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Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy
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Michael Dan a,b,∗ , Yael Yair c , Alex Samosav c , Tamar Gottesman a , Orit Yossepowich a , Orna Harari-Schwartz d , Alexander Tsivian e , Rachel Schreiber c , Uri Gophna c
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Infectious Diseases Unit, E. Wolfson Hospital, Holon, Israel Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel d Microbiology Laboratory, E. Wolfson Hospital, Holon, Israel e Department of Urology, E. Wolfson Hospital, Holon, Israel b
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Article history: Received 19 January 2014 Received in revised form 6 April 2015 Accepted 20 April 2015
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Keywords: Escherichia coli Sepsis following prostate transrectal biopsy 21 Bacteremic urinary tract infection 22 23 Q4 Virulence markers 19 20
Background: Transrectal ultrasound-guided (TRUS) prostate biopsy is a very common procedure that is generally considered relatively safe. However, severe sepsis can occur after TRUS prostate biopsies, with Escherichia coli being the predominant causative agent. A common perception is that the bacteria that cause post-TRUS prostate biopsy infections originate in the urinary tract, but this view has not been adequately tested. Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa. Methods: We compared E. coli isolates from male patients with bacteremic urinary tract infection (BUTI) to isolates of patients with post prostate biopsy sepsis (PPBS), in terms of their sequence types, determined by multi-locus sequence typing (MLST) and their virulence markers. Results: B-UTI isolates were much richer in virulence genes than were PPBS isolates, supporting the hypothesis that E. coli causing PPBS derive directly from the rectum. Sequence type 131 (ST131) strains and related strain from the ST131 were common (>30%) among the E. coli isolates from PPBS patients as well as from B-UTI patients and all these strains expressed extended spectrum beta-lactamases. Conclusions: Our finding supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. In light of the increasing prevalence of highly resistant E. coli strains, a new approach for prevention of PPBS is urgently required. © 2015 Published by Elsevier GmbH.
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Article’s main point: Bacteremic UTI isolates are much richer in virulence genes than are post-transrectal prostate biopsy sepsis isolates, supporting the hypothesis that E. coli causing post transrectal prostate biopsy sepsis derive directly from the rectum.
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Introduction
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Transrectal ultrasound-guided (TRUS) prostate biopsy is one of the most commonly performed procedures in urology. Although
∗ Corresponding author at: Infectious Diseases Unit, E. Wolfson Hospital, Holon Q3 58100, Israel. Tel.: +972 50 6396950; fax: +972 3 5016126. E-mail addresses: [email protected], [email protected] (M. Dan).
TRUS prostate biopsy is generally considered to be a relatively safe outpatient procedure, severe sepsis has been described in 0.1%–3.5% of cases after TRUS prostate biopsy, with Escherichia coli being the most commonly involved pathogen (Nam et al., 2010; Raaijmakers et al., 2002; Tal et al., 2003). Recently there has been an apparent increase in hospitalization for infectious complications after TRUS prostate biopsy (Loeb et al., 2011; Nam et al., 2010; Wagenlehner et al., 2013). The role of antimicrobial prophylaxis in preventing infectious complications after biopsy is now well established (Zani et al., 2011), although life-threatening infection still occur despite prophylaxis (Carignan et al., 2012). Moreover, a systematic review and meta-analysis of randomized controlled studies confirms that prophylaxis reduces the rate of bacteriuria following prostate biopsy, although its effectiveness in reducing symptomatic UTI and
http://dx.doi.org/10.1016/j.ijmm.2015.04.003 1438-4221/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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bacteremia is less certain (Bootsma et al., 2008). The most commonly used prophylactic antimicrobials are the fluoroquinolones (Wagenlehner et al., 2013). Several publications have recently documented alarming increase in resistance to these agents among E. coli in post-TRUS prostate biopsy infections (Feliciano et al., 2008; Zaytoun et al., 2011), as well as to other antimicrobial agents such as ampicillin, ampicillin/sulbactam, trimethoprim/sulfamethoxazole and gentamicin (Carignan et al., 2012); extended spectrum -lactamase (ESBL)–producing isolates have been increasingly recovered (Briffaux et al., 2009). The rise over the years in resistance of E. coli to ciprofloxacin (up to 90%) is accompanied by a concomitant increase in the rate of post-TRUS prostate biopsy infectious complications (Carignan et al., 2012; Feliciano et al., 2008). If this trend continues, resistance will spread to other antimicrobial agents at increasing rates. An alternative preventive approach should thus be considered. For this purpose, it is important to inquire into the pathogenesis of post-TRUS prostate biopsy infections: what is the reservoir of the infecting organisms, what is the route of their acquisition, what is the virulence arsenal of the pathogens etc. It is a common perception that the bacteria responsible for postTRUS biopsy infections originate in the urinary tract (Wagenlehner et al., 2013). It is therefore recommended to perform midstream urine culture prior to the procedure, and to choose the antimicrobial agent used for prophylaxis according to the susceptibility of the bacteria isolated from urine (Grabe et al., 2013). Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa (Carignan et al., 2012; Williamson et al., 2012). The aim of the present study was to compare E. coli strains responsible for post-TRUS prostate biopsy infections and E. coli from male patients with bacteremic urinary tract infection in terms of genetic virulence markers. Correctly identifying the source of post-TRUS prostate biopsy infections is an essential step in the development of more effective preventive measures.
