Escherichia coli clonal group A causing bacteraemia of urinary tract origin

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1 ORIGINAL ARTICLE BACTERIOLOGY Escherichia coli clonal group A causing bacteraemia of urinary tract origin L. Skjøt-Rasmussen 1, S. S. Olsen 1, L. Jakobsen 1,2, K. Ejrnæs 1,2,3, F. Scheutz 1, B. Lundgren 2,4, N. Frimodt-Møller 1,2 and A. M. Hammerum 1 1) Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen S, 2) Department of Clinical Microbiology, Hvidovre Hospital, Hvidovre, 3) Department of Pathology, Herlev Hospital, Herlev and 4) The Centre of Diagnostic Investigations, Rigshospitalet, Copenhagen, Denmark Abstract Escherichia coli clonal group A (CgA) causes disease in humans. This is the first study investigating the prevalence of CgA among E. coli from non-urine, extraintestinal infections in a northern European country. E. coli blood (n = 196) and paired urine (n = 195) isolates from the same patients with bacteraemia of urinary tract origin were analysed. The isolates were collected from January 2003 through May 2005 at four hospitals in Copenhagen, Denmark. Pulsed-field gel electrophoresis (PFGE) patterns, antimicrobial resistance and patient characteristics were determined for all CgA isolates; presence of virulence-associated genes (VAGs) and serotypes were determined for the blood CgA isolates. Thirty blood isolates (15%) belonged to CgA. CgA blood isolates were associated with female patients and sulfamethoxazole-trimethoprim resistance and they harboured a distinctive VAG profile. The blood and urine isolates from each pair were found to be related in 26 of 27 CgA blood/urine pairs, confirming a urinary tract origin of infection. Furthermore, a relationship between the PFGE patterns of CgA blood/urine isolates and CgA isolates from UTI patients in general practice and a CgA isolate from a community-dwelling human reported previously, was found, suggesting a community origin of CgA. The finding of CgA strains in 15% of the E. coli bloodstream infections with a urinary tract origin in Denmark suggests that CgA constitutes an important clonal lineage among extraintestinal pathogenic E. coli. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health. Keywords: Antimicrobial resistance, bacteraemia, clonal group A, Escherichia coli, PFGE typing, serotyping, urinary tract infection, virulence Original Submission: 10 February 2012; Revised Submission: 25 May 2012; Accepted: 4 June 2012 Editor: S. Cutler Article published online: 9 June 2012 Clin Microbiol Infect 2013; 19: /j x Corresponding author: L. Skjøt-Rasmussen, Statens Serum Institut, Department of Microbiological Surveillance and Research, Build. 47/ 217, 5 Artillerivej, 2300 Copenhagen S, Denmark lbs@ssi.dk Introduction Evidence is increasing that Escherichia coli causing urinary tract infections (UTIs) and other extraintestinal infections may be responsible for community epidemics [1,2]. A multidrugresistant clonal group, termed clonal group A (CgA), was identified as a cause of community-acquired UTI outbreaks in North America in 2001 [3,4]. CgA isolates belong to E. coli phylogenetic group D [5] and multilocus sequence typing (MLST) clonal complex 69 (CC69) [6,7], they possess a conserved virulence gene profile [3 5,8], and they are often resistant to trimethoprim-sulphonamides [5,6]. CgA has a worldwide endemic distribution with a concentration in the western world [9]. However, the distribution of CgA in Europe has received little investigation [8 11]. CgA isolates cause extraintestinal infections and especially its distribution among uropathogenic E. coli (UPEC) has been investigated [5,6,8,10,12]. Thus, assessment of the distribution of CgA isolates in Europe and especially its prevalence among complicated, extraintestinal infections such as bloodstream infections is limited. Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases

