Bacterial Diversity and Antibiotic Resistance Patterns of Community-Acquired Urinary Tract Infections in Mega Size Clinical Samples of Egyptian Patients: A Cross-Sectional Study

Background: Community-acquired urinary tract infection (UTI) is one of the most common infectious diseases nowadays. Alarming increased levels of antimicrobial resistance are developing globally which limit treatment options and may lead to life-threatening problems. Aim: Our study aimed to collect surveillance data on non-hospitalized Egyptian UTI cases and to develop strategies against multidrug-resistant pathogens (MDR). According to our knowledge, this is the first study to screen this high number (15,252 urine samples) in a short period (three months), providing valuable data on resistance profiles in non-hospitalized Egyptian UTI patients. Methods: A total of 15,252 urine samples were collected from different patients. Positive cultures were identified using a semi-quantitative method. Kirby-Bauer's disc diffusion method was used for antibiotic susceptibility testing, the double disc diffusion method was used for extended-spectrum beta-lactamases-producing strains, and the Chi-square test was used for statistical data processing. Results: The results showed 61% positive cultures, females accounted for 67.5%. Infants and elderly patients showed the highest positive cultures (74.4% and 69.2%, respectively). Despite Escherichia coli being the most common uropathogen (47.19%), Klebsiella species(24.42%) were the most MDR and extended-spectrum β-lactamase (ESBL)-producing organisms. E. coli and Klebsiella spp. displayed increased resistance to cephalosporins (75% and 81%, respectively). In contrast, both organisms displayed high sensitivity to carbapenems. Unlike Klebsiella spp., E. coli was highly sensitive (92%) to first-line treatment (nitrofurantoin) for UTI. Moreover, trimethoprim/sulfamethoxazole showed higher sensitivity rates compared to other nations. Conclusion: Despite Escherichia coli being the most often identified bacteria in our isolates Klebsiella spp. displayed higher resistance to the majority of tested antibiotics. Fortunately, trimethoprim/sulfamethoxazole significantly increased sensitivity, especially against E. coli. However, both species showed high rates of cephalosporin resistance. Moreover, It is important to promote Egypt's national action plan for antimicrobial resistance in collaboration with the World Health Organization, especially in the community to minimize the chance of bacterial resistance in the Egyptian community.


Introduction
Urinary tract infection (UTI) is now one of the most common infectious illnesses in the world, with chronic and recurring infections posing significant challenges [1].UTIs affect around 250 million individuals annually, accounting for roughly 40% of all infections globally [2].Treatment of UTI is critical since its complications can greatly impact a patient's health and level of life and have a considerable effect on healthcare and economic costs [3].The most common UTI complications, such as proteinuria, high blood pressure, and kidney damage, can all come from a lack of renal function [4].Female contraception use, personal hygiene issues, white ethnicity, a past previous UTI, a lack of water intake, neurological bladder disease, having diabetes, genitourinary instruments, being born with genitourinary abnormalities, phimosis, incomplete/infrequent urination, and prolonged constipation are all risk factors for UTI [5].Gram-negative pathogens such as E. coli and Klebsiella spp are responsible for most UTIs [6].However, an alarming level of antimicrobial resistance develops in UTI pathogens because of the indiscriminate and widespread use of antibiotics.Appropriate treatment of UTIs has become challenging due to high resistance to commonly prescribed antibiotics [1].In the African region, UTIs were reported as a highly incident risk infectious disease [7].Therefore, this study aimed to screen urine samples taken from UTI Egyptian non-hospitalized patients for positive cultures and describe antibiotic resistance profiles of the most common isolates.

Samples collection
A total of 15,252 urine samples related to different patients from various regions and governorates in Egypt were collected from outpatient laboratories from March 2022 to May 2022.Midstream urine samples were collected from infected patients of different ages in both genders.Male patients were instructed to clean around the urethra meatus to minimize the risk of contamination.In the case of uncircumcised male patients, they were advised to retract the foreskin.Female patients were instructed to part the labia and clean from the front to back.Both male and female participants were instructed to expel 15-30 ml of urine into the toilet to eliminate any bacteria colonizing at the far end of the urethra.They were then advised to collect the midstream portion of the urine stream in a sterile container with a wide opening and dispose of the remaining urine in the toilet.For infants, the instructions were to thoroughly cleanse the skin in the genital region using soap and water or cleansing wipes.Then, first, let a small amount of urine flow into the toilet and then collect the midstream portion during urination.Any contact between the sample container and the skin was instructed to be avoided.The samples collected from various branches across Egypt of a mega medical laboratory were refrigerated at 4°C and then transported in an ice pack to the main core branch of the medical laboratory for analysis within 24 hours of collection.

