Presence of the Resistance Genes vanC1 and pbp5 in Phenotypically Vancomycin and Ampicillin Susceptible Enterococcus faecalis

Abstract

Ampicillin and vancomycin are important antibiotics for the therapy of Enterococcus faecalis infections. The ampicillin resistance gene pbp5 is intrinsic in Enterococcus faecium. The vanC1 gene confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Both genes are chromosomally located. Resistance to ampicillin and vancomycin was determined in 484 E. faecalis of human and porcine origin by microdilution. Since E. faecalis are highly skilled to acquire resistance genes, all strains were investigated for the presence of pbp5 (and, in positive strains, for the penicillin-binding protein synthesis repressor gene psr) and vanC1 (and, in positive strains, for vanXYc and vanT) by using polymerase chain reaction (PCR). One porcine and one human isolate were phenotypically resistant to ampicillin; no strain was vancomycin resistant. Four E. faecalis (3/1 of porcine/human origin) carried pbp5 (MIC=1 mg/L), and four porcine strains were vanC1 positive (minimum inhibitory concentration [MIC]=1 mg/L). Real-time reverse transcriptase (RT)-PCR revealed that the genes were not expressed. The psr gene was absent in the four pbp5-positive strains; the vanXYc gene was absent in the four vanC1-positive strains. However, vanT of the vanC gene cluster was detected in two vanC1-positive strains. To our knowledge, this is the first report on the presence of pbp5, identical with the “E. faecium pbp5 gene,” and of vanC1/vanT in E. faecalis. Even if resistance is not expressed in these strains, this study shows that E. faecalis have a strong ability to acquire resistance genes—and potentially to spread them to other bacteria. Therefore, close monitoring of this species should be continued.

Introduction

Enterococci are part of the normal flora of humans and animals, but they have also been recognized as emerging pathogens that are ranked second or third in bacterial nosocomial infections.³¹ Among the more than 35 currently known species, Enterococcus faecalis are the most common causative agents of enterococcal infections (80% to 95%¹⁴). Unfortunately, increasing antibiotic resistance has been registered in the last 20 years.¹⁴ Ampicillin and amoxicillin are antibiotics of choice for the therapy of diseases caused by E. faecalis. The reserve antibiotic vancomycin is an alternative in case of penicillin allergy or resistance.¹⁸ In contrast to Enterococcus faecium, E. faecalis continue to be rarely resistant against ampicillin and vancomycin (<1.3%¹⁴). However, they are still recommended to be kept under surveillance, since enterococci have a strong tendency to acquire resistance genes.^13,53 There are several genes conferring resistance to ampicillin and vancomycin in enterococci.^2,30,54 The pbp5 gene is thought to be intrinsic in E. faecium and confers resistance to relatively low doses of ampicillin due to the expression of the penicillin binding protein 5 (PBP5), which has low affinity for penicillins.^12,40,54 The vanC1 gene, which encodes the vancomycin resistance protein C1, is thought to occur only in E. gallinarum and should therefore be applicable for species identification.³⁹ These genes are chromosomally localized and have not been found in E. faecalis until now. However, transferability of the pbp5 gene has been proven experimentally.⁴¹ This emphasizes that, contrary to popular opinion,⁶ the chromosomal location of a gene in intrinsically resistant strains does not necessarily protect against transfer into another species. Therefore, the aim of this study was to screen E. faecalis of human and porcine origin for the presence of pbp5 and vanC1 and to investigate the phenotypic resistance to ampicillin and vancomycin.

Materials and Methods

Bacterial strains

In the period 2002–2009, a total of 484 E. faecalis strains from Bavaria were isolated on Citrate Azide Tween Carbonate (CATC) Agar (VWR) and biochemically identified as previously described.⁴⁴ Of these, 238 were of porcine and 246 of human origin (Table 1). Species verification of the vanC1- and pbp5-positive isolates was performed by multiplex polymerase chain reaction (PCR) as described by Jackson et al.¹⁷ For the characterization of the genetic variability of these strains, the randomly amplified polymorphic DNA (RAPD)-PCR technique was applied by using the M13R2 primer as described by Martín et al.²⁹ Cluster analysis was performed by using the GelCompar Software V 5.10 (Applied Maths).

