Evaluation of In-vitro Susceptibility of Multidrug-Resistant Urinary Escherichia Coli Isolates to Fosfomycin

Introduction

Escherichia coli is a Gram-negative bacilli belonging to the family Enterobacteriaceae. Urinary tract infections (UTIs) are caused mainly by bacteria and are among the most common infections, with increasing resistance to antimicrobial agents.¹ The most frequent pathogen  associated with urinary tract infections causing both community and hospital-acquired UTI is E.coli. It has been noted that at least half of the female population experiences urinary tract infections during their lifetime, and among them, most suffer from recurrent urinary tract infections. According to the standard definition, an organism that is resistant to three or more drugs in different antimicrobial classes is defined as a ‘multi drug-resistant pathogen’.¹

Fosfomycin is made from a chemical called ‘phosphonic acid derivative,’ which is available in powder form and is used to treat UTIs.² Both Gram-positive and Gram negative bacteria are susceptible to fosfomycin.² The importance of fosfomycin is its ability to act against biofilm formation and multi-drug resistant pathogens causing UTI. The use of fosfomycin trometamol for uncomplicated UTIs caused by resistant uropathogens is a worthwhile choice.³

E. coli is a major pathogen responsible for UTIs which are the most common clinical conditions encountered by medical practitioners in routine clinical practice. The majority of the organisms causing UTIs are found to be multi-drug resistant (MDR), and thus, treatment options for UTIs become limited.⁴ Nowadays, most MDR gram-negative bacteria, particularly those from the Enterobacteriaceae family that are resistant to conventional antibiotics, respond well to fosfomycin when used to treat UTIs.⁵

Fosfomycin targets the bacterial cell wall, ultimately killing the bacteria. Fosfomycin trometamol is a potent antibiotic for treating UTIs due to its high urinary concentration. However, the overuse of this antibiotic must be controlled to prevent increased resistance among urinary isolates, particularly E. coli. Cross resistance is uncommon due to fosfomycin’s distinctive mode of action, allowing for an integrated approach when combined with other antibiotics, especially in polytherapy.^6,7

Materials and Methods

This prospective observational descriptive study was conducted in the Microbiology laboratory at a tertiary care center in Mangalore for a period of one year. Ethical clearance (CCM/218/2022) was obtained for the same before the commencement of the study. Adult (≥18 years of age) in-patients, with signs and symptoms of lower UTI, with the presence of significant pus cells as found on direct microscopy, and significant colony counts of only MDR strains of E. coli obtained in culture, were included in the study. All in-patients aged below 18 years, as well as isolates other than E. coli and those sensitive to more than three classes of antibiotics, were excluded from the study. Eighty-four urinary MDR E. coli isolates were considered, and the sample size requirement was calculated using the following formula,¹

n = z² p(1-p) / e², Z_α = 1.96

at 95% confidence interval, p = 98%, e = 3% (allow- able error) as per the study by Hareendranath et al.¹

After cleansing the external genitalia with soap and water, mid-stream urine sample was collected in a sterile wide-mouthed container from the patients and the sample was aseptically inoculated onto enriched media like sheep blood agar, differential media like MacConkey’s agar and UTI chrome agar. The petri dishes were then placed in an incubator for 18 to 24 hours at 37 °C. The morphological characteristics of the suspected colonies, including size, shape, color, and hemolytic properties, were recorded. A colony count of E. coli ≥ 105 CFU/mL was taken as a significant growth. Routine biochemical identification was done to identify the suspected E. coli colonies. The Clinical and Laboratory Standards Institute (CLSI) guidelines for the Kirby-Bauer disc diffusion sensitivity testing method were followed.8 The following antibiotics were tested on Mueller Hinton agar: ampicillin (10 µg), cefazolin (30 µg), cefotaxime (30 µg), cotrimoxazole (1.25/23.75 µg), gentamicin (10 µg), nitrofurantoin (300 µg), ciprofloxacin (5 µg), levofloxacin (5 µg) and piperacillin/tazobactam (100/10 µg).

All MDR E. coli isolates were then subjected to testing using fosfomycin (200 µg) disc by the disc diffusion method. Fosfomycin was said to be sensitive for E. coli if the zone diameter was more than or equal to 16 mm.⁸ The batch quality control (QC) of the fosfomycin disc was performed using the ATCC standard strain of E. coli (25922).⁸ The QC zone size was found to be within the acceptable range of 22-30 mm. The demographic details and brief relevant clinical history of the patients with MDR E. coli were recorded.

Results

A total of 84 uropathogenic MDR E. coli were analyzed to check the susceptibility with fosfomycin. Thirty-two (38%) isolates were from males, and 52 (61%) were from females. The male to female ratio was 1:1.6. The  different age groups of patients from whom the isolates were obtained are depicted in Table 1.

The Kirby Bauer antibiotic sensitivity testing of the E.coli strains against various antibiotics showed strain sensitivity of 79.76% to nitrofurantoin and 53.57% to piperacillin-tazobactum. E. coli strains tested against all the other antibiotics like cefotaxime (30 µg), ampicillin (10 µg), cotrimoxazole (1.25/23.75 µg), and fluoroquinolones (5 µg) were found to be > 60% resistant, and 75% of them were extended-spectrum beta-lactamase (ESBL) producers. All urinary MDR E. coli strains that were tested with fosfomycin (200 µg) were found to be susceptible. The detailed sensitivity pattern is depicted in Table 2.