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Materials and methods
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Patients
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Patients with post-TRUS prostate biopsy sepsis (PPBS) Twenty-one men with febrile sepsis due to E. coli following TRUS prostate biopsy were prospectively enrolled during the period of January 2010 through August 2012 at the E. Wolfson Hospital Holon, Israel. Inclusion criteria included recent TRUS prostate biopsy, fever (temperature, >38.0 ◦ C), and positive blood and/or urine cultures for E. coli. Additionally, rectal samples for cultures were obtained from all patients after signing an informed consent. All patients presented symptoms within less than a week from the procedure, and over 90% presented them within 48 h of the biopsy.
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Patients with bacteremic urinary tract infection (B-UTI) During the same period, 24 men with bacteremic urinary tract infection due to E. coli were prospectively enrolled at the same institution. Inclusion criteria included male gender, fever (temperature, >38.0 ◦ C), and positive blood and urine cultures for E. coli. None of the patients included in this study had a Foley catheter. The E. Wolfson Hospital Research Ethics Committee approved the study protocol.
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Blood, urine and rectal cultures
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Blood samples were drawn aseptically from a peripheral vein. Each blood sample was inoculated into a set of blood culture bottles. Bottles were incubated in the BacT/Alert system (BioMerieux,
Table 1 Prevalence (%) of E. coli virulence genes typical to E. coli strains from male UTI and PPBS isolates versus B-UTI isolates (number of isolates noted in parentheses). Gene
% Positive (PPBS) N = 20
% Positive (B-UTI) N = 22
FDR-corrected (p-value* )
focG Sfa papC iron malX ompT
0 (0) 0 (0) 10 (2) 25 (4) 50 (10) 65 (13)
27 (6) 32 (7) 50 (11) 41 (9) 86 (19) 91 (20)
0.0310 0.0276 0.0276 0.3376 0.0310 0.0747
* Fisher’s exact test, adjusted for multiple hypothesis by the Hochberg and Benjamini false discovery rate correction (Rohwer and Thurber, 2009).
Inc, Durham, NC) until flagged positive or for 7 days. A sample from a positive bottle was Gram stained and subcultured. A single E. coli colony was saved for further studies. Urine was cultured semiquantitatively on blood agar and cysteine–lactose–electrolyte–deficient agar plates using the calibrated loop technique. A single colony was retained for further study. Fecal swabs were inoculated on one quadrant of a MacConkey’s agar plate, then streaked for isolation in the remaining three quadrants with a sterile loop and incubated overnight at 37 ◦ C. Lactose- and indole-positive Gramnegative bacilli with a consistent colonial morphology similar to E. coli. From each plate with Gram-negative bacterial growth a representative colony of each distinct morphotype (ranging from one to five morphotypes) was selected for identification using the Vitek 2 instrument (BioMerieux, USA) GN identification cards, and verified as E. coli. The E. coli isolate mix was subsequently re-streaked for isolation and transferred to the Department of Molecular Microbiology and Biotechnology, where a single colony was analyzed at random. Based on past studies (Lidin-Janson et al., 1978), this procedure has over 85% probability of identifying the dominant clone. Serum survival growth assays A single colony was picked and grown overnight in 2 ml of Davis minimal medium supplemented with 0.2% glucose. The overnight culture was then diluted 1:10 in 10 ml of Davis minimal medium supplemented with 0.4% glucose. After 3–4 h, when the cultures reached the end of logarithmic growth phase (O.D 595 of ∼2) 20 l of culture were transferred to 96-well microplates containing 40 l of concentrated Davis minimal medium (×5) supplemented with 2% glucose in addition to either 140 l of dd H2 O (control) or Human Serum (Sigma human serum H-4522, to 70% final concentration), to a final volume of 200 l. E. coli K-12 MG1655 was used as negative control, since it is rapidly killed in the presence of serum, while avian pathogenic strain 789, which is highly serum-resistant (Ideses et al., 2005) was used as a positive control. The O.D. of the cultures in the plates were continuously measured in an ELX 800 Absorbance Microplate Reader (BioTek) at 37◦ with continuous shaking. Virulence marker identification Virulence genes presence (Table 1) was determined using PCR using primers from (Rodriguez-Siek et al., 2005), (Supplementary table S1). All PCR reactions were run in a Biometra T Personal thermocycler, using 25 l reaction volumes. One microliter from 1:50 dilutions of overnight bacterial culture in ddH2 O pre-heated for 5 min. at 96 ◦ C were used as template and 28 amplification cycles with an annealing temprature of 63◦ . Each reaction consisted of 0.2 l Biotaq DNA Polymerase [5 U/l, (Bioline)], 0.5 l of 10 M solution of the forward and reverse primers, 1.5 l of 3 mM MgCl2, and a 2.5 l dNTP mix of 2.5 mM each.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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Multiple locus sequence typing
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For 28 of the isolates, the 7 loci used for multiple locus sequence typing in E. coli, were amplified by PCR, using the primers and protocols described in http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/. 1 l from a suspension of a bacterial overnight culture diluted 1:50 in water, as described above for virulence marker identification, was used as template. The proof-reading polymerase used was Phusion Hot-start II High Fidelity (Thermo Scientific, USA). The PCR amplicons were purified using illustra ExoProStar (GE Healthcare), and sequenced by Sanger sequencing. For the remaining 14 strains genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen, USA), following the manufacturer’s protocol and whole genome sequencing was performed using Illumina HiSeq platform with 2 × 100 base reads, yielding an average 157X coverage. Draft genomes were assembled using Edena V3.131028 (Hernandez et al., 2008) and BLASTN (Altschul et al., 1990)was used to identify the gene sequences corresponding to the 7 MLST loci.
