Nitrofurantoin Drug Resistance Among Gram-Negative Bacteria Isolated from Urinary Tract Infections

Introduction

Nitrofurantoin is a synthetic antimicrobial drug belonging to the class of furans, with an added nitro group (Nitro furans) and a side chain containing hydantoin, which gives the drug its name.1 This drug is effective against various Gram Negative Bacteria (GNB), like Escherichia coli, Enterobacter, Klebsiella, which are common urinary tract pathogens. It acts by increasing its concentration in the lower urinary tract while maintaining a low serum concentration.¹ Recently there has been an emergence of resistance against Enterobacteriaceae (GNB) and this indicates exten-sively drug resistant (XDR) phenotype that makes the treatment of uncomplicated UTI challenging.^1,2 The mechanism of action of Nitrofurantoin is by reducing it to reactive intermediates by bacterial flavoproteins which in turn inactivates or alters the bacterial riboso-mal proteins.¹ E. coli, one of the commonly isolated pathogens in UTI has been found to produce nitro reductase enzyme - type-I-oxygen sensitive, type-II-oxygen insensitive. Resistant strains have been mapped to type-I nitro reductase gene, nfsa gene.³ The causative agents of UTI can be either gram negative or gram positive bacteria; however the symptoms remain same for both which include, dysuria, frequency, urgency, suprapubic pain.⁴ The world Health Organisation has identified UTI as “one of the greatest threats to human health”, becoming a common infection in community as well as hospital settings.⁵

Uncomplicated UTI is usually seen in young, healthy females without any abnormalities. The most commonly isolated organisms are E. coli and Klebsiella pneumo-niae, with a small proportion of cases caused by Proteus mirabilis.⁶ The UTI can be classified based on signs & symptoms, anatomy, and the complications associated, as follows.⁷

A. Upper Urinary Tract Infection - This involves kidneys and ureter. This is accompanied by pyelonephritis, with associated signs of fever with chills and flank pain.⁷

B. Lower Urinary Tract Infection - This involves the inflammation of bladder causing cystitis. Increased frequency and urgency of urination are the common symptoms.⁷

C. Lower Urinary Tract Infection Involving urethra - This causes urethritis and the most common symptom is burning micturition or dysuria.⁷

 Classification Based on Complications⁷

A. Complicated UTIs are caused due to stones, vesico-ureteric reflux, perinephric abscess. This is associated with pyelonephritis and prostatitis.⁷

B. Uncomplicated UTIs are caused due to coloniza-tion of urethra, bladder mucosa and ureter, which results in cystitis and urethritis.⁷

The most common organism responsible for UTI (complicated & uncomplicated) is the uropathogenic E. coli (UPEC). The common Gram negative bacteria in complicated and uncomplicated UTI as per the order of prevalence is listed in Table 1.⁸

 Nitrofurantoin has been used as a common drug in the treatment of uncomplicated UTIs and has shown considerable efficacy against carbapenem-resistant infections.⁹

The bacterial reductases activate nitrofurantoin to form reactive electrophilic intermediates.⁹ These intermediates in turn bind to the ribosomal proteins and rRNA of bacteria, inhibiting the total protein production by damaging the DNA.⁹ Nitrofurantoin drug is effective against both Gram positive and Gram negative bacteria making the drug an approved anti-biotic in the treatment of uncomplicated lower UTIs.¹⁰ This drug offers several advantages, including a low risk of developing drug resistance, a reduced likelihood of allergic reactions, minimal interactions with other drugs, and consistent efficacy against both EsβL-producing and non-ESβL producing strains.¹⁰ Nitrofurantoin is a preferred choice of antibiotic for treating UTIs caused by vancomycin-sensitive and vancomycin-resistant strains, including cases associated with catheter use.¹¹ This drug has been the popular choice for prophylaxis before surgery and can be used in children above the age of 12 years.¹¹

Materials and Methods

Sample collection

Clean Catch Midstream Urine sample (MSU) was collected in a sterile container. The patients were given instructions prior to the collection of sample to clean the external genitalia with soap and running water.

The first portion of the urine adequately flushes out the normal urethral flora.

Male patients were advised to retract the foreskin of glans penis and female patients were asked to keep the labia apart. The first portion of the urine was allowed to pass and without interrupting the urine flow, mid portion of the stream was collected.

Catheter specimen

Urine sample should not be collected directly from the collection bag; instead should be collected directly from the catheter. The catheter should not touch the container. Catheterization for the purpose of urine sample collection is not advisable.

Transport and Storage

The sample was transported to the laboratory immediately after collection, for further processing. If any delay is expected, the samples were refrigerated at 4°C until the processing.

Sample Processing

Microscopy: Wet mount of the sample was observed under low power and high power microscopes. Organisms, pus cells, parasites (if any), other structures (stones, cell casts, RBC etc.) were noted.