Table 1.

Ampicillin and Vancomycin Minimum Inhibitory Concentrations of Investigated Enterococcus faecalis (n=484)

Ampicillin	MIC≤2 mg/L (susceptible)^a, n (%)	Intermediate-resistant, n (%)	MIC>8 mg/L (resistant), n (%)
Porcine strains	203 (85.3)^b	34 (14.3)	1 (0.4)
Human strains	245 (99.6)^c	–	1 (0.4)

Vancomycin ^d	MIC≤1 mg/L, n (%)	MIC=2 mg/L, n (%)	MIC=4 mg/L, n (%)
Porcine strains	146 (61.3)^e	79 (33.2)	13 (5.5)
Human strains	144 (58.5)	102 (41.5)	–

Breakpoints according to DIN. Breakpoints according to EUCAST (available at: www.eucast.org/eucast_disk_diffusion_test/breakpoints) deviate slightly (S≤4; R>8).

Three isolates were pbp5 positive.

One isolate was pbp5 positive.

According to DIN (S≤4; R>8 mg/L) and to EUCAST (S≤4; R>4 mg/L), all isolates were susceptible to vancomycin.

Four isolates were vanC1 positive.

MIC, minimum inhibitory concentration; DIN, Deutsches Institut für Normung (German Institute for Standardization).

Antimicrobial susceptibility testing

Phenotypic resistance characteristics were tested by the broth microdilution method according to DIN 58940-81 (2002) as previously described.^9,44 Resistance testing was repeated for the pbp5- and vanC1-positive isolates. Incubation time for the vanC1-positive isolates was additionally prolonged to 48 hr, since Hirakata et al.¹⁵ recognized a shift to resistance dependent on inoculation time.

Detection of pbp5 and vanC1 by PCR

DNA was extracted from three colonies of pure cultures growing on Standard I Nutrient Agar (Merck), containing 7% defibrinated sheep blood (Fiebig) with the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. Primers were synthesized by TIB MOLBIOL Syntheselabor GmbH. PCR protocols for the detection of pbp5 and vanC1 are given in Table 2. Each PCR cycle contained one positive control (pbp5: E. faecium No. 51²¹; vanC1: Enterococcus gallinarum BM 4174; Institut Pasteur) and one negative control (master mix without template DNA). Reaction products were visualized under UV light after electrophoresis through a 1.5% agarose gel (BioRad) containing 0.285 μg/ml ethidium bromide (Sigma-Aldrich). Extraction and PCR runs were repeated twice for each pbp5- or vanC1-positive result in order to verify the results and to exclude contamination.

Table 2.

Primers and Polymerase Chain Reaction conditions

Primers	Sequence (5′—3′) ^a	Primer volume (μl)	Amplicon size (bp)	Annealing temp./time (°C/sec)
pbp5FW	AGTGGCGGTTATTTTTACTA	0.8	900	51/60^b
pbp5RV	ACCGTCTGTATCTGTGAGGC
vanC1FW	GGTATCAAGGAAACCTC	0.5	822	54/60^b
vanC1RV	CTTCCGCCATCATAGCT
psrFW	TTTGAAGCCCCGTATTATGC	1.0	206	58/8^c
psrRW	CTCCCAAAGTCCCCTTCTTC
vanXYcFW	GCAAAACAATGGGAACGACT	1.0	246	56/10^c
vanXYcRV	ATCTCGAAAATGAGGGCAGA
vanTFW	CTCAAAGACAGCCCTTTTGC
vanTRV	TTCCGCAATCGAATACCTTC	1.0	189	57/10^c
pbp5rtFW	ATTAGCAACAACCGGCAAAC
pbp5rtRV	GCCGTCTACTTCTTGGATGG	1.0	238	58/10^c
vanC1rtFW	GGTATCAAGGAAACCTC
vanC1rtRV	CTTCCGCCATCATAGCT	1.0	822	54/10^c

Primers were designed with the help of Primer3 (v. 0.4.0), available at http://frodo.wi.mit.edu/; vanC1 primers were designed as described elsewhere.¹¹

Each mixture for endpoint PCR (total volume=25 μl) consisted of 3.5 μl of 10× PCR buffer, 1.5 μl MgCl₂, 1.0 μl dNTPs, 0.25 μl Taq-Polymerase (Qiagen), forward and reverse primer (variable), and 1.0 μl template DNA.

