Antibiotic, ESBLs (Extended Spectrum Beta Lactamase), OPD& IPD, UTI, Spectrum of Uropathogens.

One of the most prevalent bacterial illnesses affecting people globally, urinary tract infections (UTIs) (Flores-Mireles et al., 2015; Pardeshi, 2018) greatly increase morbidity and medical expenses. Because broad-spectrum antibiotics are frequently used empirically to treat UTIs. (Tan and Chlebicki 2016) resistant bacterial strains have emerged as a result (Nicolai et al., 2021). The synthesis of enzymes known as Extended Spectrum Beta Lactamases (ESBLs), (Ghafourian et al., 2015) which provide resistance to a variety of beta-lactam antibiotics (Toussaint and Gallagher 2015) such as cephalosporins and penicillins, is one of the most alarming resistance mechanisms. Escherichia coli and Klebsiella pneumoniae are the two bacteria that produce ESBLs in particular.

Continuous local monitoring of uropathogen susceptibility patterns to both new and traditional antimicrobials is required to determine the best empirical treatment because antibiotic resistance in a wide range of pathogenic organisms can vary, even for brief periods of time, and can also vary by geographic location (Moghadam et al., 2020, Seifu and Gebissa 2018; Kumar et al., 2022). 

Screening and assessing the precise proportion of ESBL producers among urinary pathogens, as well as the variations in the antimicrobial susceptibility patterns of ESBL and non-ESBL producers in patients who are inside and outdoors, are further objectives of the study. This would aid in recommending the best antibiotic to give in both situations for the best treatment of such ESBL producers.

Selection of Patients:

Sample collection. Urine samples were  collected from kidney stone hospital of 45 patients in OPD and 70 patients of IPD, Patients urine were taken at the time of OPD and IPD  (under clean catch method) in a  sterilized label urine container. A total of 115 clinically suspected cases of UTI were enrolled in the study. Among them 70 of them were indoor patients (20 male, 20 old age, 30 female) and 45 (16 male, 11 old age, 18 female) were from outpatient department (Fig. 1).

Table 1: Distribution of clinically suspected cases of UTI.

Table 2: Number of organism producing ESBL.

Of a total of 115 patients, E. coli (45 from OPD and 70 from IPD) was isolated.  ESBL production was observed in 47.82% of E. coli (55/115). Among the outpatients 44.4% (20/45) of the isolates were ESBL producers while inpatients constituted 50% (35/70) isolates (Fig. 2).

Table 3: Number of Organism Non-ESBL Producing.

Of a total of 115 patients,  E. coli (45 from OPD and 70 from IPD) was isolated.  NON -ESBL production was observed in 52.18 % of E. coli (60/115). Among the outpatients 55.6 % (25/45) of the isolates were NON-ESBL producers while inpatients constituted 50% (35/70) isolates (Fig. 3).

Samples were collected from patients of the following departments:

• Medicine

• OPD

• Surgery

• Nephrology

• Obstetrics & Gynaecology

• Urology

Specimen Collection

In IPD and OPD 

The urine culture test takes three days to complete. On the first day, the patient's urine was cultivated on culture medium, which were then stored at 37°C for twenty-four hours to check for the development of bacteria or yeast.

Microscopy was used to determine if microorganisms had grown on the culture medium on the second day. Following Gram's staining, it was determined to be either yeast or bacteria. 

Bacterial growth in the culture medium was assessed on the second day. Bacterial colonies were reinoculated on Mac Conkey agar media after being mixed with distilled water or with the use of a culture swab. The plates were then stored at 37°C for a full day. A zone of inhibition was seen in the plates when the antibiotic disc-containing plates were taken out of the incubator on the third day, whether the antibiotics were effective or not was proven.

If the pure culture report has an antibiotic with a 'S' before it, it indicates that the antibiotic is sensitive and capable of killing the bacterium. 

All suspected UTI OPD patients had 20 clean midstream urine samples obtained, while all suspected UTI IPD patients had 35 clean midstream urine samples collected using a sterile screw-capped container. In order to isolate the uro-pathogens later on, the urine samples were processed and cultivated. 

