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J Med Microbiol 56 (2007), 236-240; DOI: 10.1099/jmm.0.46778-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Polymorphisms of the pbp5 gene and correlation with ampicillin resistance in Enterococcus faecium isolates of animal origin

Patricia Poeta1,2,3, Daniela Costa1,2, Gilberto Igrejas4, Yolanda Sáenz2, Myriam Zarazaga2, Jorge Rodrigues1,3 and Carmen Torres2

1 Departamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

2 Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain

3 Centro de Estudos de Ciências Animais e Veterinárias, Vila Real, Portugal

4 Departamento de Genética e Biotecnologia, Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

Correspondence
Carmen Torres
carmen.torres{at}daa.unirioja.es

Received 12 June 2006
Accepted 5 October 2006

The C-terminal region of the pbp5 gene was sequenced in 11 ampicillin-resistant and 5 ampicillin-susceptible Enterococcus faecium isolates of animal origin, and compared with a pbp5 reference sequence (GenBank accession no. X84860). Eight different pbp5 alleles (designated A–H) were detected when amino acid changes in the region 461–629 were considered. Three of these alleles (A–C) were detected in ampicillin-susceptible isolates (MIC range 1–8 µg ml–1), and included the changes 470H->Q, 471V->I, 487Q->L, 581I->V, 595E->A or 622E->D. The remaining five alleles (D–H) were found in ampicillin-resistant isolates (MIC range 32–256 µg ml–1); three of these alleles (F–H) presented a serine insertion at position 466', in addition to other important amino acid changes (485M->A, 496N->K, 499A->T, 525E->D, 586V->L or 629E->V). The other two alleles presented the amino acid changes 496N->K and 629E->V (allele D), and 470H->Q (allele F). A correlation between deduced amino acid changes in PBP5 and ampicillin MICs was detected in animal E. faecium isolates.

Abbreviations: PBP5, penicillin-binding protein 5.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Enterococcus faecium is an important human pathogen whilst also being a commensal bacterium of the intestinal tract of humans and animals. E. faecium is intrinsically resistant to moderate levels of ampicillin, with MICs typically ranging from 1 to 16 µg ml–1 (Kak & Chow, 2002). This natural ampicillin resistance of E. faecium was demonstrated to be associated with expression of penicillin-binding protein 5 (PBP5), which has a low affinity for ß-lactams (Fontana et al., 1983, 1994; Rice et al., 2001). The pbp5 gene is located in the bacterial chromosome and is considered to be intrinsic to the species, although recently it has also been reported as a transferable determinant (Rice et al., 2005). Recently, an increase in ampicillin resistance has been observed in clinical E. faecium isolates, which could compromise therapy in life-threatening infections caused by this micro-organism (Klare et al., 2003). The emergence of high-level ampicillin resistance in E. faecium may be due to either increased production of PBP5 or mutations in the pbp5 gene resulting in lower affinities for ampicillin (Kak & Chow, 2002; Rice et al., 2001; Sifaoui et al., 2001; Williamson et al., 1983; Zorzi et al., 1996). Specific amino acid changes in the C-terminal region of PBP5 (especially at aa 466', 485, 496, 499, 525, 586 and 629, around the active-site region of PBP5) have been associated with ß-lactam resistance (Jureen et al., 2003; Ligozzi et al., 1996; Rice et al., 2001, 2004; Rybkine et al., 1998; Sauvage et al., 2002; Zorzi et al., 1996), and 12 different alleles of this gene have previously been demonstrated in clinical E. faecium isolates when this C-terminal region of pbp5 gene was analysed (Jureen et al., 2003).

As far as we know, all studies of the characterization and detection of mutations in pbp5 have been performed on E. faecium isolates of human origin and not on animal isolates. Thus, the pbp5 gene of ampicillin-resistant (AmpR) and ampicillin-susceptible (AmpS) E. faecium isolates recovered from healthy animals was sequenced in the present study. The deduced amino acid changes were found to correlate with their specific ampicillin MICs.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial isolates. Antimicrobial resistance was analysed by the disc-diffusion method in a previous study in enterococcal isolates recovered from faecal samples of healthy poultry and pets (Poeta et al., 2006). A total of 11 E. faecium isolates of that study showed an AmpR phenotype, comprising 10 faecal samples from poultry and 1 faecal sample from a dog. These 11 AmpR E. faecium isolates, as well as 5 AmpS E. faecium isolates (4 from poultry and 1 from a dog), were included in the present study.