Thermocycler settings: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 1 min, annealing (variable), elongation at 72°C for 1:30 min; final extension at 72°C for 10:00 min.

Each mixture for real-time PCR (total volume=20 μl) consisted of 1.6 μl MgCl₂, 2 μl SYBR Mix (Roche), and 2 μl template DNA.

LightCycler^® settings: initial denaturation at 95°C for 10 min; 35 cycles of denaturation at 95°C for 10 sec, annealing (variable), elongation at 72°C for 8/10/8/10/33 sec (psrfm/vanXYc/vanT/pbp5rt/vanC1rt); cooling at 40°C for 30 sec.

PCR, polymerase chain reaction.

Detection of psr, vanXYc, and vanT by real-time PCR

The pbp5-positive strains were tested for the presence of the penicillin-binding protein synthesis repressor gene psr⁵⁶ (gi|790434). The vanC1-positive strains were tested for the presence of the D,D-dipeptidase-carboxypeptidase encoding vanXYc gene¹ (gi|9294728). The vanC1-positive strains were also tested for vanT, which encodes a membrane-bound serine racemase¹ (gi|9294728). For primer sequences and PCR conditions, see Table 2. For positive and negative controls, see “Detection of pbp5 and vanC1 by PCR”.

Analysis of gene expression by real-time reverse transcriptase-PCR

For quantitative reverse transcriptase (RT)-PCR, bacterial suspensions of the pbp5- and vanC1-positive strains were adjusted to optical density (OD)₆₀₀=0.8. Total RNA was extracted with the NucleoSpin^® RNAII kit (Macherey-Nagel) using 5 mg/ml lysozyme (Sigma-Aldrich). For primer sequences (pbp5rt and vanC1rt) and PCR conditions, see Table 2. For positive and negative controls, see “Detection of pbp5 and vanC1 by PCR”. DNA contamination of the RNA extracts was excluded by performing a pooled “no-RT” control.

Sequence analysis

The bands of two pbp5-positive strains (one of porcine and one of human origin) were excised from the gel and sent for sequencing (Sequiserve). One vanC1 and one vanT bands were also sent for sequencing. Sequence similarity was compared by using the NCBI BLAST database (Basic Local Alignment Search Tool; www.ncbi.nlm.nih.gov).

Results

Table 1 shows that two of the 484 isolates were phenotypically resistant to ampicillin (minimum inhibitory concentration [MIC]>8 mg/L), whereas no vancomycin-resistant strain was observed. The ampicillin-resistant strains originated from a stool sample of a diseased pig and from a stool sample of a person without clinical disease; none of them contained pbp5. All isolates that were intermediately resistant to ampicillin, as well as all isolates with MIC=4 mg/L for vancomycin originated from pig manure samples.

In total, four E. faecalis with an MIC of 1 mg/L for ampicillin were positive for the presence of the pbp5 gene. The strains of porcine origin (n=3) were isolated from stool samples of diseased pigs from different farms. The human strain originated from a stool sample of a healthy person. The sequence analysis confirmed the PCR results. According to the NCBI BLAST database, the nucleotide sequences of the two strains had 99% similarity to a section of the “E. faecium pbp5 gene (D63r)” (GenBank: X84860.1). In the porcine strain, one point mutation was present on position 882 of the PCR product. The A-to-C polymorphism is a transversion that leads to a neutral amino acid substitution at position 1,482 in the PBP5 polypeptide. This change is consistent with the substitution of an isoleucine residue with leucine. In the human strain, two point mutations were present: the T-to-C polymorphism at position 301 leads to a substitution of tyrosine with histidine; the T-to-C polymorphism at position 481 leads to a substitution of cysteine with arginine. The psr gene was not detectable in the pbp5-positive strains.

