Abstract
We compared the virulence properties of a collection of asymptomatic bacteriuria (ABU) Escherichia coli strains to urinary tract infection (UTI) strains isolated from pregnant women in a university hospital over 1 year. The in vitro and in vivo studies suggest that ABU strains presented a virulence behavior similar to that of strains isolated from cases of cystitis.
TEXT
Urinary tract infections (UTIs) are bacterial infections (cystitis or pyelonephritis) frequently observed during pregnancy. Urinary tract colonization or asymptomatic bacteriuria (ABU) refers to situations where microorganisms are present at a significant rate (bacteriuria ≥105 CFU/ml) in the urinary tract without clinical signs. This situation is very common during pregnancy (3 to 8%), with a peak incidence between the 9th and 17th weeks (26). Escherichia coli is the main pathogen found in this situation (27). Several concepts are based on acquired facts about ABU. First, microorganisms are the same in terms of species and virulence compared to ABU in nonpregnant women (24). Second, the main risk of the ABU is the occurrence of pyelonephritis in 30 to 40% of cases (10, 23). Therefore, the ABU must be systematically sought because of the risks of upper tract infection and fetal damage, mainly in the form of premature delivery (17, 25). A properly treated pyelonephritis will heal in a few days without sequelae. Finally, if ABU is not supported in time, it may progress to septic shock and/or a progressive deterioration of renal function to renal failure itself (9, 28). Proof of links between ABU and pyelonephritis are weak and based primarily on observational data. To date, few studies have focused on the virulence of E. coli isolated from UTIs or ABU in pregnant women (8, 24). This work has been conducted to assess the in vitro and in vivo virulence of uropathogenic E. coli (UPEC) isolated from pregnant women in different situations (ABU, acute cystitis, acute pyelonephritis, and urosepsis).
The prospective study was initiated on 1 January 2007 and carried out until 31 December 2007 in a French university hospital. During the follow-up of pregnancy, all women with a first episode of an ABU, an acute cystitis, an acute pyelonephritis, or an urosepsis in whom E. coli was detected were included. ABU was defined as urine specimens with an E. coli culture of ≥105 CFU/ml without clinical signs. The cystitis and pyelonephritis strains have been isolated in monocultures from urine specimens of patients diagnosed with acute cystitis and acute pyelonephritis, respectively. The urosepsis isolates were cultured from the blood of patients suffering from UTI-derived sepsis. The genus and species were determined biochemically with the Vitek 2 identification card (bioMérieux, Marcy l'Etoile, France). Susceptibility to antimicrobial agents was tested by using the Vitek 2 card (bioMérieux). Strains were classified as susceptible, intermediately resistant, or resistant to the antibiotics tested according to the recommendations of the Antibiotic Susceptibility Testing Committee of the French Society for Microbiology (http://www.sfm.asso.fr/nouv/general.php?pa=2). Extended-spectrum β-lactamase (ESBL) production was confirmed by the double-disc synergy test (11). The genotypic characterization of ESBL resistance mechanisms was determined by PCR assays targeting blaTEM, blaSHV, and blaCTX-M and identified by sequencing the PCR products (18). Phylogenetic grouping and serotyping of E. coli isolates were determined by a PCR-based method (3, 4). The in vitro virulence potential of E. coli strains was evaluated by PCR (5, 12–15) with the following genes: papG alleles I, II, and III; papA; papC; papE; sfaS; focG; afa-draBC; fimH; hlyA; cnf1; iutA; irp2; iroN; kpsMT II; kpsMK I; traT; and malX. Macrorestriction analysis of XbaI-digested chromosomal DNA was performed by pulsed-field gel electrophoresis (PFGE) with the CHEF DRII system (Bio-Rad) and analyzed with Gel Compar computer software (Applied Math, Kortrijk, Belgium) (19). The in vivo virulence of E. coli strains was investigated by the Caenorhabditis elegans model. The nematode infection assay using Fer-15 worms was carried out as described by Lavigne et al. (20). All experiments were conducted in triplicate and repeated at least 5 times for each selected strain. E. coli virulence was assessed using the nematode survival curve and calculating the LT50 and LT100, the times required to kill 50% and 100% of the worms, respectively. Bacterial counts in the C. elegans digestive tract were carried out as described by Garsin et al. (7). For each, virulence factors (VFs), the number of genes (aggregated score), and comparisons between the different groups (ABU, cystitis, pyelonephritis, and urosepsis) were evaluated by using the χ2 test or Fisher's exact test and the Kruskal-Wallis test, respectively. To assess the utility of combining several virulence markers to predict UTIs, we used a logistic regression with a backward procedure to select the most relevant markers; only markers for which the area under the curve for the receiver-operator characteristic (AUCROC) was greater than 0.80 were initially entered as explanatory variables in the regression analysis. A ROC curve was then generated for the combination derived from the regression model and its area compared with that of every single virulence marker by a nonparametric method adapted to paired data (6). A P value of ≤0.05 was considered as reflecting statistical significance. A log rank test was used to compare the entire survival curves in nematode-killing assays. The analysis was carried out using SAS/ETS software release 8.1 (SAS Institute Inc., Cary, NC).
