Fosfomycin: A Substitute in Therapeutic Options for Extended Spectrum Beta-lactamase Producing Uropathogens

Background: The emergence of antibiotic resistance among pathogens causing urinary tract infections (UTI) has made treatment options limited. The use of fosfomycin along with other drug combination can significantly address this problem. Our study aimed to identify the rate of resistance among uropathogens and their susceptibility patterns to fosfomycin along with other antibacterial agents. Methods: The retrospective study was conducted at Jinnah Sindh Medical University in collaboration with Dr. Tahir Laboratory, Karachi. A total of 146 urine samples were included which were processed for antibacterial susceptibility testing by Kirby-Bauer disk diffusion method and rate of resistance for antibacterial agents especially fosfomycin were recorded. The statistical analysis was performed by using Chi squared tests and p>0.05 was considered statistically significant. Results: The study reported lowest rate of resistance for fosfomycin among Escherichia coli 3(5.3%), Klebsiella pneumoniae 7(14%) and Pseudomonas aeruginosa 9(22.5%) in comparison with ampicillin, which showed resistance in 43(76.8%), 41(82%) and 39(97.5%) cases of E. coli, K. pneumoniae and P. aeruginosa respectively. The subgroup carbapenem resistant Enterobacteriaceae (CRE) and extended spectrum β-lactamases (ESBLs) producers were seen noticeably high in P. aeruginosa. Overall, the female to male ratio was 1.4:1 (87/59), showing female preponderance (p=0.02). A majority of patients belonged to adult age group (61.6%) followed by senior adults (23.2%, p=0.05). Conclusion: High levels of resistance to commonly used antibiotics were observed. The increasing rate of resistance among Enterobacteriaceae to cephalosporin and ampicillin is an alarming situation. In this context, fosfomycin is an interesting alternative option in treatment of complicated and uncomplicated urinary tract infections.


INTRODUCTION
The antibacterial resistance has become an exasperating yet challenging issue globally. The irrational use of antibiotics in the field of human and veterinary medicine, agriculture and farming has brought the epoch of antibacterial drugs, nearly to an edge. The antibiotic resistance has shown an Fosfomycin: A Substitute in Therapeutic Options for Extended Spectrum Beta-lactamase Producing Uropathogens https://doi.org/10.36283/PJMD10-4/006 increment of 9% from 2014 to 2017 in United Kindom 1 . In United State of America, the multidrug resistant uropathogens have been reported in 7% to 13% cases in last decade 2 . The condition in Asian countries is even more serious, the prevalence of resistant uropathogens is reported as 38.3% by SMART (Study for Monitoring Antimicrobial Resistance Trends) program 2 . The bacterial resistance has become an alarming sign in urological practices in general, and urinary tract associated infections in particular. The extended spectrum beta lactamase (ESBL) producing Enterobacteriaceae in context to uropathogens have become a complex issue when treatment modalities are concerned.
The ESBL bacteria exhibit resistance towards third generation cephalosporins, penicillins and monobactams. The ESBL E. coli, K. pneumonia, Pseudomonas species, Proteus species and other uropathogens have shown decreased activity against cephalosporins, quinolones, and aminoglycosides and even with cephamycins 3 . Trimethoprim-sulfamethoxazole has also become ineffective in ESBL pathogens and treatment of urinary tract infections by a single potent drug is becoming difficult day by day. The scenario is more complicated with hospital associated urinary tract infections where resistant strains of Pseudomonas and Acinetobacter baumannii are found to be involved. Therefore, the advent and practice of newer antibacterial drugs is current era's most pivotal requirement.