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Statistical methods
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Statistical analyses were performed using SPSS v. 14 (IBM Software, USA) and GraphPad Prism (Graphpad Software, USA). For diversity differences and ST131 complex abundance, a two-tailed Fisher’s exact test was used. For virulence gene comparisons a onetailed Fisher’s exact test was used (since the hypothesis is that their abundance would be lower in PPBS), corrected for multiple hypothesis testing by the Benjamini and Hochberg false discovery rate correction (Benjamini and Hochberg, 1995).
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Characteristics of patients
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Patients with post-TRUS prostate biopsy sepsis (PPBS). The 21 patients were admitted to hospital because of fever and rigors within 0.75, Fisher’s exact test). Taken together these data strongly suggest that most PPBS strains differ genotypically from B-UTI strains, but that the ST131 group has become so widespread that it is more or less evenly distributed between these different patient populations.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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Discussion Escherichia coli is the most common cause of UTI in men (Lipsky, 1999; Ulleryd et al., 1994) and of sepsis after TRUS prostate biopsy (Tal et al., 2003; Williamson et al., 2013). While several studies have examined the virulence characteristics and clonal background of E. coli isolates recovered from men with UTI (Andreu et al., 1997; Johnson et al., 2005; Kanamaru et al., 2003; Mitsumori et al., 1999; Otto et al., 2001; Ruiz et al., 2002; Terai et al., 1997; Ulleryd et al., 1994), little is known about the characteristics of E. coli causing sepsis after prostate biopsy. Phylogenetic and pathotypic analysis of E. coli isolates recovered from men with febrile UTI, showed that such strains are richly endowed with diverse extraintestinal virulence factors and derive from virulence-associated phylogenetic groups, largely independent of host compromise status. Comparing pathogenic E. coli isolates causing UTI to E. coli colonizing the rectum of the same patients led Johnson et al. (2005) to conclude that men are naturally highly resistant to febrile UTI, and considerable virulence capacity is required for an E. coli strain to cause it. Our results, demonstrating distinct differences between B-UTI strains and those that cause PPBS, further support that conclusion. The finding that B-UTI isolates were much richer in virulence genes than were PPBS isolates, supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. The ompT gene was found to be the virulence factor most closely associated with bacteremia (not necessarily related to the urinary tract) in a comparison of bloodstream versus rectal E. coli isolates among male veterans (Sannes et al., 2004), and also highly associated with male febrile UTI (Johnson et al., 2005). This virulence gene was also the most prevalent virulence factor in our study among isolates from B-UTI (91%). Similarly, in the PPBS group ompT was detected in 88% of isolates in patients with positive urine and blood cultures (data not shown). Thus, we may conclude that ompT may have a role in the pathogenesis of bacteremia, even when these are not associated with the urinary tract, potentially because it can confer resistance to diverse host antimicrobial peptides (Hui et al., 2010; McCarter et al., 2004; Stumpe et al., 1998). Recently a pandemic of a particular emergent E. coli clonal group, sequence type 131 (ST131, serotype O25b:H4) has been described (Rogers et al., 2011). Such ST131 isolates have distinct virulence profiles and are extensively resistant to antimicrobial agents, including their hallmark resistance to fluoroquinolones (Rogers et al., 2011). ST131 was implicated as the major culprit in bloodstream infections following transrectal prostate biopsy in New Zealand (Williamson et al., 2012), and accounted for 70% of fluoroquinolone-resistant rectal E coli isolates among men undergoing transrectal prostate biopsy in southern California (Liss et al., 2013). In Israel fluoroquinolone-resistance is highly prevalent among E. coli isolated from patients with PPBS (>80% of isolates) [Tal et al., 2003; Dan M, unpublished data, 2015], and E coli sequence type 131 has recently been documented in Israel (Karfunkel et al., 2013). ST131 strains were common among the E. coli isolates in the present study, and ST131 was found in 7/20 (35%) strains from PPBS patients and 6/22 (27%) strains from B-UTI patients. All ST131 strains were of community origin and expressed ESBL, exemplifying the important antimicrobial resistance associated with ST131, and the inadequacy of current prophylactic regimen for patients undergoing prostate biopsies. With the mounting evidence that PPBS results from E. coli that is directly introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa on one hand and the alarming increase in antimicrobial resistance on the other, a new approach of prevention which is not based on the use of