2 CMI Skjøt-Rasmussen et al. Escherichia coli CgA causing bacteraemia 657 The aim of the study was to investigate the prevalence of CgA among E. coli isolates from cases of bacteraemia with a urinary tract origin in a Northern European country. Also, the study of paired blood and urine isolates from the same patients could provide further insight into the pathogenesis of E. coli bacteraemia with a urinary tract origin. Methods Bacterial isolates and patients E. coli blood (n = 196) and urine (n = 195) isolates collected from January 2003 through May 2005 from 195 adult patients with both bacteraemia and bacteriuria admitted to four hospitals in Copenhagen, as reported elsewhere [13], were studied. From one patient, two E. coli blood isolates were cultured. Epidemiological, clinical and laboratory data for each infection episode were extracted from the patients medical records and the hospital administrative database. Bacteraemia episodes were considered to be community acquired when they were diagnosed within the first 48 h of hospitalization and were considered hospital acquired after this period. Sampling of the isolates was approved by the Scientific Ethics Committee for the Capital Region of Denmark [(KF) ] [13]. Phylogenetic analysis The phylogenetic background (A, B1, B2 and D) of the blood isolates [13] and the corresponding urine isolates was determined by triplex PCR [14]. Classification of the isolates into the four major E. coli phylogenetic lineages or non-typeable (NT) isolates was undertaken according to Gordon et al. [15]. Clonal group A The phylogenetic group D isolates were screened by PCR for CgA-associated single-nucleotide polymorphisms (SNPs) in fumc and gyrb [12]. The presence of these SNPs identified strains belonging to CC69 and excluded strains belonging to the closely related CC394. Virulence genotyping The blood isolates were previously screened for the presence of 29 VAGs with known or suspected relevance to ExPEC pathogenesis [13]. Antimicrobial resistance Minimum inhibitory concentrations (MICs) of ciprofloxacin, nalidixic acid, sulfamethoxazole and trimethoprim for the blood isolates and the corresponding urine isolates were determined by a micro broth dilution method as described in Skjøt-Rasmussen et al., 2012 [13]. Pulsed-field gel electrophoresis The CgA blood and urine isolates and the CgA reference strain ATCC BAA-457 were typed by pulsed-field gel electrophoresis (PFGE) with XbaI using the protocol made available by the CDC s PulseNet database [16,17]. The PFGE patterns were compared using BioNumerics 6.6 (Applied Maths, Kortrijk, Belgium). Isolates were considered to be related if their Dice similarity index was 85%. Also included in the analysis was E. coli from animals (Danish broiler chickens, n = 5), meat (Danish broiler chicken meat, n = 4) and humans (Danish community-dwelling humans, n = 5; UTI patients from general practice, n = 11) that were PFGE typed as described earlier by Jakobsen et al. [10]. Serotyping O, K and H antigens of the CgA blood isolates were detected using standard methods [18]. Statistical analysis Comparisons of proportions were based on the chi-square test or, when expected numbers were <5, Fisher s exact test (two-tailed). In order to reduce the risk of type-1 error with multiple comparisons, a p-value <0.01 was considered statistically significant. Results Among the 196 blood and 195 urine isolates, 44 phylogroup D blood isolates and 42 phylogroup D urine isolates were found. Of the investigated phylogroup D isolates, 30 blood isolates and 28 urine isolates belonged to CgA. Of these, 27 blood and urine CgA isolates, respectively, came from the same 27 patients. Thus, CgA was found to be responsible for 15% (30/196) of the bloodstream infections. The median age of the 30 patients experiencing CgA bacteraemia was 78 years (range, years). Of the CgA blood isolates, 97% were from women whereas only 63% of the non- CgA blood isolates were from women (p <0.001) (Table 1). Among the 196 blood isolates, 21 isolates were from hospital-acquired infections and 175 isolates were from community-acquired infections. CgA was responsible for 17% (29/ 175) of the community-acquired and 5% (1/21) of the hospital-acquired bloodstream infections with a urinary tract origin (Table 1). Total 30-day mortality did not differ among the CgA bacteraemia episodes and the non-cga episodes (Table 1).

