Abstract
Background:
Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) strains is one of the most important community-acquired infections in the world. The presence of virulence factors is closely related with the pathogenesis of UTI.
Methods:
The present study was conducted on 150 isolates of UPEC obtained from symptomatic and asymptomatic cases of UTIs with significant counts (≥105 CFU/ml) during 1 year. UPEC isolates were studied for hemolysis on 5% sheep blood agar, mannose-sensitive hemagglutination (MSHA), mannose-resistant hemagglutination (MRHA), and biofilm formation by recommended methods. Patients with UTI due to UPEC showing virulence factors were evaluated for the treatment received and the outcome of treatment. These were compared with the outcomes of patients whose culture samples grew UPEC without demonstrable virulence factors.
Results:
The study showed hemolysin production in 40% of the isolates. Forty percent of the isolates showed the presence of P fimbriae (MRHA) and 60% showed Type 1 fimbriae (MSHA). Biofilm formation capacity of all UPEC isolates was classified into three categories, strong biofilm producers (4%), moderate biofilm producers (88%), and nonbiofilm producers (8%). Patients harboring all three virulence factors showed 76% recovery compared to patients harboring strains with no demonstrable virulence factors, who showed 100% recovery.
Conclusion:
The present study has shown the production of various virulent factors and developing drug resistance in UPEC. Treatment outcomes of patients harboring strains with no virulence factors seem to be better than the ones which contain multiple virulence factors. UPEC occurs because of multiple virulence factors. Biofilm formation and MRHA are more likely to be seen in catheterized patients. The drug resistance among UPEC is on rise; therefore, the selection of appropriate antibiotics (after antibiotic susceptibility testing) is must for proper treatment of patients and to avoid emergence of drug resistance. Significant number of the UPEC isolates was sensitive to nitrofurantoin, and half of the isolates were sensitive to cotrimoxazole, so treatment is by giving these drugs orally.
Keywords: Biofilm detection, urinary tract infection, uropathogenic Escherichia coli, virulence factors, La détection de Biofilm, infection urinaire, Escherichia coli uropathogenic, virulence factorise
Résumé
Fond:
L’infection urinaire (UTI) provoquée par des tensions uropathogenic d’Escherichia coli (UPEC) est l’une des infections acquises par - le plus important de la communauté dans le monde. La présence des facteurs de virulence est étroitement liée avec la pathogénie d’UTI.
Méthodes:
La présente étude a été conduite sur 150 isolats d’UPEC obtenu à partir des cas symptomatiques et asymptomatiques d’UTIs avec les comptes significatifs (>105CFU/ml) pendant 1 an. Des isolats d’UPEC ont été étudiés pour le hemolysis sur l’agar de sang de moutons de 5%, l’hémagglutination sensible de - de mannose (MSHA), l’hémagglutination résistante de - de mannose (MRHA), et la formation de biofilm par des méthodes recommandées. Des patients avec UTI dû à UPEC montrant des facteurs de virulence ont été évalués pour le traitement reçu et les résultats du traitement. Ceux-ci ont été comparés aux résultats des patients dont les échantillons de culture ont élevé UPEC sans facteurs démontrables de virulence.
Résultats:
L’étude a montré la production d’hémolysine dans 40% des isolats. Quarante pour cent des isolats ont montré que la présence des fimbriae de P (MRHA) et de 60% a montré les fimbriae de type 1 (MSHA). La capacité de formation de Biofilm de tous les isolats d’UPEC a été classifiée dans trois catégories, producteurs forts de biofilm (4%), producteurs modérés de biofilm (88%), et producteurs de nonbiofilm (8%). Les patients hébergeant chacun des trois facteurs de virulence ont montré la récupération de 76% comparée aux patients hébergeant des tensions sans les facteurs démontrables de virulence, qui ont montré la récupération 100%.
Conclusion:
La présente étude a montré la production de divers facteurs virulents et de résistance au médicament se développante dans UPEC. Les résultats de traitement des patients hébergeant des tensions sans des facteurs de virulence semblent être meilleurs que ceux qui contiennent des facteurs multiples de virulence. UPEC se produit en raison des facteurs multiples de virulence. La formation de Biofilm et les MRHA sont pour être vus dans les patients cathétérisés. La résistance au médicament parmi UPEC est sur la hausse ; donc, la sélection des antibiotiques appropriés (après qu’essai antibiotique de susceptibilité) est nécessité pour le traitement approprié des patients et pour éviter l’émergence de la résistance au médicament. Le nombre significatif des isolats d’UPEC était sensible au nitrofurantoin, et la moitié des isolats étaient sensible au cotrimoxazole, ainsi le traitement est en donnant ces drogues oralement.