antimicrobial agents should be considered. The aim of such preventive measure should be to reduce the bacterial load of the rectal mucosa at the site of biopsy and hence lessen the bacterial inoculum introduced during the biopsy procedure. Although the role of pre-biopsy rectal cleansing enemas has been recently assessed (Zani et al., 2011), no significant benefit over prophylactic antibiotic was documented, and this technique is not routinely recommended (Zani et al., 2011). Rectal disinfection with agents such as chlorhexidine or povidone–iodine has been also evaluated as adjunct to antibiotic prophylaxis, but no solid data supporting the efficacy of such approach are currently available (Duplessis et al., 2012). With the alarming increase in ST131 prevalence, further research and development of innovative techniques that do not rely on conventional antimicrobial agents is urgently required. Acknowledgements This work was supported by the German-Israeli Project Cooperation (DIP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2015.04. 003 References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Andreu, A., Stapleton, A.E., Fennell, C., Lockman, H.A., Xercavins, M., Fernandez, F., Stamm, W.E., 1997. Urovirulence determinants in Escherichia coli strains causing prostatitis. J. Infect. Dis. 176, 464–469. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc., B Met. 57, 289–300. Bootsma, A.M., Laguna Pes, M.P., Geerlings, S.E., Goossens, A., 2008. Antibiotic prophylaxis in urologic procedures: a systematic review. Eur. Urol. 54, 1270–1286. Briffaux, R., Coloby, P., Bruyere, F., Ouaki, F., Pires, C., Dore, B., Irani, J., 2009. One preoperative dose randomized against 3-day antibiotic prophylaxis for transrectal ultrasonography-guided prostate biopsy. BJU Int. 103, 1069–1073 (discussion 1073). Carignan, A., Roussy, J.F., Lapointe, V., Valiquette, L., Sabbagh, R., Pepin, J., 2012. Increasing risk of infectious complications after transrectal ultrasound-guided prostate biopsies: time to reassess antimicrobial prophylaxis? Eur. Urol. 62, 453–459. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Duplessis, C.A., Bavaro, M., Simons, M.P., Marguet, C., Santomauro, M., Auge, B., Collard, D.A., Fierer, J., Lesperance, J., 2012. Rectal cultures before transrectal ultrasound-guided prostate biopsy reduce post-prostatic biopsy infection rates. Urology 79, 556–561. Feliciano, J., Teper, E., Ferrandino, M., Macchia, R.J., Blank, W., Grunberger, I., Colon, I., 2008. The incidence of fluoroquinolone resistant infections after prostate biopsy – are fluoroquinolones still effective prophylaxis? J. Urol. 179, 952–955 (discussion 955). Grabe, M., Bjerklund-Johansen, T.E., Botto, H., C¸ek, M., Naber, K.G., Pickard, R., Tenke, P., Wagenlehner, F.M., Wullt, B., 2013. Guidelines on urological infections. Eur. Q7 Assoc. Urol. Hernandez, D., Francois, P., Farinelli, L., Osteras, M., Schrenzel, J., 2008. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 18, 802–809. Hui, C.Y., Guo, Y., He, Q.S., Peng, L., Wu, S.C., Cao, H., Huang, S.H., 2010. Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides. Microbiol. Immunol. 54, 452–459. Ideses, D., Gophna, U., Paitan, Y., Chaudhuri, R.R., Pallen, M.J., Ron, E.Z., 2005. A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis. J. Bacteriol. 187, 8164–8171. Johnson, J.R., Johnston, B., Clabots, C., Kuskowski, M.A., Castanheira, M., 2010. Escherichia coli sequence type ST131 as the major cause of serious multidrugresistant E. coli infections in the United States. Clin. Infect. Dis. 51, 286–294 (an official publication of the Infectious Diseases Society of America). Johnson, J.R., Kuskowski, M.A., O’Bryan, T.T., Maslow, J.N., 2002. Epidemiological correlates of virulence genotype and phylogenetic background among Escherichia coli blood isolates from adults with diverse-source bacteremia. J. Infect. Dis. 185, 1439–1447. Johnson, J.R., Scheutz, F., Ulleryd, P., Kuskowski, M.A., O’Bryan, T.T., Sandberg, T., 2005. Phylogenetic and pathotypic comparison of concurrent urine and rectal
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Rohwer, F., Thurber, R.V., 2009. Viruses manipulate the marine environment. Nature 459, 207–212. Ruiz, J., Simon, K., Horcajada, J.P., Velasco, M., Barranco, M., Roig, G., MorenoMartinez, A., Martinez, J.A., Jimenez de Anta, T., Mensa, J., Vila, J., 2002. Differences in virulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men. J. Clin. Microbiol. 40, 4445–4449. Sannes, M.R., Kuskowski, M.A., Owens, K., Gajewski, A., Johnson, J.R., 2004. Virulence factor profiles and phylogenetic background of Escherichia coli isolates from veterans with bacteremia and uninfected control subjects. J. Infect. Dis. 190, 2121–2128. Stumpe, S., Schmid, R., Stephens, D.L., Georgiou, G., Bakker, E.P., 1998. Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli. J. Bacteriol. 