in many parts of the world. These isolates appear to achieve clinically relevant levels of resistance to vancomycin that leads to treatment failure (Bhateja et al., 2005). At present, the proportion of MRSA with reduced susceptibility to vancomycin is well known. Only 21 strains have so far been reported in literature, the first VRSA and hVRSA reported from Japan were MU 50 and MU 3, respectively. (Bhateja et al., 2005). Thus, the passage-derived strains with decreased MICs of vancomycin are best explained by the mechanism of heterogeneous resistance. The hetero-VRSA is the precedent strain of VRSA. Emergence of VRSA would be the result of vancomycin selection exerted upon a hetero-VRSA strain in the hospital, and the strain would return to hetero-VRSA status when vancomycin is not used for a while and its selective pressure lifted (Cui et al., 2003). Beta-lactam antibiotics are suspected to play a role in the dissemination of hetero-VRSA. In Japan, hetero-VRSA strains were found quite frequently in clinical isolates in the late 1980s before the introduction of vancomycin (vancomycin was introduced in 1991, and teicoplanin was introduced in 1998). This indicates that the conversion of VSSA to hetero-VRSA can be achieved in association with hetero- to homoconversion of methicillin resistance caused by beta-lactam selection (Cui et al., 2003). This suggests that the use of beta-lactam antibiotics for MRSA infection is a risk factor for the emergence of hetero-VRSA, although the precise genetic mechanism remains to be clarified. (Cui et al., 2003). The unusual feature of cell wall physiology of the vancomycin stressed S. aureus makes the culprit to develop resistance easily and faster. This may challenge the clinical microbiologist more than any other antibiotics used and practiced previously.

d) Early detection of the pathogen is of utmost importance
Emergence of a truly vancomycin-resistant S. aureus seriously threatens the most important treatment option available to clinicians for infections resulting from methicillin-resistant S. aureus. In the absence of vancomycin pressure, vancomycin resistance was found to be unstable and expressed at a low level. This low-level expression of vancomycin resistance in S. aureus may be the reason why these strains are hard to detect clinically (Bozdogan et al., 2004). So, early detection of the pathogen is of utmost importance.

e) Many controversies about the method standardization for MIC values and interpretations
Vancomycin resistance can be difficult to detect in clinical microbiology laboratory. Disk diffusion sensitivity testing by standard 30µg vancomycin frequently misclassifies intermediately susceptible isolates as fully susceptible. Presently MIC determinations by broth or agar dilution or by E test are the gold standard for determining vancomycin susceptibility, but these methods are not suitable for routine use in the diagnostic laboratories (Bhateja et al., 2005).Vancomycin resistance in S.aureus is difficult to define mainly because of methodological problems in their detection (Bhateja et al., 2005). No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < r =" 0.787;"> 32µg/mL are resistant (Bhateja et al., 2005). Thus these observations lead to difficulty in interpretations.

f) Genetic analysis for Vancomycin resistance:
Complex mechanisms producing changes in cell wall content and composition generate the VRSA phenotype, but the genetic basis of these changes has not yet been determined (Avison et al., 2002). In 2002, the first two clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) possessing acquired vanA operon containing Tn1546 were recovered in Michigan and Pennsylvania (Clark et al., 2005) on conjugative plasmids with a broad host range in Gram-positive bacteria. (Bozdogan et al., 2003). The first two VRSA isolates arose from independent genetic events (Clark et al., 2005). The gene clusters are not stable genetic elements and serial passage of the strain in the absence of vancomycin led to loss of the vanA cluster and, consequently to restoration of vancomycin susceptibility (Bozdogan et al., 2003).

