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Journal of Drug Delivery and Therapeutics

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In-vitro antibacterial activity of Fosfomycin and Nitrofurantoin against Pseudomonas aeruginosa and Acinetobacter baumannii against clinical isolates collected from Indian tertiary care hospitals

Rajesh Chavan¹* , Bhushan Naphade¹ , Bhalchandra Waykar² 

¹ Department of Microbiology, Badrinarayan Barwale College, Jalna, Maharashtra, 431203, India

² Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431001, India

INTRODUCTION

The dearth in the discovery and development of newer antibiotics and rapid increase in resistance to currently available frontline antibiotics has raised serious concerns in scientific community suggesting return of pre-antibiotic era ¹. This grave situation is particularly vital for the Gram-negative pathogens such as Pseudomonas Spp., and Acinetobacter Spp. The availability of newer antibiotics is not visible in near future and rapid resistance to available resistance mechanisms to currently antibiotics is of main concern ^{2, 3}. Pseudomonas aeruginosa and Acinetobacter baumannii pathogens are involved in a broad range of nosocomial and community-acquired infections, with multidrug resistant (MDR). Both the pathogens are among top six pathogens (ESKAPE) identified by the Infectious Diseases Society of America (IDSA) ^{4, 5, 6}. Due to the scarcity of newer antibiotics in the drug development pipeline, clinicians have been forced to reconsider older underutilized antibiotic such as fosfomycin, Trimethoprime-Sulfamethoxazole, mecillinam etc. for the treatment of infections caused by MDR Gram-negative organisms ^{7, 8, 9}. Among these older antibiotics, fosfomycin is gaining more attention due to its promising activity and lower resistance rates against these pathogens (especially Pseudomonas) either standalone or in combination with other antibiotics for the treatment of various infections caused by both MDR Gram-negative and Gram-positive organisms ^10,11,12.

Fosfomycin is available in both oral (fosfomycin trometamol and fosfomycin calcium) and intravenous formulations (fosfomycin disodium). ¹³ while nitrofurantoin is available as oral formulation. Both the drugs are recomonded for the treatment of uncomplicated urinary tract infections (UTIs). Oral fosfomycin tromethamine (3g Single dose) is currently indicated for the treatment of uncomplicated urinary tract infections (UTIs) caused by E. coli and Enterococcus faecalis in women ¹⁴. However, due to the rapid emergence and spread of MDR Gram-negative pathogens, renewed interest in use of the intravenous fosfomycin to treat infections other than UTIs is growing ¹⁵. It is identified as one of the few antibiotics which possess greatest promise for the management of infections caused by MDR Gram-negative pathogens ¹⁵.   Fosfomycin and nitrofurantoin were developed at the time where principles of modern drug development were not known. During that time, antibiotic development occurred on a trial and error basis rather than the current principles of PK/PD.¹⁶. Due to this, substantial knowledge gap in the area of pharmacokinetic and pharmacodynamic properties exists for both the drugs. 

The purpose of this study is to evaluate the in-vitro antibacterial activity (MICs) of fosfomycin and nitrofurantoin against P. aeruginosa and A. baumannii, clinical isolates to understand the potential utility of both the drugs in management of MDR infections caused by these pathogens.

MATERIALS AND METHODS

Clinical isolates

The current study utilized 777 clinical isolates comprising of Pseudomonas Spp., (N=425), and Acinetobacter Spp., (N= 352). These clinical isolates were part of Wockhardt bacterial strain repository.  These pathogens were collected from sixteen tertiary care hospitals across India located in geographically distinct states of India. One isolate per patient was collected from patients who were hospitalised for minimum of 48h in the hospital in one of the surgery, medicines, burns, ICUs, transplantation units and gynaecology departments. Bacterial species were confirmed by using MALDI-TOF-based VITEKVR MS (bioMe´rieux). Before MIC determination, bacterial cultures were revived and passaged twice in tryptone soya agar (HiMedia, India) medium.