180, 4002– 4006. Tal, R., Livne, P.M., Lask, D.M., Baniel, J., 2003. Empirical management of urinary tract infections complicating transrectal ultrasound guided prostate biopsy. J. Urol. 169, 1762–1765. Terai, A., Yamamoto, S., Mitsumori, K., Okada, Y., Kurazono, H., Takeda, Y., Yoshida, O., 1997. Escherichia coli virulence factors and serotypes in acute bacterial prostatitis. Int. J. Urol. 4, 289–294 (official journal of the Japanese Urological Association). Ulleryd, P., Lincoln, K., Scheutz, F., Sandberg, T., 1994. Virulence characteristics of Escherichia coli in relation to host response in men with symptomatic urinary tract infection. Clin. Infect. Dis. 18, 579–584 (an official publication of the Infectious Diseases Society of America). Wagenlehner, F.M., van Oostrum, E., Tenke, P., Tandogdu, Z., Cek, M., Grabe, M., Wullt, B., Pickard, R., Naber, K.G., Pilatz, A., Weidner, W., Bjerklund-Johansen, T.E., 2013. Infective complications after prostate biopsy: outcome of the Global Prevalence Study of Infections in Urology (GPIU) 2010 and 2011, a prospective multinational multicentre prostate biopsy study. Eur. Urol. 63, 521– 527. Williamson, D.A., Barrett, L.K., Rogers, B.A., Freeman, J.T., Hadway, P., Paterson, D.L., 2013. Infectious complications following transrectal ultrasound-guided prostate biopsy: new challenges in the era of multidrug-resistant Escherichia coli. Clin. Infect. Dis. 57, 267–274 (an official publication of the Infectious Diseases Society of America). Williamson, D.A., Roberts, S.A., Paterson, D.L., Sidjabat, H., Silvey, A., Masters, J., Rice, M., Freeman, J.T., 2012. Escherichia coli bloodstream infection after transrectal ultrasound-guided prostate biopsy: implications of fluoroquinolone-resistant sequence type 131 as a major causative pathogen. Clin. Infect. Dis. 54, 1406–1412 (an official publication of the Infectious Diseases Society of America). Zani, E.L., Clark, O.A., Rodrigues Jr., Netto N., 2011. Antibiotic prophylaxis for transrectal prostate biopsy. Cochrane Database Syst. Rev., CD006576. Zaytoun, O.M., Vargo, E.H., Rajan, R., Berglund, R., Gordon, S., Jones, J.S., 2011. Emergence of fluoroquinolone-resistant Escherichia coli as cause of postprostate biopsy infection: implications for prophylaxis and treatment. Urology 77, 1035–1041.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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Contents lists available at ScienceDirect
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Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy
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Michael Dan a,b,∗ , Yael Yair c , Alex Samosav c , Tamar Gottesman a , Orit Yossepowich a , Orna Harari-Schwartz d , Alexander Tsivian e , Rachel Schreiber c , Uri Gophna c
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Infectious Diseases Unit, E. Wolfson Hospital, Holon, Israel Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel d Microbiology Laboratory, E. Wolfson Hospital, Holon, Israel e Department of Urology, E. Wolfson Hospital, Holon, Israel b
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Article history: Received 19 January 2014 Received in revised form 6 April 2015 Accepted 20 April 2015
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Keywords: Escherichia coli Sepsis following prostate transrectal biopsy 21 Bacteremic urinary tract infection 22 23 Q4 Virulence markers 19 20
Background: Transrectal ultrasound-guided (TRUS) prostate biopsy is a very common procedure that is generally considered relatively safe. However, severe sepsis can occur after TRUS prostate biopsies, with Escherichia coli being the predominant causative agent. A common perception is that the bacteria that cause post-TRUS prostate biopsy infections originate in the urinary tract, but this view has not been adequately tested. Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa. Methods: We compared E. coli isolates from male patients with bacteremic urinary tract infection (BUTI) to isolates of patients with post prostate biopsy sepsis (PPBS), in terms of their sequence types, determined by multi-locus sequence typing (MLST) and their virulence markers. Results: B-UTI isolates were much richer in virulence genes than were PPBS isolates, supporting the hypothesis that E. coli causing PPBS derive directly from the rectum. Sequence type 131 (ST131) strains and related strain from the ST131 were common (>30%) among the E. coli isolates from PPBS patients as well as from B-UTI patients and all these strains expressed extended spectrum beta-lactamases. Conclusions: Our finding supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. In light of the increasing prevalence of highly resistant E. coli strains, a new approach for prevention of PPBS is urgently required. © 2015 Published by Elsevier GmbH.
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Article’s main point: Bacteremic UTI isolates are much richer in virulence genes than are post-transrectal prostate biopsy sepsis isolates, supporting the hypothesis that E. coli causing post transrectal prostate biopsy sepsis derive directly from the rectum.
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Introduction
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Transrectal ultrasound-guided (TRUS) prostate biopsy is one of the most commonly performed procedures in urology. Although
∗ Corresponding author at: Infectious Diseases Unit, E. Wolfson Hospital, Holon Q3 58100, Israel. Tel.: +972 50 6396950; fax: +972 3 5016126. E-mail addresses: [email protected], [email protected] (M. Dan).