VanA-type resistance is characterized by high-level inducible resistance to both vancomycin and teicoplanin and is mediated by Tn1546 or closely related elements. Tn1546, which belongs to the Tn3 family of transposons, is composed of seven genes, two of which encode a transposase and a resolvase responsible for the movements of the elements of the vanA operon, and is delineated by imperfect inverted repeats. The vanH, vanA, and vanX genes code for proteins that are necessary for the expression of resistance. VanH is a dehydrogenase that converts pyruvate to d-Lac, VanA a ligase that uses d-Lac and a d-Ala residue to synthesize the depsipeptide d-Ala-d-Lac, which is incorporated into the peptidoglycan precursors, and VanX is a d,d-dipeptidase that hydrolyzes the dipeptide d-Ala-d-Ala formed by the endogenous Ddl chromosomal d-Ala:D-Ala ligase, thus reducing the level of normal peptidoglycan precursors ending in d-Ala-d-Ala. The VanY d,d-carboxypeptidase, not essential for resistance, cleaves the d-Ala C-terminal residue of pentapeptide precursors synthesized by using d-Ala-d-Ala dipeptides that have escaped VanX hydrolysis. The vanZ gene is implicated in teicoplanin resistance by an unknown mechanism. Expression of the vanA operon is regulated by two genes, vanR and vanS, located upstream from vanH that form a two-component regulatory system (Périchon etb al., 2004).

It is difficult to rationalize a role for these mutations in the VRSA phenotype. It is well documented that Mu50 grows more slowly than N315, which may be explained by the loss of such important functions. It has been hypothesized that the basis of the VRSA phenotype in Mu50 is an increased level of peptidoglycan biosynthesis, resulting in a considerably thicker cell wall. In addition to this, there is a reduced level of glutamate amidation in the cell wall, which may be responsible for reduced cross-linking and the reduced ability of d-d-carboxypeptidases to act on the peptidoglycan network, conserving d-alanyl-d-alanine terminal dipeptides, which bind and sequester vancomycin (Avison et al., 2002).

Our analysis has identified loss-of-function mutations in genes encoding enzymes responsible, in part, for the committed stage of peptidoglycan biosynthesis and for the manufacture of intermediary metabolites that are precursors of glutamine, and hence glucosamine. These findings will inform future targeted study of the biochemistry of vancomycin resistance in S. aureus (Avison et al., 2002).

To facilitate the genetic investigation, entire genome sequences of the archetypal VRSA (Mu50), and vancomycin-susceptible MRSA strains N315, EMRSA 16 and COL were compared. The in silico analysis revealed several loss-of-function mutations in Mu50, affecting important cell wall biosynthesis and intermediary metabolism genes, not previously reported. The new findings provide further evidence for the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes, important to metabolic pathways that impinge on peptidoglycan biosynthesis (Avison et al., 2002).

The complete sequence flat-files of the genomes of Mu50 and N315 were using a specially written macro that eliminates all non-amino acid sequence information to produce a ‘proteome’ for each genome for all predicted open reading frames (ORFs) joined end to end. 164 individual differences between the proteomes of Mu50 and N315 were found. Of these, 114 are minor, i.e. single amino acid substitutions, and their effects cannot be predicted, though it cannot be assumed that they are neutral. Accordingly, each product would have to be investigated individually. DNA sequences for the remaining 50 Mu50 ORFs showing more marked differences from those in N315 were obtained. In 13 instances, the sequence in N315 was different from those in the other comparators (genome sequence data from MRSA COL), so these changes could not be linked to the VRSA phenotype. Where the Mu50 genomic copy was found to differ from those in the three MRSA strains (37 instances), many of the changes were located in ‘hypothetical’ ORFs, and genes clearly unrelated to vancomycin resistance (e.g. endotoxin genes and antigenic determinants, etc.). When these are eliminated, a total of 17 loss-of-function mutations specific to the Mu50 genome in genes encoding characterized functions were identified. A link between five of these disruptions and the biochemical differences that have been previously noted between Mu50 and MRSA strains is compelling (Avison et al., 2002).

The authors explained the complementary genes and functions for those ones that seemed to be critical to cell wall physiology related to vancomycin stress, eg., (a) murZ for murA that encodes UDP-GlcNAc-enolpyruvyl transferase, mrp for fmtB homologue, and (b) 2-ketoglutarate oxidoreductase and succinyl-CoA synthase for 2-ketoglutarate dehydrogenase (Kdh, odhA) and succinate dehydrogenase (Sdh, sdhB) respectively (Avison et al., 2002). Comparison of the PFGE patterns of the parent and passage-derived strains demonstrated that 13 of the 16 pairs shared identical banding patterns (Cui L et al., 2003). Avison et al. (2002) pointed out that Glutamine synthetase (GS) and glucosamine synthase activity in Mu50 is higher than in N315 suggesting that Mu50 is geared to direct more carbon into synthesis of GlcNAc. One side-effect of hyperproduction of GlcNAc would be a shrinking of the l-glutamine pool, because it would be used faster to produce glucosamine. This, in turn, would mean that the amount of l-glutamine available for amidation of d-glutamate in cell wall precursors would be reduced. The observed disruption of the genes odhA and sdhB, if confirmed in other VRSA strains, does fit with the hypothesis that perturbation in this branch of intermediary metabolism is important in the VRSA phenotype of Mu50 (Avison et al., 2002).