Four vancomycin-susceptible isolates (MIC=1 mg/L), all originating from stool samples of diseased pigs from two different farms, were vanC1 positive. The sequence analysis confirmed the PCR results. In detail, the nucleotide sequence of the vanC1 PCR amplicon exactly matched a section of the “E. gallinarum strain eS464 VanC1 (vanC1) gene” (GenBank: EU151772.1; 100% identities; 0 gaps). The vanXYc gene was not detectable, but the vanT gene was present in two strains. The PCR result was also confirmed by sequence analysis. In detail, the respective nucleotide sequence exactly matched a section of the “E. gallinarum vanC vancomycin-resistance gene cluster,” which harbors the vanT gene¹ (GenBank: AF162694.1; 100% identities; 0 gaps).

The species-specific multiplex PCR confirmed that the positive samples were really E. faecalis. In detail, all investigated isolates were positive for E. faecalis, but negative for E. faecium and for E. gallinarum, respectively. The Pearson correlation coefficients obtained by RAPD-PCR were between 86% and 71% in pbp5-positive strains, with the strain of human origin being the most dissimilar. Pearson correlation coefficients of vanC1-positive strains were between 93% and 73% (Fig. 1). No corresponding transcripts of the pbp5 and vanC1 genes were detectable by real-time RT-PCR in the positive strains.

FIG. 1.

Cluster analysis of (A) pbp5-positive (nos. 0134, 1357, and 0539, of porcine origin, and 2538, of human origin) and (B) vanC1-positive (nos. 1266, 1267, 1262, and 1263; all of porcine origin) Enterococcus faecalis.

Discussion

The low-affinity PBP5 in E. faecium, encoded by the pbp5 gene, confers low-level resistance to ampicillin. More than 90% of the strains isolated from German hospitals are classified as ampicillin resistant.¹⁴ On the contrary, ampicillin is still a valuable therapeutic in E. faecalis infections.²⁰ This is in accordance with the MIC results of our study, where only two isolates were resistant to ampicillin.

There is one report on the occurrence of a gene designated “pbp5” in one E. faecalis termed “56 R”⁴⁷ (gi|467805), but the sequence homology with the amplified product of the present study is <70%. Actually, the 56 R pbp5 of Signoretto et al.⁴⁷ is almost identical (99%; 0 gaps) to the pbp4 gene of E. faecalis JH2-2 as described by Duez et al.¹⁰ Subsequent investigations, working with “pbp5” containing E. faecalis, always refer to the 56 R strain.^{10,25–27,43,48–50,56} The pbp5 gene of E. faecium is chromosomally localized, but its transferability to E. faecalis has been experimentally proven.⁴¹ Nevertheless, apart from Poeta et al.³⁶ and Radimersky et al.,³⁷ who tested 2 and 36 E. faecalis strains negative for the presence of pbp5, no further screenings have been reported. This means that the present study is the first report on the detection of pbp5 in E. faecalis apart from mating experiments.

Likewise, no scientist who searched for vanC1 in E. faecalis detected the gene in this species.^{3,4,7,15,16,19,23,28,32,35,38,46,51,55} Actually, it is claimed to be even superfluous to test E. faecalis or other Enterococcus species for the presence of vanC1, since this gene is supposed to be species specific for E. gallinarum.²² There is one report on the detection of vanC1 in one E. faecalis and in one E. faecium strain,³⁴ but a subsequent analysis of the 16S rRNA sequence revealed that both vanC1-containing strains were in reality E. gallinarum.³³ In the present study, a species identification error is unlikely because the distinctly red well-developed colonies on CATC-agar are very characteristic for E. faecalis; biochemical reactions were typical, and what is more, the species-specific PCR¹⁷ confirmed the results of the conventional bacteriological investigation. Noticeably, all vanC1-positive isolates were isolated from pigs notwithstanding that vancomycin is a reserve antibiotic in human medicine and not approved for livestock use in Germany. The glycopeptide growth promoter avoparcin was banned from livestock feed in Germany in 1996 in order to reduce the risk of cross-resistance to vancomycin.⁵ This indicates that E. faecalis might have acquired the vanC1 gene from the natural vanC1 carrier E. gallinarum, which is a normal inhabitant of the porcine gut.^8,42 Normally, the three genes vanC1, vanT, and vanXYc are clustered.¹ The finding that, in our study, only two of the four vanC1-positive strains were vanT positive and none contained vanXYc might be attributable to a high instability of these genes in E. faecalis after cell division, as it has been reported, for example, for unstable elements in Staphylococcus aureus.⁵²