TRUS prostate biopsy is generally considered to be a relatively safe outpatient procedure, severe sepsis has been described in 0.1%–3.5% of cases after TRUS prostate biopsy, with Escherichia coli being the most commonly involved pathogen (Nam et al., 2010; Raaijmakers et al., 2002; Tal et al., 2003). Recently there has been an apparent increase in hospitalization for infectious complications after TRUS prostate biopsy (Loeb et al., 2011; Nam et al., 2010; Wagenlehner et al., 2013). The role of antimicrobial prophylaxis in preventing infectious complications after biopsy is now well established (Zani et al., 2011), although life-threatening infection still occur despite prophylaxis (Carignan et al., 2012). Moreover, a systematic review and meta-analysis of randomized controlled studies confirms that prophylaxis reduces the rate of bacteriuria following prostate biopsy, although its effectiveness in reducing symptomatic UTI and
http://dx.doi.org/10.1016/j.ijmm.2015.04.003 1438-4221/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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bacteremia is less certain (Bootsma et al., 2008). The most commonly used prophylactic antimicrobials are the fluoroquinolones (Wagenlehner et al., 2013). Several publications have recently documented alarming increase in resistance to these agents among E. coli in post-TRUS prostate biopsy infections (Feliciano et al., 2008; Zaytoun et al., 2011), as well as to other antimicrobial agents such as ampicillin, ampicillin/sulbactam, trimethoprim/sulfamethoxazole and gentamicin (Carignan et al., 2012); extended spectrum -lactamase (ESBL)–producing isolates have been increasingly recovered (Briffaux et al., 2009). The rise over the years in resistance of E. coli to ciprofloxacin (up to 90%) is accompanied by a concomitant increase in the rate of post-TRUS prostate biopsy infectious complications (Carignan et al., 2012; Feliciano et al., 2008). If this trend continues, resistance will spread to other antimicrobial agents at increasing rates. An alternative preventive approach should thus be considered. For this purpose, it is important to inquire into the pathogenesis of post-TRUS prostate biopsy infections: what is the reservoir of the infecting organisms, what is the route of their acquisition, what is the virulence arsenal of the pathogens etc. It is a common perception that the bacteria responsible for postTRUS biopsy infections originate in the urinary tract (Wagenlehner et al., 2013). It is therefore recommended to perform midstream urine culture prior to the procedure, and to choose the antimicrobial agent used for prophylaxis according to the susceptibility of the bacteria isolated from urine (Grabe et al., 2013). Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa (Carignan et al., 2012; Williamson et al., 2012). The aim of the present study was to compare E. coli strains responsible for post-TRUS prostate biopsy infections and E. coli from male patients with bacteremic urinary tract infection in terms of genetic virulence markers. Correctly identifying the source of post-TRUS prostate biopsy infections is an essential step in the development of more effective preventive measures.
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Materials and methods
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Patients
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Patients with post-TRUS prostate biopsy sepsis (PPBS) Twenty-one men with febrile sepsis due to E. coli following TRUS prostate biopsy were prospectively enrolled during the period of January 2010 through August 2012 at the E. Wolfson Hospital Holon, Israel. Inclusion criteria included recent TRUS prostate biopsy, fever (temperature, >38.0 ◦ C), and positive blood and/or urine cultures for E. coli. Additionally, rectal samples for cultures were obtained from all patients after signing an informed consent. All patients presented symptoms within less than a week from the procedure, and over 90% presented them within 48 h of the biopsy.
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Patients with bacteremic urinary tract infection (B-UTI) During the same period, 24 men with bacteremic urinary tract infection due to E. coli were prospectively enrolled at the same institution. Inclusion criteria included male gender, fever (temperature, >38.0 ◦ C), and positive blood and urine cultures for E. coli. None of the patients included in this study had a Foley catheter. The E. Wolfson Hospital Research Ethics Committee approved the study protocol.
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Blood, urine and rectal cultures
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Blood samples were drawn aseptically from a peripheral vein. Each blood sample was inoculated into a set of blood culture bottles. Bottles were incubated in the BacT/Alert system (BioMerieux,
Table 1 Prevalence (%) of E. coli virulence genes typical to E. coli strains from male UTI and PPBS isolates versus B-UTI isolates (number of isolates noted in parentheses). Gene
% Positive (PPBS) N = 20
% Positive (B-UTI) N = 22
FDR-corrected (p-value* )
focG Sfa papC iron malX ompT
0 (0) 0 (0) 10 (2) 25 (4) 50 (10) 65 (13)
27 (6) 32 (7) 50 (11) 41 (9) 86 (19) 91 (20)
0.0310 0.0276 0.0276 0.3376 0.0310 0.0747
* Fisher’s exact test, adjusted for multiple hypothesis by the Hochberg and Benjamini false discovery rate correction (Rohwer and Thurber, 2009).