Susceptibility testing and Interpretation of susceptibility results

Minimum inhibitory concentrations (MICs) of fosfomycin (Sigma, USA) and nitrofurantoin (Sigma, USA) were undertaken by employing agar dilution method by following Clinical and Laboratory Standards Institute (CLSI, M0&, A9) guidelines ¹⁷. MIC of fosfomycin was estimated by supplementing 25 mg/L of glucose-6-phosphate (Sigma, USA) in Mueller Hinton agar. Likewise, clinical isolates were tested for susceptibility to other antibiotics such as Piperacillin-Tazobactam (PIP-TAZ), Ceftazidime-Avibactam (CAZ-AVI), Trimethoprime-Sulfamethoxazole (SXT), Ciprofloxacin, Imipenem (IME) and Meropenem (MEM) by agar dilution MIC method in the current study. Marketed formulations of comparator drugs were used. Antibiotics were recovered from commercial formulations, and purity was determined by HPLC analysis at the Wockhardt research centre.  The lowest concentration which showed ≥80% reduction in growth compared to control (no drug) was considered as MIC. As per CLSI guidance (CLSI, M100, 29E) ¹⁸, the phenotypic resistance mechanisms such as the presence of β-lactamases (ESBL, Class C and MBL/OXA-48/181) were identified by employing marker antibiotic combinations such as ceftazidime + clavulanic acid (for ESBLs) and carbapenem + EDTA (for MBLs).

RESULTS

A total of 777 Gram-negative clinical isolates comprising of Pseudomonas Spp. [N=425] and Acinetobacter Spp., [N=352] collected during January 2016 to June 2018 from 16 Indian tertiary care hospitals were subjected to MIC determination. The number of isolates inhibited by fosfomycin [MIC (µg/mL)] is presented in Table 1. The MICs ranged from 2 - ≥512 µg/mL, for fosfomycin against Pseudomonas Spp. and Acinetobacter Spp.,. The MIC_50/90 of fosfomycin for Pseudomonas Spp. was observed to be 64/128 mg/L, whereas the MIC_50/90 of fosfomycin against Acinetobacter Spp., was 128/512 mg/L (Refer Table 1). Applying CLSI susceptibility breakpoints (E.coli), Pseudomonas Spp. showed moderate susceptibility rates (72.7%) while susceptibility rates of Pseudomonas Spp., for PIP-TAZ, CAZ-AVI, ciprofloxacin, Imipenem (IPM), and MEM were 51.8, 58.4, 40.2, 53.4, and 52.9%, respectively (Refer Table 2). In case of Acinetobacter Spp., fosfomycin along with comparator antibiotics including PIP-TAZ, SXT, ciprofloxacin, IPM and MEM showed lower susceptibility rates (< 22.4%) (Refer Table 2). Thus, taking into account all the Enterobacterales including Pseudomonas Spp., evaluated, fosfomycin retained better activity as compared to PIP-TAZ, CAZ-AVI, SXT, ciprofloxacin, IPM, and MEM. Such kind of activity of fosfomycin was not noted against Acinetobacter spp.

The susceptibility of Pseudomonas Spp., expressing various ESBL, ESBL and class C as well as MBL including OXA-48/181 enzymes to fosfomycin and other antibiotics were also analysed. Irrespective of Pseudomonas isolates expressing ESBL, ESBL and class C as well as MBL including OXA-48/181, fosfomycin showed promising susceptibility in the range of 70 to 80%. Against CAZ-S and ESBL producing strains, fosfomycin (MIC_50/90: 64/128µg/mL) susceptibility rate was in the range on 70.4 to 74.3% which was moderate in comparison with comparator drugs (87.4 to 95.1%). However fosfomycin demonstrated superior activity (Susceptibility rates: 72.7 to 80%) against Pseudomonas strains producing class C and MBL, or OXA-48/181like enzymes. The susceptibility rates for comparator drugs PIP-TAZ, ciprofloxacin and carbapenems (IPM & MEM) was in the range of 13.6 to 61.4% for class C β-lactamase producing strains while for same drugs it was less than 7.5% against MBL/OXA-48/181 producing strains (Refer Table 3).

The susceptibility of Acinetobacter Spp., expressing various ESBL, ESBL and class C as well as MBL including OXA-48/181 enzymes to fosfomycin and other antibiotics were also analysed. Irrespective of Acinetobacter isolates identified as CAZ-Sensitive or MBL including OXA-48/181 producing, fosfomycin showed susceptibility in the range of 11.4 and 24.4%. The susceptibility rates for comparator antibiotic for CAZ-S strains were superior (> 87%) than fosfomycin while against MBL-OXA-48/181 producers it was even poor compared to fosfomycin (Refer Table 4).