Ampicillin-susceptibility testing. The MIC of ampicillin (Eli Lilly) was determined in our E. faecium isolates using the agar-dilution method according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards, 2005). Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were used as quality-control strains.

Analysis of ß-lactamase activity. The production of ß-lactamase was tested by streaking the bacterial colonies over nitrocefin discs (Cefinase; Becton Dickinson Microbiology Systems) (Marshall et al., 1995). After 5–10 min at room temperature, a change in colour from yellow to purple was indicative of a positive reaction.

PCR amplification of pbp5 and DNA sequence analysis. Total DNA from E. faecium isolates was obtained using an InstaGene matrix (Bio-Rad) and 10 µl DNA was used in each PCR, with 3.5 mM MgCl2. The C-terminal region of the pbp5 gene was amplified by PCR in all 16 E. faecium isolates included in this study using primers PBP5F-1 (5'-AACAAAATGACAAACGGG-3') and PBP5R (5'-TATCCTTGGTTATCAGGG-3'), producing an amplicon of 779 bp. PCR conditions were as follows: 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 2 min, with a final 7 min extension period at 72 °C (Jureen et al., 2003). PCR products were detected on ethidium-bromide-stained agarose gels and purified using a QIAquick gel extraction kit (Qiagen), according to the manufacturer's instructions. The purified products were sequenced on both strands using an Applied Biosystems 3730 DNA sequencer, using primers PBP5F-2 (5'-GACAAACGGGATCTCACAAG-3') and PBP5R. Duplicated sequences were obtained from independent PCRs and compared with that of the pbp5 gene reference sequence (GenBank accession no. X84860).

The complete pbp5 gene was also amplified by PCR in six of our E. faecium isolates (two AmpS and four AmpR) using primers PBP5F-3 (5'-AAAAATCGAACAACAGGCGCTTA-3') and PBP5R (amplicon size 1916 bp), using TaKaRa DNA polymerase (Takara Bio) under conditions recommended by the manufacturer. Amplicons were purified and sequenced by primer walking. The sequences obtained from both strands were also compared with that of the reference sequence.


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Polymorphisms of the pbp5 gene were studied in a series of E. faecium isolates of animal origin that showed different susceptibilities to ampicillin. None showed ß-lactamase activity when nitrocefin discs were used for screening purposes.

C-terminal region of the pbp5 gene

The sequence of the C-terminal region of pbp5 was analysed in our 11 AmpR and 5 AmpS E. faecium isolates and was correlated with their specific ampicillin MIC values. The deduced amino acid changes detected between aa 461 and 634 of these enterococci are shown in Table 1Go, with amino acid positions considered to be important for ampicillin resistance indicated in bold.


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  		Table 1. Polymorphisms in the C-terminal region of pbp5 in 16 E. faecium isolates of animal origin and correlation with their specific ampicillin MIC values

 
Eight different pbp5 alleles, designated here as A–H, were detected when amino acid changes in this region were taken into account, none of which showed 100% identity with the reference sequence (GenBank accession no. X84860). Our five AmpS isolates (MICs 1–8 µg ml–1) were classified as only three of these alleles (A–C) and included changes at positions not previously considered to be important for ampicillin resistance (allele A 470H->Q, 581I->V, 595E->A; allele B 470H->Q, 595E->A; allele C 470H->Q, 471V->I, 487Q->L, 622E->D) (Table 1Go). The remaining five alleles (D–H) were found in our 11 AmpR isolates. It is important to stress that three of these alleles (F–H) presented a serine insertion at position 466' in addition to other important amino acid changes: alleles F and H (485M->A, 496N->K, 499A->T, 525E->D and 629E->V) and allele G (485M->A, 496N->K, 499A->T, 525E->D, 586V->L and 629E->V). Allele F, identified in seven AmpR isolates (MICs 32–256 µg ml–1), was the most frequent allele in our study. Allele D was found in one AmpR isolate (MIC 32 µg ml–1) and showed amino acid changes at two principal positions (496N->K and 629E->V). The remaining allele (E) was detected in one AmpR isolate (MIC 64 µg ml–1), recovered from a dog, and showed only one amino acid change (470H->Q) with respect to the reference sequence. This specific substitution was also found in all of our isolates, both AmpR and AmpS, and has not previously been associated with increased ampicillin MICs by other authors (Jureen et al., 2003).