During the studied period, 114 E. coli strains were isolated from 114 women (median age, 28.5 [range, 19 to 42]): 37 (32.5%) with ABU, 26 (22.8%) with cystitis, 14 (12.3%) with pyelonephritis, and 37 (32.5%) with urosepsis. All isolates were susceptible to carbapenems. Five strains (4.3%) were resistant to cefotaxime and produced a CTX-M-15 β-lactamase. Susceptibility was observed with amikacin (98.2%), fosfomycin (98.2%), nitrofurantoin (84.2%), ciprofloxacin (80.7%), co-trimoxazole (76.3%), amoxicillin-clavulanic acid (61.4%), and amoxicillin (51.8%). No difference was observed among the different groups. PFGE revealed a high level of genomic diversity between each strain and among each group (data not shown). The virulence profiles of strains isolated in this study are presented in Table 1. The ABU strains mainly belonged to phylotype B2 and serotype O:4, and the fimH (89.2%) and malX (86.5%) virulence factors were more frequently found in these strains. The aggregated score was 8.1. Univariate analysis showed that the profile of VFs detected in the ABU group was similar to the profile of VFs found in the cystitis group but was different from the profile of VFs found in the pyelonephritis and urosepsis groups (P < 0.001). papG allele II, papG allele III, papA, papC, papE, iroN, irp2, and malX were more frequently found for pyelonephritis and cnf1 was more frequently found for ABU (P < 0.05) as previously noted (2). The multivariate analysis selected cnf1 as independent factors linked to a decreased association of pyelonephritis (odds ratio [OR], 0.89; 95% confidence interval [CI95%], 0.54 to 0.9; P = 0.005). The AUCROC for the cnf1 gene (OR, 0.946; CI95%, 0.863 to 1.000) demonstrated an excellent tool to discriminate between ABU and pyelonephritis (sensitivity, 0.703 [standard deviation (SD), 0.025]; specificity, 1.000 [SD 0.063]). For urosepsis, papG allele II and irp2 were the most prevalent genes as previously observed (1). The low prevalence of the hlyA gene in the urosepsis strains was surprising. However, this gene was more prevalent in cystitis than in pyelonephritis (22). The multivariate analysis selected afa-draBC, hlyA, and foc as independent factors linked to a decreased association of urosepsis (OR [CI95%]: 0.69 [0.53 to 0.89], 0.77 [0.62 to 0.95], and 0.65 [0.49 to 0.88], respectively; P < 0.01). From the logistic model, the combination of afa-draBC, hlyA, and foc was the most predictive for distinguishing ABU and urosepsis: AUCROC was 0.875 (CI95%, 0.788 to 0.962). The afa-draBC and foc genes were previously described as predictors of cystitis, a result similar to our study (16). Concerning the phylotyping groups, no difference could be detected among the groups, suggesting that ABU strains were phylogenetically related to strains that cause symptomatic UTIs as previously noted in other ABU situations (21). The aggregated scores for ABU and cystitis were not different (9.2) (P = 0.24, Wilcoxon test), but those for pyelonephritis (11.2) and urosepsis were significantly different (11.5) (P < 0.001). To explain this difference, Zdziarski et al. (28) showed that the genomes of ABU strains were related to UPEC but have smaller genome sizes, with alterations in essential virulence genes. In the aim to confirm this low virulence potential of ABU strains, we used a validated in vivo nematode model. The C. elegans study showed that ABU strains belonging to the two virulent phylotypes (B2 and D) have the same virulence as the strains isolated from cystitis, pyelonephritis, and urosepsis belonging to the same phylotypes (LT50s: 3.2 to 3.3 days ± 0.2 [ABU] versus 3.1 to 3.4 days ± 0.1 [cystitis], 3.4 to 3.6 days ± 0.1 [pyelonephritis], and 3.3 to 3.4 days ± 0.1 [urosepsis]; P value nonsignificant) (Table 2). On the other hand, strains isolated from ABU, cystitis, or urosepsis and belonging to the two commensal phylotypes (A and B1) had a low virulence (LT50s: 5.1 to 5.4 days ± 0.2 [ABU], 5.0 to 5.5 days ± 0.2 [cystitis], 5.3 days ± 0.1 [urosepsis]) (Table 2). The number of E. coli CFU within the nematode gut varied around 105 bacteria per worm for each strain 72 h after ingestion without statistical difference (data not shown), confirming the ingestion and proliferation of E. coli isolates in the C. elegans intestine. These results show (i) a clear correlation between ABU strains' ability to kill C. elegans and the phylotyping origin harbored by E. coli strains, whatever the clinical manifestations, and (ii) a clear potential for virulence of the ABU strains.