INTRODUCTION
Urinary tract infections (UTIs) are one of the common bacterial infections affecting humans throughout their lifespan.[1] The pathogenesis of UTI is complex and very often influenced by the host biological and behavioral factors as well as by virulence characteristics of the infecting uropathogen.[2] Members of the family Enterobacteriaceae are the most common organisms responsible for UTI. Escherichia coli is associated with nearly 80% of community-acquired and 50% of hospital-acquired UTIs.[3] E. coli is the most prevalent Gram-negative organism in the fecal flora. The special pathogenicity theory states that specific properties that enable E. coli to overcome host defenses in a new environment are vital for its escape from the colonic milieu and establishment in a new niche devoid of bacterial competition.[4] Uropathogenic E. coli (UPEC), therefore, reside in the colon and are then introduced into the urethra.
UPEC strains utilize a variety of virulence factors to colonize and establish a UTI. Virulence factors of UPEC include the ability to adhere to uroepithelial cells, serotype O and K antigens, resistance to phagocytosis, production of hemolysis, adhesins (Type 1 fimbriae, Pfimbriae), colicins, aerobactin, cytotoxic necrotizing factor, and cell surface hydrophobicity.[5] Biofilm formation is another universal bacterial strategy for survival adopted by UPEC. They form intracellular bacterial communities such as biofilms within the bladder epithelium and in urinary catheters.[6]
The present study was done to study the outcomes of patients infected by UPEC demonstrating various phenotypic virulence factors. The utility of phenotypic assays in determining virulence factors expressed by UPEC was assessed. The emerging drug resistance pattern in UPEC was also studied as increasing drug resistance among uropathogens has been posing a challenge in treating UTIs since the past decade. The emergence of extended-spectrum beta-lactamase (ESBL)-producing strains and the increasing incidence of carbapenem resistance among uropathogens have forced clinicians to relook at treatment options available for both community-acquired as well as hospital-acquired UTIs.
METHODS
The study was conducted in a 1200-bed tertiary care hospital in Kochi, Kerala, India, from November 2016 to October 2017. The study was done on 150 isolates of UPEC obtained from symptomatic cases of UTIs with significant bacteriuria (≥105 CFU/ml). These isolates were maintained by inoculating into nutrient agar slants and storage at 4°C.
Patient demographics
Details of clinical symptoms, treatment summary, and other relevant information were obtained from hospital information system and laboratory reports.
Urine culture
Clean-catch midstream urine samples and catheterized urine samples were included in the study. The samples were plated on blood agar and MacConkey agar using a calibrated loop. Semi-quantitative analysis of colony counts was done. Urinary antimicrobial activity was assessed by inoculating a drop of urine on a lawn culture of ATCC E. coli 25293.
Hemolysin production
The ability of the UPEC isolates to induce hemolysis on blood agar was noted. The bacteria were inoculated into 5% sheep blood agar and incubated overnight at 37°C. Hemolysin production was detected by the presence of a complete clearing of the erythrocytes (β-hemolysis) around the colonies.
Hemagglutination assay
Hemagglutination was detected by clumping of erythrocytes by fimbriae of bacteria in the presence of d-mannose. This test was carried out as per the direct bacterial hemagglutination test performed by the slide method and mannose-sensitive hemagglutination (MSHA) and mannose-resistant hemagglutination (MRHA) tests. Briefly, the strains of E. coli were inoculated into 1% nutrient broth and incubated at 37°C for 48 h to achieve full fimbriation. A panel of red blood cells (RBCs) was selected by obtaining blood from sheep and humans (blood group “O”). The RBCs were then washed thrice in normal saline and made up to a 3% suspension in fresh saline. They were used immediately or within a week when stored at 3°C–5°C. The slide hemagglutination test was carried out on a multiple-concavity slide. One drop of the RBC suspension was added to a drop of the broth culture and slide was rocked to and at room temperature for 5 min. The presence of clumping was taken as positive for hemagglutination. MSHA was detected by the absence of hemagglutination in a parallel set of test in which a drop of 2% w/v d-mannose was added to the red cells and a drop of broth culture. MRHA was detected by the presence of hemagglutination of 3% “O” group human RBC in the presence of 2% mannose.