It is important to note that the insertion of serine at position 466' was detected in most of our AmpR animal E. faecium isolates, and was generally associated with other amino acid changes. Insertions of aspartic acid or serine at position 466' have also been detected previously in strains with an increased level of ampicillin resistance (Jureen et al., 2003; Rybkine et al., 1998; Zorzi et al., 1996). According to the previously reported PBP5 crystal structure (Sauvage et al., 2002), when PBP5 protein of E. faecium (PBP5fm) forms a complex with benzylpenicillin, the active-site cavity of this protein is delimited by the ßC3 strand on one side, by residues 461–465 on the opposite side and by residues 537–541 on the bottom of the cavity. Residues 461–465 form part of a loop (aa 451–465) that is well conserved in the B1 subgroup of class B high-molecular-mass PBPs. Thus, the substitution 461Q->K found in most of our AmpR strains could be implicated in lower affinity for the antibiotic. The residue V465 points into the active site, being close to the ß-lactam ring; therefore, the insertion of a residue at position 466' may slightly displace V465 inside the active site, reducing its accessibility for ampicillin (Rice et al., 2004; Sauvage et al., 2002). The serine insertion was found in the strains with high-level ampicillin resistance and seems to be an essential determinant of resistance, affecting antibiotic recognition.

As in the findings of other authors (Rybkine et al., 1998; Zorzi et al., 1996), the change of methionine to alanine at aa 485 was observed in isolates with higher MICs (128–256 µg ml–1), although two isolates showed a MIC of 32 µg ml–1. The bulky side chain of M485 located behind the K425 side chain is substituted by the small alanine molecule, obtaining more conformational space for lysine and resulting in a lower affinity for the ß-lactam antibiotic. Although the substitution 485M->A seems to be important for ampicillin resistance, it has been reported that this change by itself does not confer high-level ampicillin resistance (Sifaoui et al., 2001), as observed in our study.

We identified only one AmpR isolate (MIC 128 µg ml–1) that showed the 586V->L substitution. However, Ligozzi et al. (1996) suggested that the region from aa 558 to 586 might play an important role in the ß-lactam-binding site of PBP5, as changes in this region occurred in the highly resistant strains. Residue 629 is close to one edge of the active site and the 629E->V substitution causes a change of a hydrophilic charged residue for a hydrophobic one, which is unfavourable for the overall stability of the protein and may decrease the ability of the protein to bind to ß-lactam by restricting mobility (Leszczynski & Rose, 1986; Rice et al., 2004).

In a comparison of the 8 alleles of the pbp5 gene detected in our animal isolates with the 12 alleles reported by Jureen et al. (2003) in human isolates, it is important to note that only one of our alleles (allele E with the single change 470H->Q) corresponded to one of those determined by Jureen and co-workers (designated allele 3). Allele 3 of pbp5 was detected particularly among AmpS human isolates and also in a few AmpR isolates (Jureen et al., 2003). In our study, allele E was found in one AmpR E. faecium isolate (P42, MIC 64 µg ml–1) recovered from a dog faecal sample.