Inclusion criteria
Non-hospitalized, urinary tract-infected Egyptian patients from the community with documented age, gender, and UTI symptoms such as dysuria, frequent urination, and whose urine was cloudy or bloody with a strong odor were included in the study.Confirmation of a urinary tract infection (UTI) was established through a positive culture, specifically when the colony count exceeded 10 5 CFU/ml.Only samples collected following the Centers for Disease Control and Prevention (CDC) urine collection guidelines were included in the study.

Exclusion criteria
Non-Egyptian patients were not included in the study.Frozen urine samples or those provided in non-sterile containers are also excluded.Patients previously taken part in this study, currently using antibiotics at the time of the specimen collection and catheterized patients were ineligible to participate in this research.

Phenotypic identifications of microorganisms
The pathogenic bacterial strains were isolated from the positive collected urine samples and identified using the semi-quantitative method.Briefly, collected urine samples (1 μL -10 μL) were used to inoculate MacConkey agar and nutrient agar plates supplemented with 5% sheep blood and incubated for 24 hours at 37°C.All microorganisms isolated from positive cultures (≥10 5 CFU/mL) were identified using Gram staining, cultural characteristics, and different biochemical tests [8].

Ethical statement
Our study was approved by the Institutional Review Board (IRB) (No.202203m1) at the Suez Canal University and consent was obtained by all participants in this study.

Positive growth culture distribution
Of 15,252 urine samples collected from patients, 9317 showed a positive bacterial culture (61%).A higher percentage of positive culture was reported in women 67.5% (7691/11389) than that collected from men's urine samples 42.1% (1626/3863) and these results were statistically significant at p < 0.05 (Table 1).Age groups under one year and older than 64 years displayed a high percentage of a positive culture with 74.4% and 69.2%, respectively.In contrast, the childhood age group (1-17 years) showed the lowest rate of positive cultures among other groups (Figure 1).

Distribution of microorganisms in the collected positive cultures
The results revealed that Gram-negative bacteria were the most common cause of urinary tract infections (UTIs) in the collected positive samples (81.1%).The Enterobacteriaceae family accounts for 92.2% of Gramnegative bacteria with a high prevalence of E. coli and Klebsiella spp.Other microorganisms, such as Enterobacter spp and Citrobacter spp were recovered in lower percentages (Figure 2 and Table 2).The growth of E. coli and Klebsiella spp showed different demographic variables where much higher incidences of females in both pathogens (86.4%, and 84.1%, respectively) than in males.Moreover, the prevalence of E. coli in UTI samples was higher than that of Klebsiella spp in all female age groups.The results of the antimicrobial susceptibility test showed a higher prevalence of MDR isolates in Klebsiella spp than in E. coli (43.4% Vs. 30.3%, respectively) and these results were statistically significant at p < 0.05.The tested isolates of E. coli and Klebsiella spp were highly resistant to cefuroxime (70.2%, 75.3%), cefazoline (75.3%, 81%), ceftazidime (69.4%, 74.4%), and ampicillin (74.7%, 81.3%); respectively.On the other hand, E. coli and Klebsiella spp showed less resistance rates against imipenem (0.38%, 9.7%), meropenem (0.7%, 11.4%), ertapenem (0.7%, 12.1%), amikacin (1.2%, 8.3%); respectively.Moreover, nitrofurantoin as a firstline treatment of UTIs remains effective for E. coli with a susceptibility level of 92%, whereas its sensitivity level was only 49% in Klebsiella spp (Figure 3).E. coli was sensitive to sulfamethoxazole/trimethoprim with a percentage of 64%.The male infants infected by E. coli or Klebsiella spp were the most resistant age group to carbapenem, cephalosporins, and nitrofurantoin, while female infants infected by E. coli were most resistant to amikacin, gentamycin, and tazobactam/piperacillin antibiotics.Patients of both genders above 64 years old were the most resistant to levofloxacin and ampicillin (Table 4).The percentage of ESBL production in E. coli and Klebsiella spp were as high as 59% and 63%, respectively with a statistically significant association at a p-value equal to 0.001.