The in-vitro activity of nitrofurantoin was also evaluated against Pseudomonas spp. and Acinetobacter Spp. The number of isolates inhibited by nitrofurantoin [MIC (µg/mL)] is presented in Table 1. The MICs ranged from 4/8 - ≥512 µg/mL, for nitrofurantoin against Pseudomonas Spp. and Acinetobacter Spp.,.The high MIC_50/90 values for nitrofurantoin against Pseudomonas Spp. and Acinetobacter Spp., suggest its weakest activity (Table 1 & 2). The MIC_50/90 value of nitrofurantoin was 256µg/mL for both Pseudomonas Spp. and Acinetobacter Spp., based on the CLSI breakpoint, >97.4% of Pseudomonas Spp. and Acinetobacter Spp., were resistant to nitrofurantoin. Additional analyses of nitrofurantoin activity pertaining to various β-lactamases expressed also demonstrated higher resistant rates for strains expressing various β-lactamase. Overall, fosfomycin demonstrated better in-vitro activity against Pseudomonas Spp. compared to comparator antibiotics including PIP/TAZ, CAZ-AVI, quinolone (ciprofloxacin) and carbapenems (IPM & MEM) while in-vitro activity against Acinetobacter Spp., was not evident. The susceptibility rates for nitrofurantoin were < 1.2% for both the Spp., suggesting its poor activity against Pseudomonas Spp., and Acinetobacter Spp.

Table 1: Summary of the in-vitro activity of fosfomycin and nitrofurantoin against Pseudomonas Spp., and Acinetobacter Spp.

MIC₅₀, MIC₇₅,   MIC_90: Concentration inhibiting 50%, 75% and 90 of the isolates, respectively; MICs are reported in µg/mL

Table 2: In-vitro antibacterial activities of fosfomycin, nitrofurantoin and comparator antibiotic against Pseudomonas Spp., and Acinetobacter Spp.

PIP-TAZ: Piperacillin-Tazobactam, CAZ-AVI: Ceftazidime-Avibactam, IPM: Imipenem, MEM: Meropenem, SXT: Trimethoprim-sulfamethoxazole; MIC₅₀, MIC₇₅, MIC₉₀: Concentration inhibiting 50, 75 and 90% of the isolates, respectively; MICs are reported in µg/mL 

Table 3: In-vitro activity of fosfomycin, nitrofurantoin and comparator agents against Pseudomonas Spp., expressing various resistance mechanisms

PIP-TAZ: Piperacillin-Tazobactam, CAZ-AVI: Ceftazidime-Avibactam, IPM: Imipenem, MEM: Meropenem, SXT: Trimethoprim-sulfamethoxazole; MIC₅₀, MIC₇₅, MIC₉₀: Concentration inhibiting 50, 75 and 90% of the isolates, respectively; MICs are reported in µg/mL 

Table 4: In-vitro activity of fosfomycin, nitrofurantoin and comparator agents against Acinetobacter Spp., expressing various resistance mechanisms

PIP-TAZ: Piperacillin-Tazobactam, CAZ-AVI: Ceftazidime-Avibactam, IPM: Imipenem, MEM: Meropenem, SXT: Trimethoprim-sulfamethoxazole; MIC₅₀, MIC₇₅, MIC₉₀: Concentration inhibiting 50, 75 and 90% of the isolates, respectively; MICs are reported in µg/mL 

DISCUSSION AND CONCLUSION

Emergence of MDR among Gram-negative pathogens including P. aeruginosa and A. baumannii, there has been renewed interest in older antibiotics such as fosfomycin, polymixins, aminoglycosides etc. Considering the promising activity along with minimal resistance rates, fosfomycin offers best option for the management of infections caused by MDR pathogens. The current study examined the in-vitro antibacterial activity of fosfomycin as well as nitrofurantoin against clinical isolates of P. aeruginosa and A. baumannii, including MDR isolates. The results of in-vitro antibacterial profiling in present study revealed that, fosfomycin demonstrated promising activity against Pseudomonas Spp., irrespective of multiple resistance mechanisms, while fosfomycin did not showed activity against Acinetobacter Spp., The another drug nitrofurantoin was also evaluated in the current study demonstrated poor activity against both the Spp., Based on the results observed in the current investigation, the discussion shall focus ONLY on fosfomycin activity against Pseudomonas Spp.,

According to MIC results of fosfomycin against Pseudomonas, significant variable antipseudomonal activity of fosfomycin is reported in the literature and this variability in activity is considered to be population dependent ¹⁹. Hence it is important to understand the local susceptibility patterns of fosfomycin for its optimal use. The current study evaluates the susceptibility of P. aeruginosa isolates to fosfomycin within an Indian population which is represented by collecting strains from 16 tertiary care hospitals from various states of India. Based on the total 425 Pseudomonas clinical isolates evaluated, we found that 309 of 425 (72.7%) clinical isolates were considered susceptible to fosfomycin based upon the chosen breakpoints (64µg/mL); while 112 of 140 (80%) of only MDR isolates were susceptible to fosfomycin. The majority of the clinical isolates evaluated in the current investigation (including MDR strains) were fosfomycin susceptible, with a significant proportion being intermediate or resistant to fosfomycin. 