All pbp5-positive strains were susceptible to ampicillin. The same is true for vanC1; the vancomycin MIC remained as low as 1 mg/L for the vanC1-positive isolates even after prolonged incubation (48 hr), as recommended by Hirakata et al.¹⁵ The phenotypic susceptibility can be explained by the fact that the resistance genes pbp5 and vanC1 were not expressed in the investigated strains, as shown via real-time RT-PCR. The regulating mechanisms of expression of pbp5 are unknown, but it has been suggested that the upstream open reading frame (ORF) psr (penicillin-binding protein synthesis repressor) might have a negative impact on PBP5 production.²⁴ On the contrary, Rice et al.⁴⁰ suggest that PSR does not serve as a repressor of pbp5 transcription. Further investigations are necessary to clarify these questions.

Nevertheless, even if the strains of the present study were phenotypically susceptible to ampicillin and vancomycin, it is a cause for concern that E. faecalis are able to naturally acquire pbp5 and vanC1 from the bacterial community, since the possibility that complete gene clusters and functional genes will be transferred and expressed cannot be ruled out. This study underlines the need to continue being aware that E. faecalis are emerging nosocomial pathogens on the one hand³¹ and very potent resistance gene collectors—and possibly donors—on the other hand.^13,44,45 These risk factors affirm that enterococci should always be closely monitored, and this applies not only to the human clinical isolates, but also to the commensals from their diverse habitats.

Footnotes

Acknowledgments

This work was supported by the Bavarian State Ministry of the Environment, Public Health and Consumer Protection and by the Bavarian State Ministry of Agriculture and Forestry. The authors thank Heike Kliem and technicians for providing the laboratory and equipment for real-time RT-PCR. We also thank Dr. Ingo Klare (Robert Koch Insititut, Wernigerode, Germany) for kindly providing the reference strains.

Disclosure Statement

No competing financial interests exist.

References

Arias

, Courvalin

, Reynolds

P.E.

2000. vanC cluster of vancomycin-resistant Enterococcus gallinarum BM4174. Antimicrob. Agents Chemother., 44:1660–1666.

Arthur

, Courvalin

1993. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob. Agents Chemother., 37:1563–1571.

Bell

, Turnidge

, Coombs

, O'Brien

1998. Emergence and epidemiology of vancomycin-resistant enterococci in Australia. Commun. Dis. Intell., 22:249–252.

Bell

J.M.

, Paton

J.C.

, Turnidge

1998. Emergence of vancomycin-resistant enterococci in Australia: phenotypic and genotypic characteristics of isolates. J. Clin. Microbiol., 36:2187–2190.

BfR. 1996. Bundesinstitut für Risikobewertung, Pressedienst. Avoparcin als Futterzusatzstoff in der Tierernährung vorläufig verboten. www.bfr.bund.de/cd/780(Online.) (In German.)

CDC. 2010. Centers for Disease Control and Prevention. Healthcare-associated Infections (HAI's). Vancomycin-resistant Enterococci (VRE) and the Clinical Laboratory. www.cdc.gov/HAI/settings/lab/VREClinical-Laboratory.html(Online.)

Christidou

, Gikas

, Scoulica

, Pediaditis

, Roumbelaki

, Georgiladakis

, Tselentis

2004. Emergence of vancomycin-resistant enterococci in a tertiary hospital in Crete, Greece: a cluster of cases and prevalence study on intestinal colonisation. Clin. Microbiol. Infect., 10:999–1005.