Discussion
UTIs are serious public health problem that occurs in both males and females in all age stages.Globally, empiric therapeutic options for UTIs are limited by the emergence of multi-drug resistant uro-pathogens which may lead to treatment failures [10].The results showed a high prevalence of E. coli and Klebsiella spp in the collected positive cultures isolated from UTI patients (47.19%, and 24.42%, respectively) followed by Enterococcus spp and Candida spp (approximately 8% each) as reported by other studies in different countries including Egypt [11] and Iran [12] with the percentage of E. coli (42.25% out of 232 isolates in Egypt, 58.82% out of 64 isolates in Iran) and Klebsiella spp (19.4% out of 232 isolates in Egypt, 19.12% out of 64 isolates in Iran).
The incidence of infection in our study was higher in females than in males, where the percentage of growth of E. coli and Klebsiella spp in females' urine samples were 86.4% and 84.1%, respectively compared to 13.5% and 15.8%, respectively in males.These results may be attributed to several clinical factors including anatomical differences between both males and females, hormonal effects, behavior patterns, and the female urethra's physiological and anatomical properties [13].In agreement with our results, females were more susceptible to UTI than males in Egypt and Portugal [11,14].Males were less susceptible to UTIs because the male urethra is longer, creating an obstacle that keeps bacteria out of the urinary bladder [15].
In our study, the urine samples of people above 64 years old and infants (less than one year) had the highest percentage of a positive culture (69.2% and 74.4%, respectively).Infants accounted for 57.4% of the positive cultures in another study [16].It was reported that old patients were the most affected by UTIs, with a frequency of 56.9% [14].Moreover, Xie et al. reported that the Chinese senior population over 60 years had the highest percentage of UTIs [17].The prevalence of UTIs may be increased in old age due to decreased immunity, reduced estrogen levels, and decreased water intake in old age [13,18].Infant UTI may be attributed to congenital blockage, Phimosis, ureterovesical valve failure, undeveloped host defenses, and exposure to microorganisms that can enter the urinary system by fecal soiling [19].It is worth mentioning that in our study, the male infants showed a higher total number of positive cultures (56%) than female infants (44%).The difference might be attributed to young males' lack of circumcision at this age [20].Interestingly, our statistical results showed that Egyptian infants (less than one year) were equally susceptible to E. coli and Klebsiella spp (36% and 38%, respectively).In contrast, there was a significant difference between the percentage of E. coli (68.6% out of 242 samples) and Klebsiella spp (10.3% out of 242 samples) in urine samples of infected Bahrani infants [5].This difference may be due to different research designs, environmental conditions, or patient selection criteria.
The antibiotic sensitivity test was demonstrated to investigate the possible therapeutic options of UTI patients in different test groups.The results showed that 30.3% of the E. coli isolates and 43.4% of Klebsiella spp could be described as multi-drug-resistant isolates.This agreed with a study in Ethiopia, the MDR pattern was higher in Klebsiella spp (93.3%) than in E. coli (80.4%) [21].In addition, Klebsiella spp was more common as an ESBL-producing UTI pathogen than E. coli (63.4% and 59.1%, respectively) in our study.This finding agreed with other studies in different countries such as Ethiopia [21], and Saudi Arabia [22] by which Klebsiella spp was the most ESBL producer gram-negative microorganism with percentages of Klebsiella spp (86.7%) vs E. coli (52.2%), and Klebsiella spp (48.7%) vs E. coli (41.9%), respectively.There are limited oral options for treating ESBL-producing bacteria associated with UTI.Therefore, in this study, a wide margin of samples of E. coli and Klebsiella spp (4379 and 2275, respectively) was included for the first time in Egypt which led to realistic outputs.In this study, high resistance rates were recorded against the typical antibiotic used to treat UTIs in the Egyptian community.A markedly increased resistance of E. coli and Klebsiella spp to different cephalosporin generations (75% and 69%, respectively) and ampicillin (74% and 81%, respectively) were obtained.Our results were alarming the high resistance pattern of both E. coli and Klebsiella spp towards the commonly used generations of cephalosporins, which were higher than that in other countries, as the average resistant pattern was 60% in another study conducted in Egypt [11], but only 54% in Nepal [23],40% in South Africa [10], and 24% in Taiwan [24].Moreover, the Egypt national action plan for antimicrobial resistance https://www.who.int/publications/m/item/egypt-national-action-plan-forantimicrobial-resistance reported that the average resistant pattern in hospitalized patients was about 86% for both E. coli and Klebsiella spp.These findings may be due to high prescriptions of cephalosporins in Egypt, either by physician or pharmacist recommendations [25].However, in Klebsiella spp and E. coli, the addition of beta-lactamase inhibitors only resulted in an 8% and 6% increase in ampicillin sensitivity, respectively.In comparison to sulbactam/ampicillin, tazobactam/piperacillin, not available in Egyptian community pharmacies showed 94% sensitivity in E. coli and 82% sensitivity in Klebsiella spp [25].In addition, we observed a high resistance pattern of Klebsiella spp of 51% to nitrofurantoin, a first-line treatment for UTIs.This may limit its use in the treatment of UTI for Egyptian patients.The male infants infected by E. coli or Klebsiella spp were the most resistant age group to carbapenem, cephalosporins, and nitrofurantoin, while female infants infected by E. coli were most resistant to amikacin, gentamycin, and tazobactam/piperacillin antibiotics.These results may be due to vertical transmission of antibiotic resistance from mothers to their offspring and consumption of antibiotics in early life [26].In the current study, surprisingly there was a higher sensitivity to trimethoprim/sulfamethoxazole than in other countries, especially in E. coli (64%), this is higher than in a study from Taiwan, which had a 51% sensitivity pattern [24], and Turkey's 42% [27].This may be explained by the low prescription frequency of trimethoprim/sulfamethoxazole in Egypt [25].Therefore, we emphasized the role of trimethoprim/sulfamethoxazole in empirical antibiotics because of the recent decrease in the resistance rates in Egyptian governorates as the sensitivity pattern in a study conducted in 2009 in Egypt was 42 % [28].However, it may not be possible to reuse the drug worldwide within the next several years, and close observation of surveillance data will be required.
Both isolates have a high sensitivity to carbapenems, with an average sensitivity pattern of 99.4% for E. coli and 89% for Klebsiella spp.These results are closely comparable to those obtained in Egypt and other nations, where the average sensitivity was 91% for Klebsiella spp and 98.5% for E. coli in Egypt [11], 84% for Klebsiella spp and 98% for E. coli in Turkey [27], and 92% for Klebsiella spp and 94% for E. coli in Taiwan [24].Amikacin in our study is thought to be the most effective aminoglycoside, showing a sensitivity pattern of 91.7% in Klebsiella spp and 98.7% in E. coli, which is consistent with the results in Taiwan [24], where amikacin showed a sensitivity of 96.9% in Klebsiella spp and 98.8% in E. coli.Patients of both genders above 64 years old were the most resistant to levofloxacin and ampicillin and this may be because older individuals have a significant impact on the spread of multidrug-resistant organisms [29].