Due to the various testing methods employed such as disk diffusion, agar dilution, broth microdilution and Etest and lack of uniform interpretive MIC breakpoints, it is very difficult to compare the susceptibility rates of P. aeruginosa to fosfomycin observed in the current study. Falagas et al., in his thorough review of almost 23 studies found that ≥90% of MDR P. aeruginosa isolates were susceptible to fosfomycin in 7 of 19 studies ¹⁴, while 4 studies reported susceptibility rates in the range of 50 – 90%. However, when the data of these studies pooled together, the overall susceptibility rate was found to be 30.2%. The overall low susceptibility rates were primarily due to the inclusion of a study conducted in France, comprising of 1348 out of 1693 isolates that were reported to be resistant in their review. Recent studies published by Perdigão-Neto and colleagues, Maraki and colleagues etc. have reported wide range of susceptibility rates (7 to 89%) to fosfomycin but they did not revealed whether isolates evaluated were MDR or non-MDR ^20-29. These results indicate need of uniform testing methods and knowledge on pharmacodynamics of fosfomycin against P. aeruginosa so that proper breakpoints can be established by EUCAST or CLSI ^{17, 18}.

The activity of any antibacterial agent is measured by its MIC, however MIC do not provide information on time course of antimicrobial activity which is generally evaluated by performing studies involving effect of higher drug concentration and duration of persistent effect which help in designing of dosage regimes. There is discordance among scientific community whether fosfomycin displays concentration or time dependent activity. In time dependent activity, effect of saturating concentration on rate of killing is minimal while concentration dependent activity displays higher rate and extent of killing at higher concentrations. For fosfomycin, recent literature suggests organism dependent activity ^{30, 31}. Previously couple of studies have reported time dependent activity by fosfomycin against P. aeruginosa and Staphylococcus aureus ^31-33 while a study by Mazzei and collegues reported concentration-dependent activity of fosfomycin against Escherichia coli and Proteus mirabilis ³¹. 

The in-vitro antibacterial activity of fosfomycin against A. baumannii was not promising, displaying > 85% of strains are resistant to fosfomycin hence limiting the therapeutic use of fosfomycin against A. baumannii. Such is the case with nitrofurantoin which has displayed > 98% resistance rates to P. aeruginosa and A. baumannii thereby suggesting its non-utility in management of infection caused by these pathogens ^32-34.

In summary, the existing study has demonstrated that majority of isolates of P. aeruginosa from Indian tertiary care hospitals were susceptible to fosfomycin while most of A. baumannii isolates were resistant. Nitrofurantoin was not active against both of these pathogens thereby limiting it use. 

Acknowledgements

We thank the Wockhardt Research Centre, India, and Dr. Mahesh Patel and Dr. Sachin Bhagwat for granting permission to conduct these studies. We also acknowledge the excellent technical assistance from Mr. Vineet Zope, Mr. Harikesh Kalonia, Mr. Prashant Joshi, Mr. Sunil Koli, Miss. Debasmita Mohanty, and Miss. Harsha Agrawal.

Conflict of Interest

The authors declare that they have no competing financial interests exist

Funding

This work was self-funded.