Biofilm formation assay
The biofilm assay described by Christensen et al. is used widely and considered as a gold standard test for detection of biofilm formation.[7] In this study, we screened all isolates for their ability to form biofilm by tissue culture plate method as described by Christensen et al. with a modification in the duration of incubation which was extended to 24 h. We had evaluated biofilm production in brain–heart infusion (BHI) broth with 2% sucrose (BHI).
Inoculum preparation
The test organism and the positive control (Klebsiella pneumonia) were inoculated into BHI broth with 2% sucrose and incubated for 24 h at 37°C in stationary condition. The cultures were then diluted by a factor of 200 with fresh medium.
Loading the microtiter plate
Sterile 96-well flat bottom microtiter plates made of polystyrene were used. Rows of 5 wells each were allotted for blank, positive control, and test isolates. About 200 μl of diluted cultures was transferred into the wells of the respective rows. About 200 μl of phosphate-buffered saline (PBS) served as the “blank.” The plates were covered with the lids and incubated at 37°C for 18–24 h without shaking. Contents of the wells were removed by inverting and gently tapping. Wells were washed twice with 200 μl of PBS (pH 7.2) to remove the unattached planktonic bacteria. The plates were allowed to dry in slanting position, overnight.
Staining
About 200 μl of 0.1% crystal violet was added into each well and allowed to stand for 10 min. Excess stain was removed by washing twice with 200 μl of PBS. Stain extraction was performed by adding equal volume of 20% diethyl ether and 95% ethanol in a tube. About 100 μl of this mixture was added into each well (not in the blank), and the volume in each well was made up to 200 μl with PBS.
Measurement of optical density value
The plates were read in an automated ELISA plate reader at 490 nm. Mean of the five optical density values was calculated for each isolate and subtracted from the mean value of blank [Table 1].
Table 1.
Interpretation of biofilm formation by tissue culture plate method
| Mean OD values | Biofilm formation |
|---|---|
| OD ≥0.29 | Strong positive |
| 0.08≤ OD ≥0.28 | Moderate positive |
| OD ≤0.07 | Negative |
OD=Optical density
Antibiotic susceptibility pattern
Antibiotic susceptibility pattern was determined by Kirby–Bauer disc diffusion method with antibiotic containing disc on Muller–Hinton agar plate. The results were expressed as susceptible or resistant according to criteria recommended by the Clinical Laboratory Standards Institute (2005). The following antibiotic discs were used (drug concentration in μg were used) amoxicillin/clavulanic acid (20 μg), ampicillin/sulbactam (10/10 μg), ciprofloxacin (5 μg), cotrimoxazole (25 μg), cefixime (5 μg), cefepime (30 μg), ceftriaxone (30 μg), cefoperazone (75 μg), amikacin (30 μg), gentamicin (10 μg), ceftazidime (30 μg), piperacillin/tazobactam (100/10 μg), nitrofurantoin (300 μg), meropenem (10 μg), and imipenem (10 μg).
Outcome evaluation
Patients with UPEC showing virulence factors were evaluated for the treatment received and the outcome of treatment from their medical records. These were compared with the outcomes of patients whose culture samples grew UPEC without demonstrable virulence factors.
RESULTS
The study was done on 150 symptomatic patients whose urine samples grew UPEC with significant counts (≥105 CFU/ml). The isolates were derived from a study population which consisted of 52% females (n = 78) and 48% males (n = 72). Of the 150 patients, 126 (84%) were outpatients. The sample collection method also varied –84% of the samples were clean-catch midstream urine, 14% were catheterized samples, and 2% were supra pubic aspirates. Urinary antimicrobial activity was positive for 2% (3) of the samples.
Virulence characteristics of uropathogenic Escherichia coli isolates
Of the 150 UPEC isolates from patients with UTI, 144 demonstrated one or more virulence factors [Table 2]. Hemolysin production was detected in 57 (38%) of isolates. Of these, three isolates (2%) showed only hemolysin as a virulence factor, whereas 54 had other virulence factors also. P fimbriae were detected in 60 isolates as they demonstrated MRHA. About 92% of the isolates showed biofilm formation capacity. Biofilm formation capacity of all UPEC isolates was classified into three categories: strong biofilm producers (4%), moderate biofilm producers (88%), and nonbiofilm producers (8%). All the virulence factors were positive for only 14% of the isolates, and 4% of the isolates did not have any of the virulence factors.