The analysis is necessarily limited in a number of ways. There may well be other DNA sequence differences in Mu50 that do not cause coding changes but do influence susceptibility to vancomycin, such as mutations in promoter regions or transcription regulatory sites that affect the expression of relevant genes. The analysis of global gene expression in VRSA strains is in progress, although limited analysis of Mu50 has yielded some interesting results. A major limitation is that, of necessity, the genetic analysis only relates to one VRSA strain, Mu50. An investigation to determine whether mutations similar to those found in Mu50 are also present in other clinical VRSA strains is underway (Avison et al., 2002).

g) RAPD-Typing:

With these views, a deliberate search should be done to find out the different sites in the chromosome of the hVISA (hetero-vancomycin intermediately resistance S. aureus), VISA (vancomycin intermediately resistance S. aureus) and VRSA (vancomycin resistant S. aureus) strains that differed with that of VSSA (vancomycin susceptible S. aureus) / MRSA (Methicillin resistance S. aureus). A series of RAPD primers can be used to locate the differences among the strains. A concurrent data is expected at least from some primers, and the selected primers can be used to predict the possible hVISA, VISA and VRSA strains.

2) Hypothesis: VSSA and VRSA have same genomic compositions


3) Procedure: (As described elsewhere)

a) Isolation of staphylococci from clinical samples collected in the Tertiary Care hospital in Nepal
b) Biochemical characterization
c) Transportation of the isolates to RLABB d) MIC determination and characterization into VSSA, hVISA, VISA and VRSA
e) Isolation of whole DNA and/or chromosomal DNA and plasmid DNA
f) Primer selection for reproducible genetic polymorphisms
g) RAPD-PCR
h) Interpretation

4) Expected outcome:
a) Different colonial morphologies
b) Different biochemical patterns
c) MIC values for VSSA, hVISA, VISA and VRSA
d) Different RAPD-PCR patterns

5) References

Bozdogan B, D Esel, C Whitener, F A. Browne and Pr C. Appelbaum (2003) Antibacterial susceptibility of a vancomycin-resistant Staphylococcus aureus strain isolated at the Hershey Medical Center Journal of Antimicrobial Chemotherapy 52, 864-868.

Cui L, X Ma, K Sato, K Okuma, F C. Tenover, E M. Mamizuka, C G. Gemmell, Kim, M-C Ploy, N. E Solh, V Ferraz, and K Hiramatsu 2003 Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in Staphylococcus aureus J Clin Microbiol. January; 41(1): 5–14.

Clark N C, L M. Weigel, J B. Patel, and F C. Tenover 2005 Comparison of Tn1546-Like Elements in Vancomycin-Resistant Staphylococcus aureus Isolates from Michigan and Pennsylvania Antimicrob Agents Chemother; 49(1): 470–472.

Bozdogan B, L Ednie, K Credito, K Kosowska, and P C. Appelbaum 2004 Derivatives of a Vancomycin-Resistant Staphylococcus aureus Strain Isolated at Hershey Medical Center Antimicrob Agents Chemother. 48(12): 4762–4765.

Périchon B and P Courvalin 2004 Heterologous Expression of the Enterococcal vanA Operon in Methicillin-Resistant Staphylococcus aureus Antimicrob Agents Chemother. 48(11): 4281–4285.

Avison M B , P M. Bennett, R A. Howe and T R. Walsh 2002 Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50 Journal of Antimicrobial Chemotherapy 49: 255-260.

Bhateja P, M T Pandya, M Fatma, T R Ashok 2005 Detection of vancomycin resistant Staphylococcus aureus: A comparative study of three different phenotypic screening methods Indian Journal of Medical Microbiology 23( 1): 52-55.