3 658 Clinical Microbiology and Infection, Volume 19 Number 7, July 2013 CMI TABLE 1. Characteristics of E. coli blood clonal group A (CgA) isolates (n = 30) compared with non-cga isolates (n = 166) from patients with bacteraemia of urinary tract origin Category, trait Occurrence of trait, no. (%) of isolates CgA (n = 30) Non-CgA (n = 166) Epidemiological classification Community-acquired 29 (97) 146 (88) Hospital-acquired 1 (3) 20 (12) 30-day mortality Yes 1 (3) 22 (13) No 29 (97) 144 (87) Sex Female 29 (97) 105 (63) <0.001 Male 1 (3) 61 (37) Antimicrobial resistance profile None 6 (20) 108 (65) <0.001 SUL 8 (27) 25 (15) TRI 0 (0) 1 (<1) SUL, TRI 15 (50) 20 (12) <0.001 CIP, NAL 0 (0) 4 (2) SUL, CIP, NAL 1 (3) 3 (2) SUL, TRI, CIP, NAL 0 (0) 5 (3) SUL, sulfamethoxazole; TRI, trimethoprim; CIP, ciprofloxacin; NAL, nalidixic acid. Sulphonamide-trimethoprim-resistant blood isolates represented 50% (15/30) of the CgA isolates as compared with only 12% (20/166) of the non-cga blood isolates (p <0.001) (Table 1). Of the total 196 blood isolates, 40 were resistant to both sulfamethoxazole and trimethoprim. Thus, the CgA isolates accounted for 38% (15/40) of the sulfamethoxazoletrimethoprim-resistant isolates from Danish patients with bacteraemia of urinary tract origin. Fluoroquinolone resistance was observed in only one of the 30 (3%) CgA blood isolates (Table 1). The CgA isolates were characterized by a distinctive profile of VAGs different from non-cga isolates that included iha, iuta and sat, and lacked focg, sfa/focde, agn43a (CFT073), agn43b (CFT073), irea, iron, iss, cnf1, hlyd, malx and usp (Table 2). Among the CgA isolates, 27 blood and 27 urine isolates were paired, i.e. they came from the same patients; of these, 26 blood/urine pairs (96%) had related PFGE patterns (Fig. 1). We compared the PFGE patterns of CgA isolates of animal origin (Danish broiler chickens and Danish broiler chicken meat) [14] with the 54 CgA blood and urine isolates, but we did not find any relationship between the E. coli of animal and human origin (data not shown). Furthermore, we compared the PFGE patterns of the 54 CgA blood and urine isolates with the PFGE patterns of CgA isolates from Danish community-dwelling humans and UTI patients from general practice [14]. Six of the CgA blood/urine pairs had PFGE patterns related to those of six UTI patients from general practice, and two of the blood/urine pairs had PFGE patterns p TABLE 2. Virulence-associated genes of E. coli blood clonal group A (CgA) isolates (n = 30) compared with non-cga isolates (n = 166) from patients with bacteraemia of urinary tract origin Category, trait related to that of one isolate from a community-dwelling human (data not shown) [14]. Serotypes of the CgA blood isolates belonging to blood/ urine pairs are shown in Fig. 1. Overall, eight different O groups, six different K antigens and 20 different O:K:H serotypes were detected (Fig. 1). Apart from the previously described CgA O groups (O11, O15, O17, O44, O73, O77 and O86) [7,8,19,20], we found one O group not previously associated with CgA, namely O125ab (n = 2). However, all motile strains except one, O11:K52:H4, were H18, including the three CgA blood isolates not belonging to a blood/urine pair, which were: O rough:k52:h18, 125ab:K52:H18 and O15:K52:H-. Discussion Occurrence of trait, no. (%) of isolates CgA (n = 30) Non-CgA (n = 166) Adhesins afa/drabc 0 (0) 5 (3) bmae 1 (3) 5 (3) fimh 30 (100) 163 (98) focg 0 (0) 44 (27) gafd 0 (0) 2 (1) iha 28 (93) 70 (42) <0.001 papah 27 (90) 113 (68) sfa/focde 0 (0) 66 (40) <0.001 Biofilm related agn43 30 (100) 147 (89) agn43a (CFT073) 1 (3) 67 (40) <0.001 agn43b (CFT073) 0 (0) 57 (34) <0.001 agn43 (K12) 8 (27) 51 (31) Iron uptake chua 30 (100) 145 (87) fyua 25 (83) 155 (93) irea 0 (0) 65 (39) <0.001 iron 0 (0) 110 (66) <0.001 iuta 29 (97) 118 (71) Protectins iss 1 (3) 45 (27) kpsm II 25 (83) 138 (83) kpsm II K2 25 (83) 144 (87) kpsmt III 1 (3) 0 (0) trat 27 (90) 107 (64) Toxins cdtb 0 (0) 18 (11) cnf1 0 (0) 56 (34) <0.001 hlyd 0 (0) 66 (40) <0.001 sat 26 (87) 62 (37) <0.001 Miscellaneous ibea 0 (0) 23 (14) malx 0 (0) 128 (77) <0.001 usp 0 (0) 133 (80) <0.001 This study shows that the E. coli CgA can cause bacteraemia with a urinary tract origin with a high prevalence (15%) in Northern Europe. To our knowledge, only two p

4 CMI Skjøt-Rasmussen et al. Escherichia coli CgA causing bacteraemia 659 FIG. 1. Pulsed-field gel electrophoresis of XbaI digested DNA from paired E. coli CgA blood (n = 27) and urine (n = 27) isolates. Isolate designation (ID) of all isolates (B: blood; U: urine) and serotypes of the blood isolates are shown on the right. The paired blood and urine isolates from patient no. 122 were the only paired isolates not having related PFGE patterns. studies have investigated CgA in Northern Europe; both studied UTI isolates [10,11]. Thus, our study is the first to describe CgA among E. coli from bloodstream infections in Northern Europe. The same percentage (15%) of CgA causing community-acquired bloodstream infections was found in a study based on strains collected during in the United States; this study was also based on bloodstream infections especially associated with preceding UTI [20].