If only the potentially significant amino acid positions (466', 485, 496, 499, 525, 586 and 629) of the C-terminal region of PBP5 were considered, all of our AmpS isolates presented the same amino acid pattern as the reference sequence. In the case of AmpR isolates, four different patterns could be identified: (i) amino acid changes at the above seven positions in one isolate (MIC 128 µg ml–1, allele G); (ii) changes at six positions in eight isolates (MICs 32–256 µg ml–1, alleles F and H); (iii) changes at two positions in one isolate (MIC 32 µg ml–1, allele D); and (iv) no changes at important positions in one isolate (P42, MIC 64 µg ml–1, allele E) (Table 1Go). Thus, it seems that specific alterations in the C-terminal part of the pbp5 gene alone could not entirely be correlated with the different levels of ampicillin resistance among our strains and other factors are probably implicated (Jureen et al., 2004; Rice et al., 2001; Sifaoui et al., 2001).

N-terminal region of the pbp5 gene

In order to determine whether the ampicillin resistance phenotype detected in E. faecium P42 (allele E) was due to amino acid changes in positions outside the C-terminal region of pbp5, the complete pbp5 gene was amplified and sequenced for this isolate, as well as for five additional AmpR and AmpS E. faecium isolates (P44, P54, P13, P2 and P11, belonging to alleles B, C, F, G and H, respectively). The amino acid changes detected in the N-terminal region of the pbp5 gene of these isolates with respect to the reference sequence are indicated in Table 2Go. It was found that E. faecium P42 and one of the AmpS isolates showed no amino acid changes with respect to the reference sequence, whilst four changes were detected in the other AmpS isolate. Nevertheless, a large number (up to 12) and variety of amino acid changes were demonstrated in this region in the three AmpR isolates (P2, P11 and P13). Zorzi et al. (1996) described most of these amino acid changes in the N-terminal domain of their studied strains, with the 68A->T and 85E->D substitutions only detected in the highly resistant penicillin strains (EFM-1 and H80721, with MICs of 90 and 512 µg ml–1, respectively). These two changes were also observed in our high-level AmpR isolates. The function of the N-terminal domain is still unclear. It has been hypothesized that it is necessary for correct folding of the C-terminal module and both domains seem to be interdependent (Sauvage et al., 2002). In fact, deletion of some segments of the N-terminal domain of PBP5 from Enterococcus hirae resulted in proteins that were unable to bind penicillin (Mollerach et al., 1996). Future studies should be carried out to determine whether the amino acid changes in the N-terminal region of PBP5 could be associated with changes in ß-lactam susceptibility in E. faecium species.


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  		Table 2. Amino acid changes detected in the N-terminal region of pbp5 in six of the E. faecium isolates of this study

 
Conclusions

The specific alterations in the C-terminal part of the pbp5 gene could be used as markers for ampicillin resistance (at least the insertion at aa 466' and the changes at position 485), as they are present in most of the AmpR but not in the AmpS strains, although, as observed by other researchers (Jureen et al., 2004; Rice et al., 2001; Sifaoui et al., 2001), it seems that other factors could be necessary for a complete explanation of the differences in levels of ampicillin resistance. High levels of resistance could be observed in strains that either overexpress PBP5 and/or present multiple mutations that decrease the affinity of PBP5 for the antibiotic. This possibility could explain the different levels of ampicillin resistance among strains classified as the same allele (allele F) or the P42 strains whose MIC values could be due simply to protein overexpression. Zorzi et al. (1996) suggested that only a single copy of the pbp5 gene was present in their analysed strains. Thus, the variations seen in the amount of PBP5 could not be due to a gene dosage effect, although the detection of pbp5 located within transferable elements could change this suggestion (Rice et al., 2005). More recently, it has been reported that expression of ampicillin resistance was higher when the pbp5 gene was located downstream of the ftsWEfm and psr genes (Rice et al., 2001). The role of the putative ftsWEfm gene product is unknown, although it has been suggested that its homologue in Escherichia coli may serve as a chaperone protein for PBP3 (Eberhardt et al., 2003). In addition, it has been proposed that overproduction of PBP5 could result from either an alteration in the psr promoter or a modification of the target site of Psr, probably located between the psr and pbp5 genes (Zorzi et al., 1996).