REFERENCES

1. Boucher HW, Talbot GH, Benjamin DK, Bradley J, Guidos RJ, Jones RN. 10 × '20 Progress—Development of New Drugs Active Against Gram-Negative Bacilli: An Update From the Infectious Diseases Society of America. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2013; 56(12):1685-1694. doi: 10.1093/cid/cit152. 
2. Bassetti M, Ginocchio F, Mikulska M, Taramasso L, Giacobbe DR. Will new antimicrobials overcome resistance among Gram-negatives? Expert review of anti-infective therapy. 2011; 9(10):909-922. doi: 10.1586/eri.11.107.
3. Freire-Moran L, Aronsson Bo, Manz C, Gyssens IC, Anthony D So, Monnet DL et al. Critical shortage of new antibiotics in development against multidrug‐resistant bacteria‐Time to react is now. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy. 2011; 14(2):118‐124. doi: 10.1016/j.drup.2011.02.003.
4. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clinical Infectious Diseases. 2009; 48(1):1-12. doi: 10.1086/595011.
5. Talbot GH, Bradley J, Edwards JE, Gilbert D, Scheld M, Bartlett JG. Bad Bugs Need Drugs: An Update on the Development Pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America. Clinical Infectious Diseases. 2006; 42(5):657-668. doi: 10.1086/499819.
6. Driscoll JA, Brody SL, Kollef MH. The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections. Drugs. 2007; 67(3):351-368. doi: 10.2165/00003495-200767030-00003.
7. Raz R. Fosfomycin: an old-new antibiotic. Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases. 2012; 18(1):4-7. doi: 10.1111/j.1469-0691.2011.03636.x.
8. Mirakhur A, Gallagher MJ, Ledson MJ, Hart CA, Walshaw MJ. Fosfomycin therapy for multiresistant Pseudomonas aeruginosa in cystic fibrosis. J Cyst Fibros. 2003; 2(1):19-24. doi: 10.1016/S1569-1993(02)00143-1.
9. Karaiskos I, Giamarellou H. Multidrug-resistant and extensively drug-resistant Gram-negative pathogens: current and emerging therapeutic approaches. Expert opinion on pharmacotherapy. 2014; 15(10):1351-1370. doi: 10.1517/14656566.2014.914172.
10. Samonis G, Maraki S, Karageorgopoulos DE, Vouloumanou EK, Falagas ME. Synergy of fosfomycin with carbapenems, colistin, netilmicin, and tigecycline against multidrug resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa clinical isolates. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 2012; 31(5):695-701. DOI: 10.1007/s10096-011-1360-5.
11. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum beta-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis. 2010; 10(1):43-50. DOI: 10.1016/S1473-3099(09)70325-1.
12. Falagas ME, Maraki S, Karageorgopoulos DE, Kastoris AC, Mavromanolakis E, Samonis G. Antimicrobial susceptibility of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Enterobacteriaceae isolates to fosfomycin. Int J Antimicrob Agents. 2010; 35(3):240- 243. DOI: 10.1016/j.ijantimicag.2009.10.019.
13. Falagas ME, Giannopoulou KP, Kokolakis GN, Rafailidis PI. Fosfomycin: Use Beyond Urinary Tract and Gastrointestinal Infections. Clinical Infectious Diseases. 2008; 46:1069-1077. DOI: 10.1086/527442.
14. Falagas ME, Roussos N, Gkegkes ID, Rafailidis PI, Karageorgopoulos DE. Fosfomycin for the treatment of infections caused by Gram-positive cocci with advanced antimicrobial drug resistance: a review of microbiological, animal and clinical studies. Expert Opinion on Investigational Drugs. 2009; 18(7):921-944. DOI: 10.1517/13543780902967624.
15. Pulcini C, Bush K, Craig WA, Frimodt-Møller N, Grayson ML, Mouton JW et al. Forgotten antibiotics: an inventory in Europe, the United States, Canada, and Australia. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2012; 54(2):268‐274. DOI: 10.1093/cid/cir838.
16. Podolsky SH. Antibiotics and the social history of the controlled clinical trial, 1950-1970. Journal of the history of medicine and allied sciences. 2010; 65(3):327-367. DOI: 10.1093/jhmas/jrq003.
17. Clinical and Laboratory Standards Institute (CLSI). 2018. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard 11th ed., CLSI document M07. CLSI., 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA.
18. 2)      Clinical and Laboratory Standards Institute (CLSI). 2020. Performance standards for antimicrobial susceptibility testing. Approved Standard 30^th ed., CLSI document M100. CLSI., 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA.