Limitations
This study exclusively focused on the culturable bacteria and yeast.The study primarily examined resistance patterns for solely the two most frequent uropathogenic bacteria, Escherichia coli and Klebsiella species, and the effect of gender and age groups on the prevalence of resistance.However, other factors contribute to the development of urinary tract infections (UTIs) as well as the prevalence of resistance, including the host's immune response, urinary tract abnormalities, virulence characteristics of microorganisms, and factors that disturb the equilibrium of the normal flora.It is important to consider these unidentified variables when interpreting the observed relationships between gender, age groups, and resistance patterns.Therefore, future research must be carried out to address these limitations.

FIGURE 2 :
FIGURE 2: Distribution of microorganisms in positive culture urine samples

FIGURE 3 :
FIGURE 3: Percentage of resistance of isolated E. coli and Klebsiella spp to different antimicrobial classes.

TABLE 1 : Number (%) of positive cultures in each age group 2024
Tarek et al.Cureus 16(1): e51838.DOI 10.7759/cureus.51838FIGURE 1: Percentage of positive culture in each age group *Significant associations exist between different age groups and positive culture at p < 0.05.

TABLE 2 : Number and percentage of microorganisms out of 9317 positive urine cultures
Females were highly susceptible to UTI by Klebsiella spp at the age of 18 to 64 years compared to other age groups.While females in age groups 1 to 17 years and 18 to 64 years were the most infected with E. coli.Infant males less than one year old were the most infected by both UTI pathogens among males in other age groups, as recorded in Table3.

TABLE 4 : The antimicrobial resistance number (%) of E. coli and Klebsiella spp isolates in different demographic variables.
Significant difference between E. coli and Klebsiella spp in antibiotic resistance result (p <0.05) *