Table 2.
Comparative analysis of various virulence factors
| Hemolysin | Hemagglutination | Biofilm | Percentage |
|---|---|---|---|
| Negative | Negative | Positive | 34% (51) |
| Negative | Positive | Positive | 24% (36) |
| Positive | Negative | Positive | 20% (30) |
| Positive | Positive | Positive | 14% (21) |
| Negative | Negative | Negative | 4% (6) |
| Positive | Negative | Negative | 2% (3) |
| Positive | Positive | Negative | 2% (3) |
Virulence factors in catheterized patients (n = 21)
Out of 150 samples, only 14% of the samples were catheterized. All these isolates showed the presence of both MRHA and biofilm production. About 43% of the remaining samples were noncatheterized of which 54.8% showed biofilm formation and only few of the isolates showed the presence of other two virulence factors (hemolysis = 26.8%; MRHA = 18.2%)
Antibiotic susceptibility pattern
Among the antibiotics tested, amoxicillin/clavulanic acid and piperacillin (80%) resistance was highest, followed by cefixime (74%) and cefaperazone (70%), ampicillin/sulbactam (70%) and ceftriaxone (70%). The isolates were sensitive to imipenem (98%), meropenem and amikacin (96%), nitrofurantoin (92%), and piperacillin/tazobactam (84%). The double-disc synergy test (DDST) showed that 56% of the isolates were positive for ESBL production. Totally 16 (32%) isolates were multidrug resistant (MDR), i.e., resistant to three or more drug groups.
Outcome evaluation
The treatment outcomes of patients infected with strains which did not show any virulence factors were good with 100% receiving the appropriate antibiotics and demonstrating recovery [Table 3]. Of the patients who grew isolates with demonstrable biofilm production, 91% recovered and 9% complained of unresolved symptoms, repeated urine culture positivity, or prolonged antibiotic use due to breakthrough symptoms. Among the patients with isolates showing hemolysin- and hemagglutination-positive isolates, 80.7% and 91.6% recovered. When all three virulence markers were seen, the recovery percentage was lower at 76.1%. Among the catheterized, biofilm-positive patients (n = 21), catheter removal in 18 patients effectively resolved the UTI episode. Of the remaining three patients, where catheter removal was not an alternative, a change in catheter and sensitive antibiotic administration was effective in treating the UTI episode. Pyelonephritis was seen in two patients whose urine samples grew UPEC with all three virulence factors.
Table 3.
Measuring the treatment outcome of patients with acute urinary tract infections due to uropathogenic Escherichia coli with various virulence factors
| Virulence factor | Appropriate antibiotic | Recovered | Complication/further treatment history |
|---|---|---|---|
| None (n=6) | 100% (6) | 100% (6) | 0% (0) |
| Biofilm (n=138) | 92.7% (128) | 91.3% (126) | 7.2% (10) |
| Hemolysin (n=57) | 84.2% (48) | 80.7% (46) | 21% (12) |
| Hemagglutination (n=60) | 66.6% (40) | 91.6% (55) | 8.4% (5) |
| All three (n=21) | 95.2% (20) | 76.1% (16) | 23.8% (5) |
DISCUSSION
UPEC differs from nonpathogenic E. coli by the production of virulence factors which enable the bacteria to adhere to uroepithelial cells and to establish UTI.[4] The occurrence of virulence factors in UPEC strains strengthens the association of UPEC with urinary pathogenicity.
In the present study, females (52%) had an increased frequency of UTI than males. As demonstrated in previous studies, these differences may be due to several clinical factors, including anatomic differences, hormonal effects, and behavioral patterns.[8]
All catheterized samples showed 100% biofilm formation and MRHA-positive isolates. Prolonged catheterization increases the chance of biofilm formation in the urinary tract during catheterization. The study of Tayal et al. suggests that catheterization and a prolonged duration of catheterization (≥7 days) correlated very well with increased chances of microorganisms to form biofilms in the urinary tract.[9] Catheter-associated UTIs (CAUTIs) are associated with increased morbidity and mortality and are most common cause of secondary bloodstream infections.[10] Risk factors for developing a CAUTI include prolonged catheterization, female gender, older age, and diabetes.[11] All the catheterized samples showed biofilms in this study and catheter removal along with appropriate antimicrobial cover resolved the UTI in 86% of cases.