19. Reffert JL, Smith WJ. Fosfomycin for the treatment of resistant gram‐negative bacterial infections. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy. 2014; 34(8):845-857. DOI: 10.1002/phar.1434. 
20. Falagas M, Kastoris A, Karageorgopoulos D, Rafailidis P. Fosfomycin for the treatment of infections caused by multidrug‐resistant non‐fermenting Gram‐negative bacilli: a systematic review of microbiological, animal and clinical studies. Int J Antimicrob Agents. 2009; 34(2):111‐120. DOI: 10.1016/j.ijantimicag.2009.03.009.
21. Perdigao-Neto LV, Oliveira MS, Rizek CF, Carrilho CM, Costa SF, Levin AS. Susceptibility to fosfomycin of multiresistant Gram-negative bacteria and performance of different susceptibility testing methods: preparing for clinical use. Antimicrob Agents Chemother. 2014; 58(3):1763-7. DOI: 10.1128/AAC.02048-13.
22. Maraki S, Samonis G, Rafailidis PI, Vouloumanou EK, Mavromanolakis E, Falagas ME. Susceptibility of urinary tract bacteria to fosfomycin. Antimicrob Agents Chemother. 2009; 53(10):4508-4510. DOI: 10.1128/AAC.02048-13. 
23. Lingscheid T, Tobudic S, Poeppl W, Mitteregger D, Burgmann H. In vitro activity of doripenem plus fosfomycin against drug-resistant clinical blood isolates. Pharmacology. 2013; 91(3-4):214-218. DOI: 10.1159/000348572.
24. Demir T, Buyukguclu T. Evaluation of the in vitro activity of fosfomycin tromethamine against Gram-negative bacterial strains recovered from community- and hospital-acquired urinary tract infections in Turkey. International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases. 2013; 17(11):e966-970. DOI: 10.1016/j.ijid.2013.04.005. 
25. McCaughey G, McKevitt M, Elborn JS, Tunney MM. Antimicrobial activity of fosfomycin and tobramycin in combination against cystic fibrosis pathogens under aerobic and anaerobic conditions. Journal of Cystic Fibrosis. 2012; 11:163-172. DOI: 10.1016/j.jcf.2011.11.003.
26. Lu CL, Liu CY, Huang YT, Liao CH, Teng LJ, Turnidge JD, et al. Antimicrobial susceptibilities of commonly encountered bacterial isolates to fosfomycin determined by agar dilution and disk diffusion methods. Antimicrob Agents Chemother. 2011; 55(9):4295‐4301. DOI: 10.1128/AAC.00349-11.
27. Samonis G, Maraki S, Rafailidis PI, Kapaskelis A, Kastoris AC, Falagas ME. Antimicrobial susceptibility of Gram-negative nonurinary bacteria to fosfomycin and other antimicrobials. Future Microbiol. 2010; 5(6):961-970. DOI: 10.2217/fmb.10.47.
28. Nadeem SG, Qasmi SA, Afaque F, Saleem M, Hakim ST. Comparison of the in vitro susceptibility of Clinical isolates of Pseudomonas aeruginosa in a local hospital setting in Karachi, Pakistan. B Jour Med Pract. 2009; 2(4):35-39.
29. Hortiwakul T, Chayakul P, Ingviya N, Chayakul V. In vitro activity of colistin, fosfomycin, and piperacillin/tazobactam against Acinetobacter baumannii and Pseudomonas aeruginosa in Songklanagarind Hospital, Thailand. J Infect Dis Antimicrob Agents. 2009; 26:91-96.
30. Roussos N, Karageorgopoulos DE, Samonis G, Falagas ME. Clinical significance of the pharmacokinetic and pharmacodynamic characteristics of fosfomycin for the treatment of patients with systemic infections. International Journal of Antimicrobial Agents. 2009; 34:506-515. DOI: 10.1016/j.ijantimicag.2009.08.013.
31. MacLeod DL, Barker LM, Sutherland JL, Moss SC, Gurgel JL, Kenney TF et al. Antibacterial activities of a fosfomycin/tobramycin combination: a novel inhaled antibiotic for bronchiectasis. The Journal of antimicrobial chemotherapy. 2009; 64(4):829‐836. DOI: 10.1093/jac/dkp282.
32. Pfausler B, Spiss H, Dittrich P, Zeitlinger M, Schmutzhard E, Joukhadar C. Concentrations of fosfomycin in the cerebrospinal fluid of neurointensive care patients with ventriculostomy associated ventriculitis. Journal of Antimicrobial Chemotherapy. 2004; 53(5):848-852. DOI: 10.1093/jac/dkh158. 
33. Grif K, Dierich MP, Pfaller K, Miglioli PA, Allerberger F. In vitro activity of fosfomycin in combination with various antistaphylococcal substances. The Journal of antimicrobial chemotherapy. 2001; 48(2):209-217. DOI: 10.1093/jac/48.2.209. 
34. Mazzei T, Cassetta MI, Fallani S, Arrigucci S, Novelli A. Pharmacokinetic and pharmacodynamic aspects of antimicrobial agents for the treatment of uncomplicated urinary tract infections. Int J Antimicrob Agents. 2006; 28 Suppl 1:S35‐41. DOI: 10.1016/j.ijantimicag.2006.05.019.