Inc, Durham, NC) until flagged positive or for 7 days. A sample from a positive bottle was Gram stained and subcultured. A single E. coli colony was saved for further studies. Urine was cultured semiquantitatively on blood agar and cysteine–lactose–electrolyte–deficient agar plates using the calibrated loop technique. A single colony was retained for further study. Fecal swabs were inoculated on one quadrant of a MacConkey’s agar plate, then streaked for isolation in the remaining three quadrants with a sterile loop and incubated overnight at 37 ◦ C. Lactose- and indole-positive Gramnegative bacilli with a consistent colonial morphology similar to E. coli. From each plate with Gram-negative bacterial growth a representative colony of each distinct morphotype (ranging from one to five morphotypes) was selected for identification using the Vitek 2 instrument (BioMerieux, USA) GN identification cards, and verified as E. coli. The E. coli isolate mix was subsequently re-streaked for isolation and transferred to the Department of Molecular Microbiology and Biotechnology, where a single colony was analyzed at random. Based on past studies (Lidin-Janson et al., 1978), this procedure has over 85% probability of identifying the dominant clone. Serum survival growth assays A single colony was picked and grown overnight in 2 ml of Davis minimal medium supplemented with 0.2% glucose. The overnight culture was then diluted 1:10 in 10 ml of Davis minimal medium supplemented with 0.4% glucose. After 3–4 h, when the cultures reached the end of logarithmic growth phase (O.D 595 of ∼2) 20 l of culture were transferred to 96-well microplates containing 40 l of concentrated Davis minimal medium (×5) supplemented with 2% glucose in addition to either 140 l of dd H2 O (control) or Human Serum (Sigma human serum H-4522, to 70% final concentration), to a final volume of 200 l. E. coli K-12 MG1655 was used as negative control, since it is rapidly killed in the presence of serum, while avian pathogenic strain 789, which is highly serum-resistant (Ideses et al., 2005) was used as a positive control. The O.D. of the cultures in the plates were continuously measured in an ELX 800 Absorbance Microplate Reader (BioTek) at 37◦ with continuous shaking. Virulence marker identification Virulence genes presence (Table 1) was determined using PCR using primers from (Rodriguez-Siek et al., 2005), (Supplementary table S1). All PCR reactions were run in a Biometra T Personal thermocycler, using 25 l reaction volumes. One microliter from 1:50 dilutions of overnight bacterial culture in ddH2 O pre-heated for 5 min. at 96 ◦ C were used as template and 28 amplification cycles with an annealing temprature of 63◦ . Each reaction consisted of 0.2 l Biotaq DNA Polymerase [5 U/l, (Bioline)], 0.5 l of 10 M solution of the forward and reverse primers, 1.5 l of 3 mM MgCl2, and a 2.5 l dNTP mix of 2.5 mM each.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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For 28 of the isolates, the 7 loci used for multiple locus sequence typing in E. coli, were amplified by PCR, using the primers and protocols described in http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/. 1 l from a suspension of a bacterial overnight culture diluted 1:50 in water, as described above for virulence marker identification, was used as template. The proof-reading polymerase used was Phusion Hot-start II High Fidelity (Thermo Scientific, USA). The PCR amplicons were purified using illustra ExoProStar (GE Healthcare), and sequenced by Sanger sequencing. For the remaining 14 strains genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen, USA), following the manufacturer’s protocol and whole genome sequencing was performed using Illumina HiSeq platform with 2 × 100 base reads, yielding an average 157X coverage. Draft genomes were assembled using Edena V3.131028 (Hernandez et al., 2008) and BLASTN (Altschul et al., 1990)was used to identify the gene sequences corresponding to the 7 MLST loci.
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Statistical methods
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Statistical analyses were performed using SPSS v. 14 (IBM Software, USA) and GraphPad Prism (Graphpad Software, USA). For diversity differences and ST131 complex abundance, a two-tailed Fisher’s exact test was used. For virulence gene comparisons a onetailed Fisher’s exact test was used (since the hypothesis is that their abundance would be lower in PPBS), corrected for multiple hypothesis testing by the Benjamini and Hochberg false discovery rate correction (Benjamini and Hochberg, 1995).
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Characteristics of patients
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Patients with post-TRUS prostate biopsy sepsis (PPBS). The 21 patients were admitted to hospital because of fever and rigors within 0.75, Fisher’s exact test). Taken together these data strongly suggest that most PPBS strains differ genotypically from B-UTI strains, but that the ST131 group has become so widespread that it is more or less evenly distributed between these different patient populations.
Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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Discussion Escherichia coli is the most common cause of UTI in men (Lipsky, 1999; Ulleryd et al., 1994) and of sepsis after TRUS prostate biopsy (Tal et al., 2003; Williamson et al., 2013). While several studies have examined the virulence characteristics and clonal background of E. coli isolates recovered from men with UTI (Andreu et al., 1997; Johnson et al., 2005; Kanamaru et al., 2003; Mitsumori et al., 1999; Otto et al., 2001; Ruiz et al., 2002; Terai et al., 1997; Ulleryd et al., 1994), little is known about the characteristics of E. coli causing sepsis after prostate biopsy. Phylogenetic and pathotypic analysis of E. coli isolates recovered from men with febrile UTI, showed that such strains are richly endowed with diverse extraintestinal virulence factors and derive from virulence-associated phylogenetic groups, largely independent of host compromise status. Comparing pathogenic E. coli isolates causing UTI to E. coli colonizing the rectum of the same patients led Johnson et al. (2005) to conclude that men are naturally highly resistant to febrile UTI, and considerable virulence capacity is required for an E. coli strain to cause it. Our results, demonstrating distinct differences between B-UTI strains and those that cause PPBS, further support that conclusion. The finding that B-UTI isolates were much richer in virulence genes than were PPBS isolates, supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. The ompT