Fosfomycin-promethazine is a synthetic, broad spectrum, bactericidal antibiotic that works by inhibiting pyruvyl-transferase during bacterial cell wall synthesis, and it decreases bacterial adherence to epithelial cells in the urinary tract. Fosfomycin is considered as first line of therapy for uncomplicated UTI 4 . Fosfomycin has shown efficacy in both in vitro and in vivo studies for treating UTIs caused by ESBL-producing Enterobacteriaceae and vancomycin resistant enterococci 5 . In addition, it is an alternative option for multidrug resistant (MDR) organisms, allergies where the intravenous antibiotics cannot be recommended 6 . The recent studies have reported its activity against methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae 7 .
In developing countries like Pakistan, where health care facilities are inadequate, the emergence of resistant bacterial strains is no lesser than havoc. Data from Pakistan regarding susceptibility patterns towards fosfomycin is limited and newer studies are required in order to get awareness of current scenario in Pakistan. Therefore, this study was designed to get insight of fosfomycin susceptibility patterns among local population.

METHODS
This retrospective study was conducted at Jinnah Sindh Medical University after getting ethical approval from Institutional Review Board (JSMU/IRB/2019/274) and Dr. Tahir laboratory, Hamdard University and hospital. A total of 146 urine samples which showed positive bacterial isolates collected between the time periods of July 2019 to July 2020, were included in the study. The samples were collected and processed in Dr. Tahir Laboratory. The data regarding patient's age, gender and registration number and isolated uropathogens belonging to Enterobacteriaceae were recorded. The cases with incomplete data were excluded from study.
The urine samples were collected in sterile screw capped containers and was properly labeled. The samples were inoculated on Blood, MacConkey's and cysteine lactose electrolyte deficient (CLED) agar. The identification of bacterial isolates was made by appropriate biochemical tests and was categorized as extended spectrum beta lactamases (ESBLs) and carbapenem resistant Enterobacteriaceae (CRE) based on sensitivity guidelines provided by Clinical Laboratory Standard Institute (CLSI, United States of America, 2020). The organisms which showed resistance towards third generation cephalosporins (ceftazidime, ceftriaxone and cefixime), monobactams (aztreonam) and were inhibited by β Lactamase inhibitors (clavulanate, sulbactam and tazobactam) were termed as ESBLs 8 . On the other hand, the Enterobacteriaceae, which mediated resistance towards carbapenems (ertapenem, meropenem and imipenem), were termed as CREs 9 .
The screening test for ESBL was performed by the method proposed by CLSI 10 . E. coli ATCC 25922 was used as quality control strains. The bacterial isolates were supposed to be ESBL, when the zone of inhibition for ceftazidime was ≤22mm and for cefotaxime was ≤ 27mm respectively. The double disc synergy test was used to confirm ESBL producers. In this test, amoxicillin-clavulanic acid (20/10μg) was placed on Muller-Hinton agar plate, which was already streaked with testing organism. The discs for ceftazidime (30μg) and cefotaxime (30μg) were placed about 20mm apart from amoxicillin-clavulanic acid discs. Those phenotypes, which showed phenomenon of synergism, were confirmed as ESBL producers.
The CREs were detected by performing sensitivity testing of imipenem, ertapenem and meropenem on Muller-Hinton agar by Kirby Bauer disc diffusion method. The break point for imipenem was ≤19mm while for ertapenem and meropenem was taken ≤ 21mm (proposed by CLSI) 10 . The data were recorded in Statistical Package for Social Sciences (SPSS, IBM USA, version 22). The descriptive statistics and chi square tests were used to analyze the data. The data was presented in the form of tables, graphs and figures.

RESULTS
The study included 146 bacterial isolates of E. coli, K. pneumoniae and P. aeruginosa belonging to Enterobacteriaceae. There were 56 (38.3%) E. coli strains, 50 (34.2%) K. pneumoniae isolates while in 40 (27.3%) cases, P. aeruginosa was recovered. Overall, the female to male ratio was 1.4:1 (87/59), showing female preponderance (p=0.02) as shown in Table 1. A majority of patients belonged to adult age group (61.6%) followed by senior adults (23.2%, p=0.05).The E. coli was also most commonly observed in patients falling in adult age group (73.2%), followed by geriatric group (14.3%). The female to male ratio for positivity towards E. coli was 1:0.75 (24/32). The K. pneumoniae was prevalent in adult age group (58%) and being least common in adolescents (2%). The female to male ratio for UTI caused by K. pneumoniae was found to be 1:0.6(30/20). The P. aeruginosa also showed the same trend for being commonly observed in adults (73.2%) and least prevalent among children (5.4%). The female to male ratio for K. pneumoniae was 1.6:1 (25/15).