Del Grosso

, Caprioli

, Chinzari

, Fontana

M.C.

, Pezzotti

, Manfrin

, Giannatale

E.D.

, Goffredo

, Pantosti

2000. Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy. Microb. Drug Resist., 6:313–318.

DIN 58940-81. 2002. Medical Microbiology—Susceptibility Testing of Microbial Pathogens to Antimicrobial Agents—Part 81: Microdilution; Special Requirements for Testing of Non-fastidious Bacteria. Beuth: Verlag, Berlin.

10.

Duez

, Zorzi

, Sapunaric

, Amoroso

, Thamm

, Coyette

2001. The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene. Microbiology, 147:2561–2569.

11.

Dutka-Malen

, Evers

, Courvalin

1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol., 33:24–27; Erratum in: J. Clin. Microbiol. 33:1434.

12.

Fontana

, Ligozzi

, Pittaluga

, Satta

1996. Intrinsic penicillin resistance in enterococci. Microb. Drug Resist., 2:209–213.

13.

Franklin

, Acar

, Anthony

, Gupta

, Nicholls

, Tamura

, Thompson

, Threlfall

E.J.

, Vose

, Van Vuuren

, White

D.G.

, Wegener

H.C.

, Costarrica

M.L.

Office International des Epizooties Ad hoc Group. 2001. Antimicrobial resistance: harmonisation of national antimicrobial resistance monitoring and surveillance programmes in animals and in animal-derived food. Sci. Tech. Rev., 20:859–870.

14.

GERMAP. 2008. Antibiotika-Resistenz und -Verbrauch. Bericht über den Antibiotikaverbrauch und die Verbreitung von Antibiotikaresistenzen in der Human- und Veterinärmedizin in Deutschland. www.bvl.bund.de/DE/08__PresseInfothek/00__doks__downloads/Germap__2008,templateId=raw,property=publicationFile.pdf/Germap_2008.pdf(Online) (In German.)

15.

Hirakata

, Yamaguchi

, Izumikawa

, Matsuda

, Tomono

, Kaku

, Koga

, Yamada

, Kohno

, Kamihira

1997. In vitro susceptibility studies and detection of vancomycin resistance genes in clinical isolates of enterococci in Nagasaki, Japan. Epidemiol. Infect., 119:175–181.

16.

Ieven

, Vercauteren

, Descheemaeker

, van Laer

, Goossens

1999. Comparison of direct plating and broth enrichment culture for the detection of intestinal colonization by glycopeptide-resistant enterococci among hospitalized patients. J. Clin. Microbiol., 37:1436–1440.

17.

Jackson

C.R.

, Fedorka-Cray

P.J.

, Barrett

J.B.

2004. Use of a genus- and species-specific multiplex PCR for identification of enterococci. J. Clin. Microbiol., 42:3558–3565.

18.

Kappstein

2007. Surveillance resistenter Erreger und rationaler Einsatz von Antibiotika. Krankenhaushygiene up2date., 3:197–215. (In German.)

19.

Khan

S.A.

, Nawaz

M.S.

, Khan

A.A.

, Hopper

S.L.

, Jones

R.A.

, Cerniglia

C.E.

2005. Molecular characterization of multidrug-resistant Enterococcus spp. from poultry and dairy farms: detection of virulence and vancomycin resistance gene markers by PCR. Mol. Cell. Probes., 19:27–34.

20.

Klare

, Konstabel

, Badstübner

, Werner

, Witte

2003. Occurrence and spread of antibiotic resistances in Enterococcus faecium. Int. J. Food. Microbiol., 88:269–290.

21.

Klare

, Rodloff

A.C.

, Wagner

, Witte

, Hakenbeck

1992. Overproduction of a penicillin-binding protein is not the only mechanism of penicillin resistance in Enterococcus faecium. Antimicrob. Agents Chemother., 36:783–787.

22.