Culture: A semi-quantitative method (standard loop technique) was followed for the culture. A sterile calibrated loop was used to culture a fixed volume of uncentrifuged urine, which was then streaked onto MacConkey agar and blood agar. The culture plates were subjected to incubation at 37°C for 24-48 hrs. After the completion of incubation period, colonies were examined, the number of colonies grown were counted, and the total count per mL was estimated.

The colonies on blood agar provides a quantitative measurement of bacteriuria and MacConkey agar enables the presumptive diagnosis of the bacterium.

The Kass criteria were used for the semi-quantitative analysis of the number of organisms present in the urine sample. The criteria are as follows:

Significant bacteriuria: When bacterial count is more than 105/mL of a single species.

Doubtful significance: In the range of 104-105 bacteria per mL. Specimen should be repeated for culture.

No significant growth: Less than 103 bacteria per mL; regarded as a contaminant.

Identification

The organisms were identified by colony character, Gram’s stain, motility, biochemical reactions and presumptively identified up to genus level.

Antibiotic Sensitivity Test (AST)

The organisms were subjected to AST testing as per CLSI 23 guidelines. Sensitivity and resistance pattern with special reference to Nitrofurantoin were noted.

Results

Out of the 50 samples received from the suspected cases of urinary tract infection (UTI), 78% of the processed samples showed ‘growth’, with 22% showing ‘no growth’.

Table 2 shows E. coli as the most frequently isolated organism, followed by K. pneumoniae species (17%), Pseudomonas and Acinetobacter.

Table 3 shows that, 21% of the urinary tract infections were noted in male patients, while 79% of the infections were found in female patients. The infection rate was highest in females aged 25-45 years, at a rate of 41%. Among males, the highest incidence was noted in individuals above 65 years of age, with an infection rate of 50%.

The highest resistance to Nitrofurantoin was seen among Acinetobacter species (100%), while E. coli showed the least resistance at 8.69%. Pseudomonas species did not demosntrate any resistance to Nitrofurantoin (Table 4).

Discussion

In the present study, out of the 50 samples collected from patients having symptoms of UTI, 78% of the urine samples yielded growth with >104 CFU/mL, while 22% did not show any growth.

This contrasts with a Russian study conducted by Rafalisky et al., which reported bacterial growth in 49% of patients with signs of UTI, while 50% showed no growth.⁶

The same study identified E. coli as the most common organism, with a frequency of isolation of 49.1% among the total samples that showed growth. The next most common organisms were K. pneumoniae and E. faecalis, with isolation rates of 9.5%, 8.7%, respectively.⁶

The present study showed E. coli, K. pneumoniae, Acinetobacter species and Pseudomonas aeruginosa as the most frequently isolated organisms. This was consistent with the results of the Russian study by Rafalisky et al.

In a another study conducted by Beyene et al. in south western Ethiopia, E. coli, S. saprophyticus, K. pneumoniae were the most common organisms isolated with the frequency rates of 33.%, 14.3% & 19%, respectively.¹²

E. coli, K. pneumoniae and S. saprophyticus were some of the common organisms isolated in the present study in comparison with various other studies conducted on UTI.

The present study revealed that females were more predisposed to UTIs compared to males, with 79% of female participants showing positive culture growth, compared to 21% in males.¹²

Similar results were observed in an Ethiopian study by Beyene et al., where 64% of female patients showed positive culture growth compared to 35.1% in males. In the present study, the isolation rates were 39% in the 35 year age group and 18% in the 60 year age group. In contrast, Beyene et al., reported isolation rates of 53% and 11% for these age groups, respectively.

The highest incidence of UTI was noted among patients aged less than 35 years and least incidence was noted among patients above 60 years of age.

The present study observed an 8.6% resistance rate to nitrofurantoin and a 14% resistance among K. pneumoniae strains. This differed significantly from the Ethiopian study by Beyene et al., which reported no resistance among strains of E. coli and a 50% resistance rate among K. pneumoniae strains.

Interestingly, in the present study, Acinetobacter species showed 100% resistance, K. oxytoca exhibited 50% resistance, and no resistant strains were observed among Pseudomonas isolates. A study by Garima Gautam et al., reported 2% resistance among Acinetobacter strains, and 4.6% resistance among Pseudomonas species.

Conclusion

Nitrofurantoin drug is commonly prescribed in uncomplicated UTIs and is frequently used in the treatment of multi drug resistant infections. It is very important to identify urinary tract pathogens up to species level. This avoids prescribing Nitrofurantoin in cases where organisms with increasing resistance have been identified. Prescription of Nitrofurantoin while taking in to consideration the resistance profile of the uropathogen can help maintain the sustainability of the drug while treating uncomplicated UTIs.

Conflict of Interest

Nil

Funding

Rajiv Gandhi University of Health Sciences Short Term Research Project for Undergraduates.

Acknowledgement

Thankful to Rajiv Gandhi University of Health Sciences (RGUHS)

Thankful To St. Alphonsa Institute of Allied Health Sciences.