The rate of resistance for various antimicrobial agents among Enterobacteriaceae is shown in Table 2. The E. coli strains were highly resistant to penicillin group (76.8%) followed by cephalosporins (cefaclor 48.2%, ceftriaxone 41.1% and ceftazidime 37.5%). The E. coli isolates were found to be least resistant for fosfomycin (5.3%). About 10.8% samples of E. coli were CRE and 41.78% were ESBL producers. The UTI was found to be more common among females, however relationship between gender and resistance for antimicrobial agents was not established (p>0.05). Similarly, there was no association found between age groups and drug resistance (p>0.05).
The K. pneumoniae showed highest rate of resistance for penicillins (76%). The K. pneumoniae isolates were found to be considerably resistant for quinolone group (46%). The rate of resistance was also high among cephems (ceftriaxone 58.2%, cefaclor 44%, and ceftazidime 42%). The fosfomycin came up as the most potent agent against K. pneumoniae, being 14% resistant in all cases. The frequency of ESBL producers was 50.44, while 26% isolates were CRE ( Figure 1). The rate of resistance in P. aeruginosa for penicillin group was significantly high (97.5%) which was followed by folate pathway inhibitors (67.5%).The resistance for cephalosporins was also substantially high (ceftazidime 62.5%, cefaclor 55%, ceftriaxone 47.5%). The 57% P. aeruginosa isolates were ESBL producers while 40% fall in CRE group. The resistance towards fosfomycin was observed as 22.5% being highest among all three pathogens (Figure 2).

DISCUSSION
The current study was conducted to evaluate the antibiotic susceptibility patterns of uropathogens belonging to Enterobacteriaceae for fosfomycin in particular. Urinary tract infection (UTI) is one of the commonest infectious diseases, affecting outpatients as well as hospitalized individuals. It is estimated that the lifetime incidence of uncomplicated UTI among adult and sexually active women is about 50-60% 11 . This incidence increases by 20% with increasing age in females 12 . Our study also demonstrated the increased frequency of UTI in female population in comparison with males. Similar results have been reported by Mohammed et al. and Sewify et al 13,14 . The females are more prone to contract UTI because of several factors including short urethra, sexual activity, compromised hygienic practices and low levels of estrogen after menopause 15 . According to our study, the age group, which affected most, was adult group i.e. between 20-59 years of age. Our results were in complete agreement with Tan and Chlebicki and Kabugo et al 16,17 . The reasons behind increased prevalence among adults are dependent upon sexual behaviors, use of contraceptive devices, pregnancy, urethral strictures and renal stones 18 .
Gram-negative Enterobacteriaceae, being positive in 75% to 95% cases, carries the major UTI burden 18 . The increasing rate of resistance among uropathogens has become an agonizing problem globally, which has not savaged even developed world. Pakistan is experiencing the similar serious threat of antimicrobial resistance. The present study showed the rising frequency of extended spectrum beta lactamases (ESBL) producers and carbapenem resistant Enterobacteriaceae (CRE). The most common isolated pathogens in our study were E. coli (38.3%) followed by K. pneumoniae (34.2%) and P. aeruginosa (27.3%). These results were in accordance with Yeganeh-Sefidan et al. and Qamar et al. who also documented the same results 19,20 . The penicillin group was found to be least active against aforementioned uropathogens in vitro susceptibility testing. The P. aeruginosa showed highest rate of resistance (97.5%) towards ampicillin, which was followed by E. coli (76.8%) and K. pneumoniae (76%). Yeganeh-Sefidan et al., Yekani et al. and Lake et al. also reported the highest resistance of uropathogens towards ampicillin 19,21,22 .