In conclusion, the C-terminal region of the pbp5 gene was found to be highly polymorphic among our AmpR and AmpS E. faecium isolates of animal origin and there seemed to be a correlation between specific changes located at positions close to the active site of PBP5 and the ampicillin MIC, although further studies of the mechanisms of ampicillin resistance in E. faecium are necessary.


   ACKNOWLEDGEMENTS
 
P. P. was supported by a grant with the reference SFRH/BD/17424/2004 from Fundaçao para a Ciência e a Tecnologia (FCT) of Portugal. This work has been financed in part by the Integrated Action Spain–Portugal from the Ministry of Education and Science (HP2005-0052).


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Eberhardt, C., Kuerschner, L. & Weiss, D. S. (2003). Probing the catalytic activity of a cell division-specific transpeptidase in vivo with ß-lactams. J Bacteriol 185, 3726–3734.[Abstract/Free Full Text]

Fontana, R., Cerini, R., Longoni, P., Grossato, A. & Canepari, P. (1983). Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. J Bacteriol 155, 1343–1350.[Abstract/Free Full Text]

Fontana, R., Aldegheri, M., Ligozzi, M., Lopez, H., Sucari, A. & Satta, G. (1994). Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 38, 1980–1983.[Abstract/Free Full Text]

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Jureen, R., Mohn, S. C., Harthug, S., Haarr, L. & Langeland, N. (2004). Role of penicillin-binding protein 5 C-terminal amino acid substitutions in conferring ampicillin resistance in Norwegian clinical strains of Enterococcus faecium. APMIS 112, 291–298.[CrossRef][Medline]

Kak, V. & Chow, J. W. (2002). Acquired antibiotic resistances in enterococci. In The Enterococci; Pathogenesis, Molecular Biology and Antibiotic Resistance, pp. 355–357. Washington, DC: American Society for Microbiology.

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Leszczynski, J. F. & Rose, G. D. (1986). Loops in globular proteins: a novel category of secondary structure. Science 234, 849–855.[Abstract/Free Full Text]

Ligozzi, M., Pittaluga, F. & Fontana, R. (1996). Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 40, 354–357.[Abstract]

Marshall, S. A., Sutton, L. D. & Jones, R. N. (1995). Evaluation of S1 chromogenic cephalosporin ß-lactamase disk assay tested against Gram-positive anaerobes, coagulase-negative staphylococci, Prevotella spp. and Enterococcus spp. Diagn Microbiol Infect Dis 22, 353–355.[CrossRef][Medline]

Mollerach, M. E., Partoune, P., Coyette, J. & Ghuysen, J. M. (1996). Importance of the E-46-D-160 polypeptide segment of the non-penicillin-binding module for the folding of the low-affinity, multimodular class B penicillin-binding protein 5 of Enterococcus hirae. J Bacteriol 178, 1774–1775.[Abstract/Free Full Text]

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Poeta, P., Costa, D., Rodrigues, J. & Torres, C. (2006). Antimicrobial resistance and the mechanisms implicated in faecal enterococci from healthy humans, poultry and pets in Portugal. Int J Antimicrob Agents 27, 131–137.[CrossRef][Medline]

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Rice, L. B., Bellais, S., Carias, L., Hutton-Thomas, R., Bonomo, R. A., Caspers, P., Page, M. G. P. & Gutmann, L. (2004). Impact of specific pbp5 mutations on expression of ß-lactam resistance in Enterococcus faecium. Antimicrob Agents Chemother 48, 3028–3032.[Abstract/Free Full Text]

Rice, L. B., Carias, L. L., Rudin, S., Lakticová, V., Wood, A. & Hutton-Thomas, R. (2005). Enterococcus faecium low-affinity pbp5 is a transferable determinant. Antimicrob Agents Chemother 49, 5007–5012.[Abstract/Free Full Text]

Rybkine, T., Mainardi, J. L., Sougakoff, W., Collatz, E. & Gutmann, L. (1998). Penicillin-binding protein 5 sequence alterations in clinical isolates of Enterococcus faecium with different levels of ß-lactam resistance. J Infect Dis 178, 159–163.[Medline]

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