gene was found to be the virulence factor most closely associated with bacteremia (not necessarily related to the urinary tract) in a comparison of bloodstream versus rectal E. coli isolates among male veterans (Sannes et al., 2004), and also highly associated with male febrile UTI (Johnson et al., 2005). This virulence gene was also the most prevalent virulence factor in our study among isolates from B-UTI (91%). Similarly, in the PPBS group ompT was detected in 88% of isolates in patients with positive urine and blood cultures (data not shown). Thus, we may conclude that ompT may have a role in the pathogenesis of bacteremia, even when these are not associated with the urinary tract, potentially because it can confer resistance to diverse host antimicrobial peptides (Hui et al., 2010; McCarter et al., 2004; Stumpe et al., 1998). Recently a pandemic of a particular emergent E. coli clonal group, sequence type 131 (ST131, serotype O25b:H4) has been described (Rogers et al., 2011). Such ST131 isolates have distinct virulence profiles and are extensively resistant to antimicrobial agents, including their hallmark resistance to fluoroquinolones (Rogers et al., 2011). ST131 was implicated as the major culprit in bloodstream infections following transrectal prostate biopsy in New Zealand (Williamson et al., 2012), and accounted for 70% of fluoroquinolone-resistant rectal E coli isolates among men undergoing transrectal prostate biopsy in southern California (Liss et al., 2013). In Israel fluoroquinolone-resistance is highly prevalent among E. coli isolated from patients with PPBS (>80% of isolates) [Tal et al., 2003; Dan M, unpublished data, 2015], and E coli sequence type 131 has recently been documented in Israel (Karfunkel et al., 2013). ST131 strains were common among the E. coli isolates in the present study, and ST131 was found in 7/20 (35%) strains from PPBS patients and 6/22 (27%) strains from B-UTI patients. All ST131 strains were of community origin and expressed ESBL, exemplifying the important antimicrobial resistance associated with ST131, and the inadequacy of current prophylactic regimen for patients undergoing prostate biopsies. With the mounting evidence that PPBS results from E. coli that is directly introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa on one hand and the alarming increase in antimicrobial resistance on the other, a new approach of prevention which is not based on the use of
antimicrobial agents should be considered. The aim of such preventive measure should be to reduce the bacterial load of the rectal mucosa at the site of biopsy and hence lessen the bacterial inoculum introduced during the biopsy procedure. Although the role of pre-biopsy rectal cleansing enemas has been recently assessed (Zani et al., 2011), no significant benefit over prophylactic antibiotic was documented, and this technique is not routinely recommended (Zani et al., 2011). Rectal disinfection with agents such as chlorhexidine or povidone–iodine has been also evaluated as adjunct to antibiotic prophylaxis, but no solid data supporting the efficacy of such approach are currently available (Duplessis et al., 2012). With the alarming increase in ST131 prevalence, further research and development of innovative techniques that do not rely on conventional antimicrobial agents is urgently required. Acknowledgements This work was supported by the German-Israeli Project Cooperation (DIP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm.2015.04. 003 References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Andreu, A., Stapleton, A.E., Fennell, C., Lockman, H.A., Xercavins, M., Fernandez, F., Stamm, W.E., 1997. Urovirulence determinants in Escherichia coli strains causing prostatitis. J. Infect. Dis. 176, 464–469. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc., B Met. 57, 289–300. Bootsma, A.M., Laguna Pes, M.P., Geerlings, S.E., Goossens, A., 2008. Antibiotic prophylaxis in urologic procedures: a systematic review. Eur. Urol. 54, 1270–1286. Briffaux, R., Coloby, P., Bruyere, F., Ouaki, F., Pires, C., Dore, B., Irani, J., 2009. One preoperative dose randomized against 3-day antibiotic prophylaxis for transrectal ultrasonography-guided prostate biopsy. BJU Int. 103, 1069–1073 (discussion 1073). Carignan, A., Roussy, J.F., Lapointe, V., Valiquette, L., Sabbagh, R., Pepin, J., 2012. Increasing risk of infectious complications after transrectal ultrasound-guided prostate biopsies: time to reassess antimicrobial prophylaxis? Eur. Urol. 62, 453–459. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Duplessis, C.A., Bavaro, M., Simons, M.P., Marguet, C., Santomauro, M., Auge, B., Collard, D.A., Fierer, J., Lesperance, J., 2012. Rectal cultures before transrectal ultrasound-guided prostate biopsy reduce post-prostatic biopsy infection rates. Urology 79, 556–561. Feliciano, J., Teper, E., Ferrandino, M., Macchia, R.J., Blank, W., Grunberger, I., Colon, I., 2008. The incidence of fluoroquinolone resistant infections after prostate biopsy – are fluoroquinolones still effective prophylaxis? J. Urol. 179, 952–955 (discussion 955). Grabe, M., Bjerklund-Johansen, T.E., Botto, H., C¸ek, M., Naber, K.G., Pickard, R., Tenke, P., Wagenlehner, F.M., Wullt, B., 2013. Guidelines on urological infections. Eur. Q7 Assoc. Urol. Hernandez, D., Francois, P., Farinelli, L., Osteras, M., Schrenzel, J., 2008. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 18, 802–809. Hui, C.Y., Guo, Y., He, Q.S., Peng, L., Wu, S.C., Cao, H., Huang, S.H., 2010. Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides. Microbiol. Immunol. 54, 452–459. Ideses, D., Gophna, U., Paitan, Y., Chaudhuri, R.R., Pallen, M.J., Ron, E.Z., 2005. A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis. J. Bacteriol. 187, 8164–8171. Johnson, J.R., Johnston, B., Clabots, C., Kuskowski, M.A., Castanheira, M., 2010. Escherichia coli sequence type ST131 as the major cause of serious multidrugresistant E. coli infections in the United States. Clin. Infect. Dis. 51, 286–294 (an official publication of the Infectious Diseases Society of America). Johnson, J.R., Kuskowski, M.A., O’Bryan, T.T., Maslow, J.N., 2002. Epidemiological correlates of virulence genotype and phylogenetic background among Escherichia coli blood isolates from adults with diverse-source bacteremia. J. Infect. Dis. 185, 1439–1447. Johnson, J.R., Scheutz, F., Ulleryd, P., Kuskowski, M.A., O’Bryan, T.T., Sandberg, T., 2005. Phylogenetic and pathotypic comparison of concurrent urine and rectal
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Please cite this article in press as: Dan, M., et al., Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.04.003
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