Table 1.
VFs associated with E. coli strains causing ABU, cystitis, pyelonephritis, and urosepsis in pregnant womena
| VF | Association [no. present (%)] with strains causing: |
Pb |
||||||
|---|---|---|---|---|---|---|---|---|
| ABU (n = 37) | C (n = 26) | P (n = 14) | U (n = 37) | Total (n = 114) | ABU vs C | ABU vs P | ABU vs U | |
| Genes | ||||||||
| Adhesins | ||||||||
| papG allele I | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |||
| papG allele II | 13 (35.1) | 10 (38.5) | 12 (85.7) | 29 (78.4) | 64 (56.1) | 0.004 | 0.005 | |
| papG allele III | 9 (24.3) | 7 (26.9) | 0 (0) | 3 (8.1) | 19 (16.7) | 0.005 | 0.05 | |
| No papG allele | 15 (40.6) | 9 (34.6) | 2 (14.3) | 5 (13.5) | 31 (27.2) | |||
| papA | 12 (32.4) | 9 (34.6) | 10 (71.4) | 25 (67.6) | 56 (49.1) | 0.03 | ||
| papC | 7 (18.9) | 6 (23.1) | 9 (64.3) | 23 (62.2) | 45 (39.5) | 0.005 | 0.01 | |
| papE | 9 (24.3) | 6 (23.1) | 8 (57.1) | 22 (59.5) | 45 (39.5) | 0.04 | 0.04 | |
| sfaS | 3 (8.1) | 3 (11.5) | 0 (0) | 3 (8.1) | 9 (7.9) | |||
| focG | 13 (35.1) | 7 (26.9) | 9 (64.3) | 0 (0) | 29 (25.4) | <0.001 | ||
| afa-draBC | 12 (32.4) | 8 (30.8) | 6 (42.9) | 2 (5.4) | 28 (24.6) | 0.01 | ||
| fimH | 33 (89.2) | 25 (96.2) | 13 (92.9) | 34 (91.9) | 105 (92.1) | |||
| Toxins | ||||||||
| hlyA | 23 (62.2) | 16 (61.5) | 11 (78.6) | 6 (16.2) | 56 (49.1) | <0.001 | ||
| cnf1 | 11 (29.7) | 11 (42.3) | 0 (0) | 3 (8.1) | 25 (21.9) | 0.01 | 0.04 | |
| Siderophores | ||||||||
| iutA | 23 (62.2) | 17 (65.4) | 9 (64.3) | 27 (73.0) | 76 (66.7) | |||
| irp2 | 24 (64.9) | 21 (80.8) | 14 (100) | 35 (94.6) | 94 (82.5) | 0.02 | ||
| iroN | 17 (45.9) | 21 (80.8) | 11 (78.6) | 29 (78.4) | 78 (68.4) | 0.02 | 0.01 | |
| Capsules | ||||||||
| kpsMT II | 23 (73.0) | 21 (80.8) | 12 (85.7) | 32 (86.5) | 88 (77.2) | |||
| kpsMTK I | 15 (40.5) | 15 (57.7) | 10 (71.4) | 6 (16.2) | 46 (40.4) | 0.04 | ||
| Miscellaneous | ||||||||
| traT | 18 (48.6) | 11 (42.3) | 9 (64.3) | 25 (67.6) | 63 (55.3) | |||
| malX | 32 (86.5) | 25 (96.2) | 14 (100) | 37 (100) | 93 (81.6) | |||
| Aggregated gene mean (SD, min-max)c | 8.1 (3.04, 2-15) | 9.2 (3.11, 3-16) | 11.2 (2.15, 6-15) | 10.5 (2.35, 5-15) | ND | 0.001 | 0.01 | |
| Phylotypes | ||||||||
| A | 6 (16.2) | 1 (3.8) | 0 (0) | 0 (0) | 7 (6.1) | |||
| B1 | 4 (10.8) | 2 (7.7) | 0 (0) | 3 (13.5) | 9 (7.9) | |||
| B2 | 18 (48.6) | 12 (46.2) | 9 (64.3) | 13 (32.4) | 52 (45.6) | |||
| D | 9 (24.3) | 11 (42.3) | 5 (35.7) | 21 (45.9) | 46 (40.4) | |||
| Serotypes | ||||||||
| O:4 | 14 (37.8) | 12 (46.2) | 5 (35.7) | 22 (59.4) | 53 (46.5) | |||
| O:6 | 6 (16.2) | 3 (11.5) | 7 (21.9) | 9 (24.3) | 25 (21.9) | |||
| O:18 | 5 (13.5) | 2 (7.7) | 0 (0) | 0 (0) | 7 (6.1) | |||
| O:75 | 3 (8.1) | 6 (23.1) | 0 (0) | 0 (0) | 9 (7.9) | |||
| O:2 | 2 (5.4) | 3 (11.5) | 0 (0) | 0 (0) | 5 (4.4) | |||
| Others | 7 (18.9) | 0 (0) | 2 (14.3) | 6 (16.2) | 15 (13.2) | |||
C, cystitis; P, pyelonephritis; U, urosepsis.