According to our results, hemolysin production was seen in 40% of the isolates and 60% showed no hemolysis. In studies conducted by Tabasi et al., hemolysin production was detected in 34% of the isolates and 66% isolates showed no hemolysis.[12] Similarly, Saikia et al. observed that hemolytic activity was shown by 35.71% isolates.[13] In UPEC, the virulence factor is beta-hemolysin; therefore, hemolytic activity may indicate virulence of the strains. However, as beta-hemolysis is not seen in more than half of the isolates, it should not be used as a single marker for determination of UPEC. While our study showed that 21% of patients whose urinary isolates showed hemolysin as a virulence factor underwent prolonged treatment, all these cases had isolates which also produced biofilms or MRHA. Isolates with hemolysin alone as a virulence factor were associated with good recovery with appropriate therapy.
In the present study, 40% of the isolates showed the presence ofPfimbria as they demonstrated MRHA. Similar findings were seen in studies of Tabasi et al. (37.2% isolates showed the presence ofPfimbriae [MRHA]) and Kausar et al. (30% isolates were MRHA positive and 36% MSHA positive).[5,12] However, studies of Mittal et al. and Saikia et al. showed that the rate of MRHA-positive E. coli isolates was 45.5% and 35.71%, respectively.[1,13] The possession of MRHA by UPEC can be considered as one of the important virulence factors in the pathogenesis of UTIs. Hemagglutination is mediated by fimbriae. Virulence factors such asP fimbriae and Type 1 fimbriae are more frequent in UPEC. Patients with these isolates showed good outcome when treated with appropriate antibiotics.
As seen in our study from the biofilm formation assay, 4% were strong positive, 88% were moderate positive, and 8% were negative. These do not correlate with the studies of Tabasi et al. (strong biofilm producers [17.3%], moderate biofilm producers [18.6%], weakly biofilm producers [49.4%], and nonbiofilm producers [14.7%]) and Murugan et al. (strong positive [9.4%], positive [34.4%], weakly positive [40.6%], and negative [15.6%]).[12,14] Biofilm production is one of the most important virulence factors possessed by UPEC. It can bring out a wide variety of pathological events such as antibiotic tolerance and increased resistance to host defense mechanisms.
In this study, the occurrence of multiple virulence factors was observed in several isolates. All the virulence factors were positive for 14% of the isolates, and 4% of the isolates did not show any of the virulence factors. Pyelonephritis was seen in patients harboring multiple virulence factors. Therefore, a detection of these markers in complicated UTIs can help physicians in delineating patients who would require prolonged antibiotic therapy and follow-up.
Biofilm-producing and nonbiofilm-producing E. coli isolates showed similar antibiotic susceptibility pattern. This result agrees with the study of Tayal et al.[9] Among the antibiotics tested, amoxicillin/clavulanic acid and piperacillin (80%) resistance was highest. This could be due to dissemination of MDR strains in hospital settings, and the different combination of antibiotics resulted in varying degree of resistance among the biofilm-producing UPEC.[15] The isolates showed good susceptibility to nitrofurantoin (94%) which further proves it can be used for empirical treatment of uncomplicated UTI. All the catheterized samples were sensitive to amikacin, cotrimoxazole, and nitrofurantoin. In this study, the DDST showed that 56% of the isolates were positive for ESBL production and 44% were negative for ESBL. Totally 32% isolates were MDR. Based on the results of the current study, antibiotics such as nitrofurantoin remain effective oral agents for treatment of UTI patients. A significant number of the UPEC isolates were sensitive to nitrofurantoin and half of the isolates were sensitive to cotrimoxazole.
CONCLUSION
The present study has shown the production of various virulence factors and developing drug resistance in UPEC. While UPEC occurs because of multiple virulence factors, additional antibiotic resistance may provide a substantial advantage to the survival of the pathogen. The early detection of MDR and ESBL-producing organisms is important to restrict their spread in community. The drug resistance among UPEC is on rise; therefore, the selection of appropriate antibiotics (after antibiotic susceptibility testing) is must for proper treatment of patients and to avoid emergence of drug resistance. Outcome of patients with multiple virulence factors is often associated with complications. Therefore, appropriate clinical follow-up of these cases may have a role to play in the prevention of complications in these cases.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
We would like to thank Mrs. Sumi, Technical Supervisor, for excellent technical support provided during the project.
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