The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis.
BACKGROUND
Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model.


RESULTS
Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region


CONCLUSIONS
The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.
Department of Internal Medicine, Division of Infectious Diseases, Center for the Study of Emerging and Reemerging Pathogens, Houston, TX, USA.
Diana
D
Panesso
Maria C
MC
Montealegre
Sandra
S
Rincón
Maria F
MF
Mojica
Louis B
LB
Rice
Kavindra V
KV
Singh
Barbara E
BE
Murray
Cesar A
CA
Arias
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D016984:Enterococcus faecium / Q000235:genetics / Q000472:pathogenicity*; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D010538:Peritonitis / Q000382:microbiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins
J Clin Microbiol. 2002 Sep;40(9):3326-33
12202574
BMC Genomics. 2010 Apr 14;11:239
20398277
Biochem J. 2006 Oct 15;399(2):241-7
16822234
Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):996-1001
11792842
J Clin Microbiol. 2004 Oct;42(10):4473-9
15472296
J Bacteriol. 2006 Apr;188(8):2875-84
16585749
Antimicrob Agents Chemother. 2009 Oct;53(10):4240-6
19667280
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4658-63
11296296
Antimicrob Agents Chemother. 1994 Sep;38(9):1899-903
7810996
Plasmid. 2007 Mar;57(2):131-44
16996131
Appl Environ Microbiol. 2006 Jan;72(1):334-45
16391062
Proteins. 1994 Dec;20(4):347-55
7731953
J Clin Microbiol. 2009 Apr;47(4):896-901
19193843
Future Microbiol. 2009 Nov;4(9):1125-35
19895216
J Infect Dis. 2003 Feb 1;187(3):508-12
12552437
Virulence. 2010 Jul-Aug;1(4):236-46
20676385
J Bacteriol. 2007 Nov;189(22):8233-40
17827282
Emerg Infect Dis. 2005 Jun;11(6):821-8
15963275
J Infect Dis. 2009 Feb 1;199(3):342-9
19049434
Antimicrob Agents Chemother. 2010 Jun;54(6):2660-5
20385861
J Infect Dis. 2009 Oct 1;200(7):1162-5
19702507
Int J Med Microbiol. 2008 Jul;298(5-6):515-27
17977789
J Infect Dis. 1998 Nov;178(5):1416-20
9780263
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1996-2001
12574517
J Clin Microbiol. 1995 Jan;33(1):24-7
7699051
Int J Med Microbiol. 2011 Feb;301(2):165-75
20951641
Microbiology (Reading). 2008 Oct;154(Pt 10):3212-3223
18832326
J Clin Microbiol. 1995 May;33(5):1434
7615777
Mol Microbiol. 2003 Mar;47(6):1733-47
12622825
J Infect Dis. 2009 Nov 15;200(10):1566-73
19821720
FEBS Lett. 1991 Nov 18;293(1-2):160-3
1959653
Infect Immun. 2008 Sep;76(9):4120-8
18591236
J Clin Microbiol. 2010 May;48(5):1562-9
20220167
J Med Microbiol. 2010 Apr;59(Pt 4):505-507
20075113
J Bacteriol. 1986 Mar;165(3):831-6
3005240
Infect Control Hosp Epidemiol. 2008 Nov;29(11):996-1011
18947320