The pure cultures that were isolated were cultivated on Mac Conkey agar. The susceptibility of the identified species was then tested by exposing them to certain antibiotics (Sedhain et al., 2019; Almutawif & Eid 2023).

Culture: The urine sample was inoculated on Mac Conkey agar and Nutrient agar media by standard loop method and incubated  aerobically at 37˚C for 18 to 24 hours.

• Mac Conkey Agar is a selective and differential media used to isolate non-fastidious Gram negative rods (primarily, the family Enterobacteriaceae and the genus Pseudomonas) and differentiate the bacteria on their ability to ferment lactose.

• Preparation of Mac Conkey Agar -

• Suspended 49.53 gm of dehydrated medium in 1000 mili litre of purified or distilled water.

• Heated it to boiling to dissolve the medium thoroughly.

• Sterilized it by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

• Cooled it to 45-50°C.

• Mixed it well before pouring it into sterile petriplates.

Analysis of bacterial isolates in detail: The following common microbiological methods were used to analyse bacterial isolates in detail: 

I. To differentiate between Gram-positive and Gram-negative bacteria, the morphology of the bacterium is studied using Gram’s staining.

II. By using the "hanging drop method," locomotion was identified. 

III. Colony Morphology: The colony's form, size, borders, surface, edge, elevation, colour, structure, and consistency were all noted as an eye lens was used to study it.

The conventional Mac Conkey petri test was used to record the capacity to ferment lactose. 

An isolated colony's Gram-stained smear was performed and evaluated. 

The results of a routine oxidase test were conducted and examined for Gram-negative rods.

Based on the bacteria that were isolated, the following biochemical assays were performed:

For lactose fermenters that are Gram-negative: 

(b) The Indole Test was conducted using peptone water. 

(c) The Urease Test was conducted using Christensen's urease medium.

For Gram-negative non-lactose fermenters, Pseudomonas species were ruled out by first testing the bacterial isolates for Oxidase Reaction. A glucose phosphate peptone water medium for the Methyl Red Test (MR) and the Voges-Proskauer Test (VP) were used for further assessment. 

(b) Indole test using peptide water culture; (c) Urease test using Christensen's urease medium; and (d) Triple Sugar Iron agar (TSI agar) test for the fermentation of glucose, lactose, and sucrose and the generation of H2S (e) The phenylalanine deaminase assay for phenyl pyruvic acid (PPA) synthesis

• For Gram-positive organisms

These organisms, which were identified as Gram-positive bacteria or Candida spp., were all found to develop on nutritional agar medium but not on MacConkey agar media. They were then tested for Staphylococcus aureus using the Catalase and Coagulase tests.

RESULTS

The study involved the collection of urine samples from fifteen distinct UTI patients. Of the patients, 22 were boys (40%), 18 were girls (32.7%), and 15 were elderly (27%). Between 60 and 70 years old of the patients, 72.7% were male and female patients between the ages of 10 and 30. All patients (100%) were admitted to the hospital and were in the outpatient department. A 100% sterile urine sample was collected using the clean catch method (by taking a urine sample midstream) in a urine container with a label.

In 21.00%, 5.90%, 35.80%, 15.80%, 7.60%, 47.60%, and 16.90% of the half cases (42.5%), fever, dysuria, frequency, stomach discomfort, vomiting, malodorous urine, and anorexia were observed, respectively. Sonography results showed that individuals with hydronephrosis and hydroureter were about twice as likely to have bladder hypertrophy (13.70% vs. 7.80%). The most frequent risk factor for UTI in our analysis was a history of using antibiotics during the previous three months (50.50%). 

The patient's history of urinary tract infections, constipation, use of immunosuppressive medications, vesicoureteral reflux, and urinary tract stones were additional risk factors for UTIs.   The patient's history of urinary tract infections, constipation, use of immunosuppressive medications, vesicoureteral reflux, and urinary tract stones were additional risk factors for UTIs. According to our research, a history of prior UTI and fever are linked variables.