Discussion

Fosfomycin is derived from the bacterial culture of Streptomyces spp. and has structural differences from other antibiotics, placing it in a unique antimicrobial group.⁷ The discovery of fosfomycin in the late 1960s revitalized clinical knowledge due to its high effectiveness against various microbes. This drug, once forgotten, is now at the forefront of therapy for cystitis.¹ The high concentration of fosfomycin in the urinary tract can be attributed to its greater impact, and alternatively, its limited impact on the gastrointestinal microbial flora minimizes the risk of developing resistance.¹

Fosfomycin is principally available as a single 3 g oral dose; however, intravenous preparations are also available, but reserved only for specific use. Fosfomycin is contraindicated among breastfeeding women and in children below 12 years. The currently available European Committee on Antimicrobial Testing (EUCAST) version (2016) document does not mention the disk diffusion breakpoints for fosfomycin; therefore, the guidelines of the Clinical and Laboratory Standards Institute (CLSI) were used to interpret fosfomycin susceptibility results.⁶

Fosfomycin has potent action against bacteria that are resistant to carbapenem like Acinetobacter baumannii, especially in combination with Colistin, although it has an intrinsically low activity when used in stand-alone therapy.⁷ However, fosfomycin does show intrinsic resistance to Bacteroides spp., Stenotrophomonas maltophila, Burkholderia cepacia, Staphylococcus capitis, Staphylococcus saphrophyticus, and Mycobacterium tuberculosis.⁷

Among the 84 MDR urinary E. coli strains isolated, 52 (61.9%) were from females, and 32 (38%) were from males. This implies that females are more commonly affected by UTIs compared to males.^4,9 The increasing   resistance to antibiotics among E. coli is a cause for concern. However, fosfomycin has proven effective against urinary tract infections caused by E. coli, particularly multidrug-resistant strains, despite some reports of documented resistance.¹⁰ The property of fosfomycin is its dissimilarity in structure and no cross resistance when compared with other antibiotics.¹¹

There were no resistant strains to fosfomycin, although lower resistance patterns were observed for nitrofura- ntoin among MDR urinary E. coli isolates in our study. Similar findings were reported by Gopichand et al.¹² The threat of antimicrobial resistance (AMR) in any form is a warning to the community health, invariably reducing the effect of targeted therapies and conversely increasing the cost and hospital stay.¹³ The safety and efficacy of fosfomycin in co-morbid patients and its use for extended periods are commendable. However, there could be issues of poor action against Klebsiella species than E. coli, according to Matthews et al.¹⁴ Apart from fosfomycin, nitrofurantoin is also gaining ground as a drug of choice for initial treatment of cystitis with no underlying complication, and studies suggest that these drugs do not develop resistance quickly, especially for uropathogenic E. coli.¹⁵ However, microbiological cure rates with fosfomycin for MDR E. coli causing UTI were found to be high.²

The resistance pattern of antimicrobials has been on the rise based on factors such as time, class of drug and geographical regions, according to a study conducted in Germany by Naber et al.¹⁶ A similar study in Ontario showed that orally administered fosfomycin was effective in treating UTIs, particularly in cases that harbour multidrug-resistant uropathogens.¹⁷ Even in the current study, we found fosfomycin to be susceptible in-vitro; however, the clinical outcome was not assessed, as it was beyond the purview of our objective.

In Korea, for lower urinary tract infections, especially the catheter-associated UTIs, nitrofurantoin and temo- cillin, in addition to fosfomycin, were found to have excellent bactericidal effects.¹⁸ In the current study, susceptibility of fosfomycin to MDR E. coli causing UTI revealed that fosfomycin is still an effective drug in treating UTIs with MDR E. coli. The research work conducted by Maraki et al. and Batra et al. also endorses this fact.^19,20 It is also appropriate to investigate the minimum inhibitory concentration (MIC) of this drug as a reference for future studies and to potentially establish susceptibility breakpoints.²¹ Fosfomycin was also found effective against AmpC-producing strains according to the reports of James et al., and thus is a suitable alternative for the resistant uropathogens.²² However, in our study, we did not conduct any detailed investigation on AmpC-producing strains. In acute uncomplicated or complicated cystitis or in cases of lower UTIs, a three-dose regimen demonstrated good resolution of infection, as per the reports of Qiao et al.²³ In our study, the treatment aspect of the patients was not taken into consideration.

In the present study, among the patients who were admitted, patients aged above 61 years were found to be most affected with UTIs caused by MDR E. coli. The findings of Sardar et al., also indicate that MDR E. coli strains were more common among in-patients aged above 60 years.⁹ In our study, a high level of resis- tance was observed against routine first and second line antibiotics. The routine first-line antibiotics like ampicillin, cephalosporins, co-trimoxazole, and f luoroquinolones were highly resistant in our observations and showed similarity to a study by Yeganesh-Sefidan et al.⁵ Thus, the in-vitro effectiveness of fosfomycin was observed to be highly beneficial, making it a viable option for both community-acquired and hospital-acquired UTIs. However, the limitations of our study include the small sample size and the lack of clinical and microbiological follow-up to assess the in-vivo action of the drug.

Conclusion

The rise of MDR bacteria has led to a renewed interest in certain old, forgotten drugs for UTIs, which, when re-introduced, have helped address the current situation. The present study illustrated that high in-vitro activity and potency of fosfomycin against urinary E. coli isolates, establishing it as a promising substitute for treating UTIs caused by MDR isolates. In this context, fosfomycin will continue to be a drug of choice for most physicians, and its judicious use can be a lifesaver.

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