Influence of bacterial morphotype on urine culture and molecular epidemiological differences in Escherichia coli harboring bacterial morphotype-induced urinary tract infections

Abstract

Bacteria that adhere to epithelial cells, form intracellular bacterial communities (IBCs), or transition to filamentous forms are referred to as morphotype-positive bacteria. Escherichia coli (E. coli) with this morphotype plays a critical role in urinary tract infections (UTIs), yet its impact on urine culture outcomes and molecular epidemiological characteristics remains unclear. In this retrospective study, we assessed the effect of bacterial morphotype on urine culture results and investigated the molecular differences between E. coli strains with and without this morphotype, using PCR and whole genome sequencing (WGS). We observed that E. coli with the morphotype-positive phenotype frequently appeared in urine sediments, leading to fewer colony-forming units (CFUs) in culture and contributing to false-negative results. However, vortexing the urine samples significantly increased CFUs, improving culture sensitivity. Additionally, E. coli with the positive morphotype carried more adhesion-related virulence genes (VGs), with the majority belonging to phylogenetic group B2. Whole genome sequencing further revealed a broader array of virulence genes in these strains. Our findings demonstrate that vortexing is an effective method to enhance urine culture positivity by releasing intracellular bacteria, and that morphotype-positive E. coli harbors a diverse set of virulence factors, indicating their potential high pathogenicity. These results highlight the importance of detecting bacterial morphotypes in urine samples for accurate UTI diagnosis and emphasize the need for increased attention to these highly virulent strains.

Importance: Uropathogenic Escherichia coli (UPEC) is widely acknowledged as the primary pathogen responsible for urinary tract infections (UTIs). Following adherence to the epithelium, UPEC undergoes periodic morphological changes, such as filamentation, which not only contribute to immune evasion but also lead to false-negative results. This study focuses on three transient stages of UPEC morphological changes: adherence to the epithelium, formation of intracellular bacterial communities (IBCs), and the presence of filamentous UPEC. Any one of these characteristics is acceptable to classify UPEC strains as morphotype-positive UPEC. This study reported the prevalence of UPEC with the bacterial morphotype and established a direct relationship between urine culture and bacterial morphotype. The molecular epidemiological distinctions were both revealed. These findings provide further evidence of the necessary for bacterial morphotype detection, and greater attention should be given to E. coli harboring this bacterial morphotype.

Keywords: E. coli; bacterial morphotype; urinary tract infection; urine culture; virulence; whole genome sequencing.

Fig 1

A mode of morphological transformation cycle of MP UPEC. This mode includes the following three stages of MP UPEC invasion of the urine epithelium: UPEC initially adopts a rod-shaped form, adheres to epithelial cells, and penetrates them through fimbriae and other adhesins. It then replicates extensively within these cells, transitioning from a dispersed rod-shaped to a dense, clustered, spherical form known as IBCs. Once the host epithelial cells perish, numerous UPEC are released into the external environment in a filamentous form. These UPEC then revert to their original rod-shaped form, seeking new epithelia of the urinary tract to adhere to, thus completing a full cycle of morphological transformation, leading to a constant invasion of the urinary tract system. UPEC, uropathogenic Escherichia coli; IBC, intracellular bacterial community; MP, morphotype positive.

Fig 2

The morphotypes of UPEC observed in exfoliated urothelial cells by a light microscope (40×). The urine sediment was stained by Sternheimer-Malbin. Among the eight images, all except image (H) represent MP UPEC. (A, B, E, F, and G) The scattered UPEC adheres to urinary epithelial cells, being an intracellular UPEC. (C) Biofilm. (D) An intracellular bacterial community (IBC), a large number of bacteria that accumulate and form compact clusters of bacteria that can be observed in epithelial cells. (H) No UPEC was observed within the epithelial cells, which was classified as MN bacteria. UPEC, uropathogenic Escherichia coli; MP, morphotype positive; MN, morphotype negative.

Fig 3

Comparison of the CFU counts before and after vortexing between the urine with morphotype-positive and negative UPEC. Urine samples with the morphotype-positive and negative UPEC were all subjected to urine culture after vortexing. CFU values increased in 59.62% of the MP UPEC and in 40.00% of the MN UPEC. A significant difference was observed between the two groups, indicating that the MP UPEC affected the CFU value greatly (χ² = 5.089, P = 0.024). Statistical analysis was performed using the Chi-square test, and analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). P < 0.05 marked as *. UPEC, uropathogenic Escherichia coli; MP, morphotype positive; MN, morphotype negative.

Fig 4

Comparison of the phylogenetic group between the morphotype-positive and negative UPEC. The figure displays the distribution of different phylogenetic groups (A, B1, B2, and D) among MP (n = 42) and MN (n = 58) UPEC isolates. Group A: the prevalence in MP UPEC was 4.76% compared to 6.90% in MN UPEC, with a P value of 0.750. Group B1: the prevalence in MP UPEC was 2.38% compared to 12.07% in MN UPEC, with a P value of 0.134. Group B2: the prevalence in MP UPEC was 78.57%, significantly higher than the 53.45% observed in MN UPEC, with a P value of 0.010. Group D: the prevalence in MP UPEC was 14.29% compared to 27.59% in MN UPEC, with a P value of 0.113. Statistical analysis was performed using the Chi-square test, and analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). B2, phylogenetic group B2; B1, phylogenetic group B1; A, phylogenetic group A; D, phylogenetic group D; MP, morphotype positive; MN, morphotype negative.