5 660 Clinical Microbiology and Infection, Volume 19 Number 7, July 2013 CMI We found CgA to be significantly associated with women; an explanation for this could be the origin of the studied bacteraemia cases being the urinary tract because UTI among adults is most common among females [21]. As in other studies [3 5,9,22], we identified a strong association of CgA with sulphonamide-trimethoprim resistance. We found CgA to account for 38% of the sulfamethoxazole-trimethoprim-resistant isolates from Danish patients with bacteraemia of urinary tract origin. This is consistent with studies conducted on UTI isolates; several studies have found CgA to account for up to 50% of sulfamethoxazole-trimethoprim-resistant E. coli from women with UTI [3,4]. For many years, sulfamethizole has been one of the drugs of choice for treatment of uncomplicated UTI in Denmark. Despite a reduction in the use of sulfonamides in humans, sulphonamide resistance has remained relatively constant during the last years (around 35 40%) [23]. The use of trimethoprim, also used for treatment of UTI, has increased over the last years [23], as has the level of trimethoprim resistance (around 34%) [24]. Despite neither sulphonamides nor trimethoprim are being used for treatment of bloodstream infections, we detected sulfamethoxazoletrimethoprim-resistant CgA bloodstream isolates. Thus, the presence of CgA could possibly explain the persistence of resistance despite a decrease in antimicrobial consumption [23]. To our knowledge, this is the first study to investigate the association between the presence of CgA and outcome of infection. We did not find a significant association between CgA and 30-day mortality (Table 1). A reason for this might be that our study included too few isolates needed for evaluation of this factor. Like other studies, we also found CgA isolates to be associated with a distinctive set of VAGs [3 5,8]. Thus, our results indicate that despite exhibiting extraintestinal virulence and antimicrobial resistance, CgA isolates are not responsible for higher mortality as compared with non-cga isolates from bloodstream infections. The higher mortality among non-cga clonal isolates may be related to the significantly higher frequency of the other VAGs present (Table 2). A strength of our study is the investigation of paired blood and urine isolates from the same patients with bacteraemia of urinary tract origin; furthermore, to our knowledge this is the first study to investigate such paired isolates belonging to CgA. PFGE typing of the paired CgA blood and urine isolates showed that 96% of the blood/urine pairs had related PFGE patterns. This indicates that the assumption of a urinary tract origin of the bloodstream infection is justified when patients having concurrent bacteraemia and bacteriuria are encountered. We found PFGE relatedness between CgA blood/urine pairs and UTI isolates from patients from general practice and between blood/urine pairs and one isolate from a community-dwelling human. The finding of PFGE relatedness between blood/urine isolates and the isolate from a community-dwelling human (isolate 2171C04) is further substantiated by the ability of 2171C04 to cause infection in the murine model of human UTI [10]. The finding of a relationship between Danish CgA isolates from patients with both bacteraemia and bacteriuria collected during , UTI patients from general practice collected during , and community-dwelling humans collected in 2004 suggests a common community origin of CgA. As CgA isolates have previously been detected among isolates from broiler chickens and broiler chicken meat in Denmark, food animals and food may be a common source [10]. In this study, we found CgA with a high prevalence (15%) among cases of bacteraemia with a urinary tract origin in a Northern European country. This suggests that CgA constitutes an important clonal lineage among ExPEC and represents an important public health threat. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health. Acknowledgements Karin Sixhøj Pedersen, Alexandra Medina, Susanne Jespersen and Pernille Gymoese, Statens Serum Institut, are thanked for excellent technical assistance. This study is part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP). Transparency Declaration The authors declare that there are no conflicts of interest. References 1. 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