Leclercq

, Dutka-Malen

, Duval

, Courvalin

1992. Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. Antimicrob. Agents Chemother., 36:2005–2008.

23.

Liassine

, Frei

, Jan

, Auckenthaler

1998. Characterization of glycopeptide-resistant enterococci from a Swiss hospital. J. Clin. Microbiol., 36:1853–1858.

24.

Ligozzi

, Pittaluga

, Fontana

1993. Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5. J. Bacteriol., 175:2046–2051.

25.

Ligozzi

, Pittaluga

, Fontana

1996. Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium. Antimicrob. Agents Chemother., 40:354–357.

26.

Lleò

M.M.

, Bonato

, Tafi

M.C.

, Signoretto

, Boaretti

, Canepari

2001. Resuscitation rate in different enterococcal species in the viable but non-culturable state. J. Appl. Microbiol., 91:1095–1102.

27.

Lleò

M.M.

, Pierobon

, Tafi

M.C.

, Signoretto

, Canepari

2000. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol., 66:4564–4567.

28.

J.J.

, Perng

C.L.

, Chiueh

T.S.

, Lee

S.Y

, Chen

C.H.

, Chang

F.Y

, Wang

C.C.

, Chi

W.M.

2001. Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR. Epidemiol. Infect., 126:357–363.

29.

Martín

, Corominas

, Garriga

, Aymerich

2009. Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment. J. Appl. Microbiol., 106:66–77.

30.

Murray

B.E.

1992. Beta-lactamase-producing enterococci. Antimicrob. Agents Chemother., 36:2355–2359.

31.

NRZ. Nationales Referenzzentrum für Surveillance von nosokomialen Infektionen. 2010. Krankenhaus-Infektions-Surveillance-System. Modul ITS-KISS. Referenzdaten. Berechnungszeitraum: Januar 2005- Dezember 2009. www.nrz-hygiene.de/surveillance/kiss/its-kiss/(Online.) (In language.)

32.

Oguri

, Misawa

, Nakamura

, Igari

, Ishii

, Yamaguchi

2001. Epidemiological study on vancomycin-resistant enterococci from fecal samples in the east area of Japan. Kansenshogaku Zasshi., 75:541–550.

33.

Patel

, Piper

K.E.

, Rouse

M.S.

, Steckelberg

J.M.

, Uhl

J.R.

, Kohner

, Hopkins

M.K.

, Cockerill

F.R.

3rd. , Kline

B.C.

1998. Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates. J. Clin. Microbiol., 36:3399–3407.

34.

Patel

, Uhl

J.R.

, Kohner

, Hopkins

M.K.

, Cockerill

F.R.

3rd . 1997. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci. J. Clin. Microbiol., 35:703–707.

35.

Pérez-Hernández

, Méndez-Alvarez

, Delgado

, Moreno

, Reyes-Darias

, Sierra López

, Villar

, González

, Martín Sánchez

, Macía

, Claverie-Martín

2002. Low prevalence of vancomycin-resistant enterococci in clinical samples from hospitalized patients of the Canary Islands, Spain. Int. Microbiol., 5:117–120.

36.

Poeta

, Costa

, Igrejas

, Rodrigues

, Torres

2007. Phenotypic and genotypic characterization of antimicrobial resistance in faecal enterococci from wild boars (Sus scrofa) Vet. Microbiol., 125:368–374.

37.

Radimersky

, Frolkova

, Janoszowska

, Dolejska

, Svec

, Roubalova

, Cikova

, Cizek

, Literak

2010. Antibiotic resistance in faecal bacteria (Escherichia coli, Enterococcus spp.) in feral pigeons. J. Appl. Microbiol., 109:1687–1695.

38.

Radu

, Toosa

, Rahim

R.A.

, Reezal

, Ahmad

, Hamid

A.N.

, Rusul

, Nishibuchi

2001. Occurrence of the vanA and vanC2/C3 genes in Enterococcus species isolated from poultry sources in Malaysia. Diagn. Microbiol. Infect. Dis., 39:145–153.

39.