P values (χ2 test, Fisher's exact test, Wilcoxon test) are shown where P is ≤0.05.
min, minimum; max, maximum; ND, not determined.
Table 2.
LT50 and LT100 of C. elegans infected by E. coli isolated from pregnant womena
| Status/strain | Phylotype | Serotype | No. of VFs | LT50 (SD) (days) | LT100 (SD) (days) |
Pb |
|||
|---|---|---|---|---|---|---|---|---|---|
| NECS26375 | NECS173730 | NECS690406 | NECS621464 | ||||||
| ABU | |||||||||
| NECS26375 | A | O:4 | 7 | 5.1 (0.1) | 9.7 (0.7) | NS | <0.001 | <0.001 | |
| NECS173730 | B1 | O:6 | 2 | 5.4 (0.2) | 10.0 (0.5) | NS | <0.001 | <0.001 | |
| NECS690406 | B2 | O:4 | 9 | 3.2 (0.2) | 7.4 (0.6) | <0.001 | <0.001 | NS | |
| NECS621464 | D | O:4 | 7 | 3.3 (0.1) | 7.6 (0.6) | <0.001 | <0.001 | NS | |
| Cystitis | |||||||||
| NECS992953 | A | O:2 | 7 | 5.0 (0.2) | 9.4 (0.6) | NS | NS | <0.001 | <0.001 |
| NECS698674 | B1 | O:4 | 6 | 5.5 (0.2) | 9.9 (0.9) | NS | NS | <0.001 | <0.001 |
| NECS978323 | B2 | O:4 | 15 | 3.1 (0.1) | 6.8 (0.8) | <0.001 | <0.001 | NS | NS |
| NECS55403 | D | O:4 | 7 | 3.4 (0.1) | 7.4 (0.6) | <0.001 | <0.001 | NS | NS |
| Pyelonephritis | |||||||||
| NECS914883 | B2 | O:4 | 11 | 3.4 (0.1) | 7.7 (0.7) | <0.001 | <0.001 | NS | NS |
| NECS37344 | D | O:4 | 11 | 3.6 (0.1) | 8.0 (0.5) | <0.001 | <0.001 | NS | NS |
| Urosepsis | |||||||||
| NECS123517 | B1 | O:6 | 4 | 5.3 (0.1) | 9.8 (0.8) | NS | NS | <0.001 | <0.001 |
| NECS198654 | B2 | O:4 | 7 | 3.3 (0.1) | 7.2 (0.8) | <0.001 | <0.001 | NS | NS |
| NECS924212 | D | O:4 | 11 | 3.4 (0.1) | 7.7 (0.7) | <0.001 | <0.001 | NS | NS |
| Control strain | |||||||||
| OP50 | 7.1 (0.2) | 10.8 (0.8) | <0.001 | <0.001 | <0.001 | <0.001 | |||
For each group of isolates (ABU, cystitis, pyelonephritis, urosepsis), strains representative of the results are presented. The data are representative of at least three independent trials for each group of strains. OP50 is an avirulent E. coli strain used as a control.
P value for LT50 (indicated ABU strain versus other strains). NS, not significant.
In conclusion, even if ABU strains have been described to have a reductive evolution of their genomes, the strains isolated from pregnant women remain as virulent as strains isolated from cystitis. Numerous factors (modulation of immune response, variation of adhesion and biofilm formation, environment, etc.) could influence the expression of VFs and allow the adaptation of the ABU strains to growth in the urinary tract.
Acknowledgments
This work was supported by INSERM, Université de Montpellier 1 and La Région Languedoc-Roussillon.
Fer-15 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).
Footnotes
Published ahead of print on 14 September 2011.
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