Table 4: Details the findings of the urine culture sensitivity test of different sexes as the detail  given below.

Source: Samples collected from Kidney Stone Hospital, Prayagraj U.P.

Urine samples from O.P.D. and I.P.D. were collected using the clean catch method in order to conduct a culture sensitivity test on boys, girls, and elderly patients, as shown in Table 4. We discovered that E. coli had grown in over 55 urine samples that were collected. Additionally, as we said above, we discovered colony counts of 103 C.F.U./ml in the urine sample of females aged 10–30 years, and 10-3–10-5 C.F.U./ml in the urine sample of boys aged 10–30 years. Additionally, urine samples from elderly individuals aged 60 to 70 years showed 10-3 C.F.U/ml. All of the samples underwent a 72-hour aerobic incubation period at 37 °C.

Table 5: Details of the findings of the urine culture of antibiotic sensitivity test of different sexes and their results.

Source: Sample collected from kidney Stone hospital, Prayagraj U.P.

Note:  + =10%, R = 100 %, Sensitive (S) = +, Resistance (R) = R, Low % = High sensitive, high % = Low sensitive.

Source: As per table no. 4 Note:  + = 1 to 33.3, ++ = 33.4 to 66.6, +++ = 66.7 to 99.9, R =  0 to 10

Fig. 1.

Reactive antibiotics percentage of different category in patients Fig. 1

Comparison of percentage resistance to various antimicrobial agents between ESSBL producers and ESBL non producers.

The comparison of E. coli that produce ESBL and those that do not for resistance to different antimicrobial drugs (Farfour et al., 2022) is detailed in Table 6. It was discovered that, with the exception of amikacin, ESBL-producing E. coli were much more resistant to practically all antibiotics than their non-ESBL-producing counterparts. Compared to ESBL-producing E. coli (10%), amikacin resistance was higher in non-ESBL-producing E. coli (18%). Regardless of ESBL production, none of the isolates exhibited meropenem resistance (Ding et al., 2021).

Table 6: Comparison of Percentage Resistance to Various Antimicrobial Agent Between ESSBL Producers and ESBL Non-Producers.

DISCUSSION

Of these 115 samples, 55 (47.8%) had a culture-positive UTI; 33.5% of these were significant, and 18.7% were doubtfully significant bacteriuria. The M:F ratio is 1:1.2 in the culture-positive instances.

Ten out of fifteen UTI patients with candiduria were obtained from indoor patients, and five samples were found to be grossly contaminated, indicating the importance of better patient education for sample collection. This is in proportion to the sex distribution of the samples collected, which is consistent with other studies. Several studies have reported >62% culture positivity, but our study found that the culture positivity rate was only 47.8%. This is primarily due to the history of antibiotic use prior to the patient's admission to the kidney stone hospital, which may also be the cause of increased growth of Candida spp. Twenty have been acquired from catheterised persons out of 115 indoor patients. Eleven of these instances of UTI were culture-positive. Out of 1205 UTsI patients, Catheters are a significant risk factor for nosocomial UTIs, according to our research.

Urinary tract infections can be caused by a wide variety of bacteria, although Gram-negative bacilli are by far the most prevalent, according to researchers in this field. Our findings are consistent with the overall trend in epidemiology. ESBL production was found in 47.82% of the 115 E. coli that were isolated for our investigation (45 from OPD and 70 from IPD) (55/115). Although 50% (35/70) of the isolates were from inpatients, 44.4% (20/45) of the isolates from outpatients produced ESBL.

Following Citrobacter and Klebsiella species, E. coli is the most prevalent urinary pathogen in both outpatients (39.13%) and inpatients (60.86%), according to our study. While some other studies have indicated a greater incidence (85%) of E. coli isolates, our results are consistent with earlier studies that revealed an incidence of E. coli of about 50% 41, 43. The incidence of E. coli has been observed to be lower in in-patients (32.4%) and out-patients (37.4%), respectively.