Ramotar

, Woods

, Larocque

, Toye

2000. Comparison of phenotypic methods to identify enterococci intrinsically resistant to vancomycin (VanC VRE) Diagn. Microbiol. Infect. Dis., 36:119–124.

40.

Rice

L.B.

, Carias

L.L.

, Hutton-Thomas

S.R.

, Sifaoui

, Gutmann

, Rudin

2001. Penicillin-Binding Protein 5 and Expression of Ampicillin Resistance in Enterococcus faecium. Antimicrob. Agents Chemother., 45:1480–1486.

41.

Rice

L.B.

, Carias

L.L.

, Rudin

, Lakticová

, Wood

, Hutton-Thomas

2005. Enterococcus faecium low-affinity pbp5 is a transferable determinant. Antimicrob. Agents Chemother., 49:5007–5012.

42.

Rizzotti

, Simeoni

, Cocconcelli

, Gazzola

, Dellaglio

, Torriani

2005. Contribution of enterococci to the spread of antibiotic resistance in the production chain of swine meat commodities. J. Food. Prot., 68:955–965.

43.

Robbi

, Signoretto

, Boaretti

, Canepari

1996. The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe. Microb. Drug Resist., 2:215–218.

44.

Schwaiger

, Bauer

2008. Detection of the erythromycin rRNA methylase gene erm(A) in Enterococcus faecalis. Antimicrob. Agents Chemother., 52:2994–2995.

45.

Schwaiger

, Hölzel

C.S.

, Bauer

2011. Detection of the Macrolide-Efflux Protein A Gene mef(A) in Enterococcus faecalis. Microb. Drug Resist., 17:429–32.

46.

Seong

C.N.

, Shim

E.S.

, Kim

S.M.

, Yoo

J.C.

2004. Prevalence and characterization of vancomycin-resistant enterococci in chicken intestines and humans of Korea. Arch. Pharm. Res., 27:246–253.

47.

Signoretto

, Boaretti

, Canepari

1994. Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis. FEMS Microbiol. Lett., 123:99–106.

48.

Signoretto

C. M.

, Boaretti

, Canepari P

1998. Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5. Arch. Microbiol., 170:185–190.

49.

Signoretto

, Canepari

1997. Alterations in the chemical composition of peptidoglycan of Escherichia coli DH5 alpha induced by the expression of Enterococcus faecalis penicillin binding protein 5. New Microbiol., 20:21–28.

50.

Signoretto

, Canepari

2000. Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium. Arch. Microbiol., 173:213–219.

51.

Titze-de-Almeida

R. M.

, Rollo Filho

, C.A. , Nogueira

C.A.

, I.P. , Rodrigues

I.P.

, J. , Eudes Filho

, Nascimento

R.S.

, Ferreira

R.F.

2nd. , Moraes

L.M.

, Boelens

, Van Belkum

, Felipe

M.S.

2004. Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units. Braz. J. Infect. Dis., 8:197–205.

52.

Ubeda

, Barry

, Penadés

J.R.

, Novick

R.P.

2007. A pathogenicity island replicon in Staphylococcus aureus replicates as an unstable plasmid. Proc. Natl. Acad. Sci. U S A., 104:14182–14188.

53.

WHO. 1997. The Medical Impact of Antimicrobial Use in Food Animals. Report of a WHO Meeting. Berlin, Germany. 13–17 October 1997. WHO/EMC/ZOO/97.4. WHO: Geneva.

54.

Williamson

, le Bouguénec

, Gutmann

, Horaud

1985. One or two low affinity penicillin-binding proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin. J. Gen. Microbiol., 131:1933–1940.

55.

Yang

, Lee

, Kim

, Kang

, Kim

, Ha

2007. Occurrence of the van genes in Enterococcus faecalis and Enterococcus faecium from clinical isolates in Korea. Arch. Pharm. Res., 30:329–336.

56.

Zorzi

, Zhou

X.Y.

, Dardenne

, Lamotte

, Raze

, Pierre

, Gutmann

, Coyette

1996. Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium. J. Bacteriol., 178:4948–4957.