The resistance to ciprofloxacin, trimethoprimsulfamethoxazole and gentamicin was also noteworthy. The K. pneumoniae isolates showed highest rate of resistance (44%) to ciprofloxacin in comparison with P. aeruginosa and E. coli, which showed 40%, and 26.8% resistance respectively. Reports by Reis   contradictory to our results who stipulated higher sensitivity rates of uropathogens to ciprofloxacin 24 . The resistance to ciprofloxacin is conferred by single gene mutation and colonization by ciprofloxacin resistant Enterobacteriaceae causes the treatment burden and eventually killing of ciprofloxacin susceptible starins 25 . The ciprofloxacin is one of the most commonly prescribed antibiotics for uncomplicated UTI in Pakistan; rising incidence of resistance is indeed an alarming sign.
The resistance to trimethoprim-sulfamethoxazole (co-trimoxazole) among uropathogens was also significant. The Pseudomonas were highly resistant (67.5%) to the co-trimoxazole, our results showed complete agreement with Yekani et al 21 . The aminoglycosides are also prescribed for UTI and cystitis and according to our results; highest rate of resistance to gentamicin was seen in P. aeruginosa followed by E. coli (25%) and Klebsiella (22%). Gajdács and Urbán also documented the increasing resistances for aminoglycosides 26 . On contrary, the amikacin was found to be highly sensitive drug for all uropathogens. The frequencies of resistance by E. coli, K. pneumoniae and P. aeruginosa were found to be 6.2%, 10.8% and 9.7% respectively.
The third generation cephalosporins are used worldwide to treat complicated and hospital associated infections. This leads to emergence of third generation resistant Enterobacteriaceae (3GCREB) particularly among ESBL producers 27 . Often the ESBL production is co related with resistance to trimethoprim-sulfamethoxazole, aminoglycosides and quinolones as the same plasmids encodes resistance genes, which are responsible for ESBL production 19 . In the present study, the prevalence of ESBLs in E. coli was as high as 38.4%, While in K. pneumoniae and P. aeruginosa; it was found to be 56% and 47.5% respectively. Our results are in line with Rizzo et al. who reported the resistance by E. coli to 3 rd generation cephalosporins as 33.9% and 26.9% respectively 27 . According to current study, the frequency of ESBL production among K. pneumoniae was 56% and in P. aeruginosa was 47.5%, which clearly reflects the higher incidence of ESBL producers among Enterobacteriaceae. The carbapenem resistant Enterobacteriaceae (CREs) are related significantly with morbidity and mortality. The prevalence of CRE in the present study was found to be noticeable among uropathogens.
The present study clearly demonstrates the highest rate of sensitivity of all three isolated bacterial species to fosfomycin. According to our results, overall rate of resistance among all isolates to fosfomycin was low. The P. aeruginosa showed higher rates of resistance to fosfomycin as compare to E. coli and Klebsiella species. The reason for low level of resistance among E. coli to fosfomycin is usually that fosfomycin is not prescribed for uncomplicated UTI. These stipulated results were in accordance with Yeganeh-Sefidan et al. conversely Huang et al. presented higher rates of resistance among Enterobacteriaceae to fosfomycin 19,28 . According to Cao et al., the rate of resistance among E. coli isolates was as low as 4.6%, which is comparable to the current study results 29 . The rate of resistance by K. pneumoniae in our study was found to be 6.7% so; similar results were documented by van den Bijllaardt et al 30 .

CONCLUSION
The present study highlighted the effectiveness of fosfomycin in urinary tract infections especially by E. coli, K. pneumoniae and P. aeruginosa. The findings suggest the prompt and regular vigilance of antimicrobial resistance among uropathogens so that emergence of resistance by bugs can be controlled.