The current study examined the pattern of antimicrobial susceptibility in urine isolates, paying particular attention to those that generate Extended Spectrum Beta Lactamases (ESBLs) (Tonkic et al., 2005). There were 115 clinically suspected UTI cases in all. After the investigation was finished, the following results were made: Out of the 115 urine samples that were gathered for culture, 55 (47.82%) had significant bacteriuria, whereas 60 (52.7%) had questionable significant bacteriuria. Of the remaining 115 samples, 40 were sterile, 6 had non-significant bacteriuria, 4 had severe contamination, and 10 had Candida spp. growth. For the sake of the investigation, these were thus thrown away.

It was discovered that the M:F ratio in 55 culture-positive cases was 1:1.2. The age range of 15–30 years old accounted for the greatest number of cases, encompassing both males (18) and girls (22). The age group of over 60 years old accounted for the smallest number of instances (15). Seventy instances came from the in-patient department, and forty-five cases were from the out-patient group. Of the 50 patients who had catheterisation, 32 (64%) had UTIs. Escherichia coli was the most prevalent urine pathogen in both in-patients (49.5%) and out-patients (53.3%). Citrobacter species, Klebsiella species, Enterobacter species, Acinetobacter species, Enterococcus species, Pseudomonas species, Proteus species, and Staphylococcus aureus were the next most frequent urinary pathogens, in that order. With the exception of Pseudomonas aeruginosa, the sensitivity pattern of Gram-negative bacilli revealed that amikacin, cefoperazone-sulbactam, and nitrofurantoin were extremely sensitive (64.1% to 88.8%) for both in-patients and out-patients; OPD isolates were more sensitive than IPD isolates. It was discovered that ampicillin, co-trimoxazole, and fluoroquinolones had very poor in vitro sensitivity (8.5% to 29.6%). The pattern of antibiotic susceptibility was not particular to any one bacterium. Pseudomonas aeruginosa isolates were all susceptible to cefpirome, piperacillin, meropenem, aztreonam, and piperacillin-tazobactam. Additionally, ceftazidime, amikacin, cefotaxime, and ceftazidime-clavulanic acid have demonstrated excellent susceptibility. Linezolid, vancomycin, and teicoplanin were all effective against the Gram-positive uropathogens (Pouladfar et al., 2017). There was just one Methicillin Resistant Staphylococcus aureus (MRSA) found in the patient cluster. E. coli produced ESBL in 47.82% of cases. Of the isolates in the in-patients, 63.3% were ESBL producers, compared to 36.6% in the out-patients. Therefore, a greater rate of ESBL synthesis was reported in the current investigation. Comparing E. coli that produced ESBL to those that did not, the former exhibited greater resistance to every antibiotic except cefoperazone-sulbactam, nitrofurantoin, and amikacin. Not a single isolate exhibited meropenem resistance. This trend was observed in both outpatients and inpatients. Indeed, this is a noteworthy discovery. Both OPD and IPD patients had a high resistance pattern and significant ESBL production, indicating that multidrug-resistant pathogens are widely distributed even within the population. In order to increase the organisms' susceptibility and ensure that the right antimicrobial drugs are chosen, urine culture and sensitivity must be made necessary. Therefore, the only way to stop bacterial infections from becoming more resistant is to utilise existing medicines sparingly and appropriately.

This is a noteworthy discovery, no doubt. Both IPD and OPD patients exhibited significant ESBL production and a high resistance pattern, indicating that multidrug-resistant pathogens are widely distributed even within the population. Enforcing urine culture and sensitivity is necessary to increase organism susceptibility and ensure appropriate antimicrobial agent selection. The only way to prevent the growing resistance of bacterial infections is to utilise the existing antibiotics sparingly and appropriately.

Amit Kumar Gupta, Vivek Pratap Singh and Deepak Gupta (2025). Antimicrobial Sensitivity and Resistance in Urinary Isolates: ESBL Producers vs. Non-Producers. Biological Forum, 17(7): 220-226.