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Correspondence |
1 Center for the Study of Emerging and Reemerging Pathogens, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, TX 77030, USA
2 Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA
3 Bacterial Molecular Genetics Unit, Universidad El Bosque, Bogota, Colombia
4 Department of Microbiology Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030, USA
Correspondence
Barbara E. Murray
(bem.asst{at}uth.tmc.edu)
Enterococci, like streptococci, are typically described as showing chaining in Gram-stains of clinical samples. The degree of chaining seems to vary amongst enterococcal clinical isolates; however, there are no recent studies validating chaining by enterococci since they were classified out of the genus Streptococcus nor any studies of the mechanism of cellular chain length. A marked increase in chaining seen in an Enterococcus faecalis OG1RF mutant (TX5128) has been explained by the lack of the GelE protease (gelatinase) suggesting the possibility that chaining among clinical isolates (one-third of which are gelatinase non-producers) might be associated with the absence of GelE (Singh et al., 2005; Waters et al., 2003). GelE is an enterococcal zinc-metalloprotease (Bleiweis & Zimmerman, 1964) that is capable of degrading a broad spectrum of substrates, including insoluble collagen fragments (Makinen & Makinen, 1994) and polymerized fibrin (Waters et al., 2003). Synthesis of GelE in E. faecalis is regulated by the fsr locus (Qin et al., 2001), a quorum-sensing system that positively regulates expression of gelE. The fsr locus and gelatinase production have been shown to contribute to the virulence of E. faecalis OG1RF in several models, including mouse peritonitis, Caenorhabditis elegans and biofilm formation (Mohamed et al., 2004; Qin et al., 1998, 2000; Sifri et al., 2002; Waters et al., 2003).
In the present study, a collection of clinical isolates of E. faecalis was studied to determine the degree of variation in the chaining phenotype and to establish whether the lack of GelE production or the absence of gelE or fsrB genes among clinical isolates might explain any differences found. Since a correlation between chain length and disruption of gelE in the mutant TX5128 was demonstrated in vitro, such a correlation in clinical isolates might also be helpful to predict the absence/presence of important virulence factors (i.e. GelE) or their regulatory genes (i.e. fsrB) with a simple clinical microbiology observation. Therefore, the number of cells per chain in isolates grown in broth was determined by light microscopy, and a correlation between chain length and genotype was attempted.
A total of 106 clinical isolates of E. faecalis [including blood (endocarditis and non-endocarditis), urine and community sources] from different geographical and clonal origins present in our collection and previously characterized for the presence of gelE and fsrB and for the GelE phenotype (on Todd–Hewitt agar plates with 3 % gelatin) (Roberts et al., 2004) was studied to determine the chaining phenotype (see below). E. faecalis strains OG1RF (wild-type, GelE+; Murray et al., 1993), TX5128 (a gelE disruption mutant derived from OG1RF, GelE–; Qin et al., 1998) and TX5266 (an fsrB deletion mutant that is GelE– under routine conditions; Qin et al., 2001) were used as controls in the chaining experiments. All isolates were incubated in brain heart infusion broth overnight at 37 °C with agitation (150–200 r.p.m.). Kanamycin (2000 µg ml–1) was added for TX5128 (gelE mutant) (Waters et al., 2003). The next day, the culture was diluted to obtain a turbidity equivalent to 0.5 McFarland (A625 0.08–0.10; 108 c.f.u. ml–1) and then incubated for 5 h at 37 °C with agitation (150–200 r.p.m.). Cells were harvested by centrifugation, resuspended in 5 ml 0.9 % saline and 5 µl was spotted on a glass slide, Gram-stained, covered with a coverslip and visualized under a light microscope. To evaluate the chaining phenotype, each isolate was analysed and the number of cells per chain (a chain is defined as the presence of 
1 cell) in 50 randomly chosen chains at x100 magnification (usually four different power fields) was recorded. All isolates were evaluated in a blind manner by two observers (without knowing the origin, identification of the isolate, GelE phenotype or genotype). Discrepancies between observers were resolved by performing additional blind experiments until agreement was reached.
The following criteria were used to classify every isolate: an isolate was categorized as a short chainer if 80 % of the chains (out of 50 assessed) had 
4 cells (and thus, <20 % had >4 cells); as a medium chainer if 41–79 % of chains had 4 cells; as a long chainer if 40 % of chains contained 4 cells; and as an extra-long chainer when >70 % of chains contained 10 cells. Correlations between genotype/phenotype and chain length were performed for every category, with an initial analysis comparing short and non-short chainer (i.e. the sum of all medium, long and extra-long chain-forming isolates) subgroups. A Fischer exact test was performed to establish statistically significant differences between short-chain-forming isolates and other categories; P<0.05 was considered significant.
There was a marked difference in the degree of chaining amongst clinical isolates, ranging from short chainers (Fig. 1a
; 79 % of isolates; range of the mean number of cells per chain per isolate, 1.0–3.5; mean number of cells per chain of all isolates, 2.3) to extra-long chainers (Fig. 1e; 3 % of isolates; mean number of cells per chain per isolate, >15; mean number of cells per chain, 27). Amongst the remaining isolates, 13 (12 %) were classified as medium chainers (Fig. 1b, c; range of the mean number of cells per chain per isolate, >3.5–7.5; mean number of cells per chain of all isolates, 5.0) and 5 (4 %) were classified as long chainers (Fig. 1d; range of the mean number of cells per chain per isolate, >7.5–15.0; mean number of cells per chain, 11.3). Short-chaining isolates were independently compared to each of the other chaining categories with regard to the presence (or absence) of GelE, gelE and fsrB. A similar analysis was performed comparing short-chaining isolates and the sum of the other subgroups (medium, long and extra-long chainers). There was no correlation between the absence of gelE, fsrB or gelatinase production and isolates with medium, long or extra-long chains (Table 1) or when non-short isolates (grouping isolates categorized as having either medium, long or extra-long chains) were compared to those with short chains (all P>0.05) (Table 1). Indeed, the majority of non-short isolates produced gelatinase and had the gelE or fsrB gene: 68 % for GelE, 95 % for gelE and 77 % for fsrB. Moreover, when long and extra-long chainers were grouped together, only 33, 11 and 22 % lacked GelE, gelE and fsrB, respectively. A similar percentage of short chain formers lacked GelE (36 %), gelE (12 %) or fsrB (28 %). These results indicate that chaining among clinical isolates is not dependent on the lack of gelatinase, fsrB or gelE. Under the conditions used in this study, E. faecalis OG1RF was classified as a short chainer (80 % of the chains had 4 cells), whereas both TX5128 (gelE disruption mutant) and TX5266 (fsrB deletion mutant) were categorized as medium chainers (mean number of cells per chain of all isolates, 5.0) (Fig. 1b, c).
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In summary, marked variation in chaining was found in clinical isolates of E. faecalis. However, despite a pronounced effect of disruption of the gelE gene of strain OG1RF on chaining, neither the lack of GelE production nor the absence of the gelE gene was associated with chaining among clinical isolates, with some GelE+ isolates showing marked chaining (>15 cells per chain) and some GelE– and gelE-lacking isolates showing predominantly short chains (4 cells per chain).
Acknowledgements
This work was supported by NIH grant R37 AI47923 from the Division of Microbiology and Infectious Diseases to B. E. M. We are grateful to Natalia Mendoza, Universidad El Bosque, Colombia, for statistical analysis, and Kavindra Singh and Sreedhar Nallapareddy for helpful discussions in the preparation of this manuscript.
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