Fig 5

Comparison of the prevalence of MLST types between the morphotype-positive and negative UPEC. The prevalence of nine prominent STs was assessed in two groups, with those occurring only once, grouped as "others." The most prevalent STs in the MP group were ST1193 (23.81%) and ST131 (14.29%), followed by ST73 (14.29%), ST95 (9.52%), ST83 (4.76%), and ST10 (4.76%). Similarly, in the MN group, the top ST was ST1193 (15.52%), followed by ST131 (13.79%), ST69 (10.34%), ST95 (6.90%), ST117 (5.17%), and ST12 (3.45%). ST1193 and ST131 were common in both groups, and there was no significant difference in the phylogenetic group between the two groups. Statistical analysis was performed using the Chi-square test, and analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). MLST, multilocus sequence typing; UPEC, uropathogenic Escherichia coli; MP, morphotype positive; MN, morphotype negative.

Fig 6

(A) Total number of VG harboring per strain for E. coli isolates from urine and feces. The statistical analysis was performed using the non-parametric Mann-Whitney test, and the analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). (B) Prevalence of 12 adhesion-related VGs for the E. coli isolates from urine and feces. The statistical analysis was performed using the Chi-square test, and the analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). E. coli from urine samples showed a higher prevalence of adhesion-related VGs compared to fecal samples, with median VG counts of 6 (IQR 4–10) and 4 (IQR 3–6), respectively. Genes such as papGII, papC, papA, sfa, papEF, yfcV, papB, and flu were significantly more prevalent in urine isolates. This indicates that E. coli from feces with more adhesion VGs is more likely to colonize the urinary tract and cause infections. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. VG, virulence gene.

Fig 7

(A) Total number of VG harboring per strain for E. coli isolates among the MP and MN UPEC. The statistical analysis was performed using the non-parametric Mann-Whitney test, and the analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). (B) Prevalence of 12 adhesion-related virulence genes for the E. coli isolates among the MP and MN UPEC. The statistical analysis was performed using the Chi-square test, and the analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). MP UPEC showed a higher prevalence of adhesion-related VGs compared to MN UPEC, with median VG counts of 7 (IQR 5–10) and 6 (IQR 4–7), respectively. Notably, genes such as papGII, papC, papA, papEF, yfcV, papB, and focG were significantly more prevalent in the MP UPEC. This suggests that adhesion genes associated with P fimbriae may play a more important role in the invasion of epithelial cells forming the MP UPEC. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. VG, virulence gene; MP, morphotype positive; MN, morphotype negative.

Fig 8

The result of antibiotic resistance among the MP and MN UPEC. Forty-two MP UPEC and 58 MN UPEC strains were tested for antibiotic resistance to 15 antibiotics. All strains were highly susceptible to a glycylcycline class of semisynthetic antimicrobial agent (Tigecycline) and carbapenems (Imipenem and Ertapenem), which are beta-lactams. MP UPEC strains show similar resistance as MN UPEC strains, and no significance was analyzed between the two groups, indicating that MP UPEC strains may not contribute to increased resistance rates and pose a threat to antibiotics. Statistical analysis was performed using the Chi-square test and the Fisher exact test, and all analyses were conducted using the SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). UPEC, uropathogenic Escherichia coli; IPM, imipenem; ETP, ertapenem; TGC, tigecycline; TZP, piperacillin/tazobactam; AMC, amoxicillin/clavulanic acid; AN, amikacin; FOX, cefoxitin; SFP, cefoperazone/sulbactam; CAZ, ceftazidime; FEP, cefepime; CXM, cefuroxime; CRO, ceftriaxone; SXT, trimethoprim/sulfamethoxazole; LEV, levofloxacin; AMP, ampicillin; ESBL, extended-spectrum β-lactamases; MP, morphotype positive; MN, morphotype negative.

Fig 9

Virulence genes detected by whole genome sequencing for selected strains with MP and MN UPEC. The different phylogenetic groups, VF categories, and morphotypes are color coded and illustrated at the tips. The occurrence of virulence genes is also coded by two different colors. The outer three rings in this circular diagram represent the three MP UPEC strains, which carry significantly more virulence genes compared to the three MN UPEC strains represented by the inner three rings. This indicates that the positive strains have stronger virulence than the negative strains. MP, morphotype positive; MN, morphotype negative; UPEC, uropathogenic Escherichia coli.

Fig 10

Antibiotic resistance genes detected by whole genome sequencing for selected strains with MP and MN UPEC. The various categories of resistance genes, morphotypes, and phylogenetic groups are color coded and depicted at the tips. The occurrence of antibiotic resistance genes is also coded by two different colors. The outer three rings in this circular diagram represent the three MP UPEC strains, and the inner three rings represent the MN UPEC strains. Apart from the GN531 strain, which carries the highest number of resistance genes, the MP and MN UPEC strains show similar profiles in terms of resistance genes carried. MP, morphotype positive; MN, morphotype negative; UPEC, uropathogenic Escherichia coli.