Fosfomycin , a therapeutic option for infections produced by multiple drug-resistant Enterobacteriaceae

Due to the increasing prevalence of infections caused by resistant bacteria and especially multiple drug resistance Enterobacteriaceae, availability of alternative effective antibiotics is restricted. The goal of this study was to investigate the susceptibility profile of multiple drug resistance and extensively drug resistance Enterobacteriaceae isolated from various clinical samples to fosfomycin. A total of 303 non-duplicate Enterobacteriaceae isolates were collected. Identification and susceptibility testing were done according to standard microbiological procedures and the Kirby-Bauer test, respectively. Of all isolates, 272 (89.8%) and 26 (8.6%) were detected as multiple drug resistance and extensively drug resistance strains, respectively. The most effective antibiotic (98%) was fosfomycin, when compared with other antibiotics against multiple drug resistance and extensively drug resistance Enterobacteriaceae isolates. In this study, we find high levels of resistance to commonly used antibiotics. However, fosfomycin can be a good option for treating multiple drug resistance Enterobacteriaceae.


Introduction
The Enterobacteriaceae is the largest, most diverse group of medically important Gramnegative bacilli and cause a variety of human infections, including septicemia, urinary tract, wound and gastrointestinal infections. 1 As rapidly as novel antimicrobial agents are introduced, Enterobacteriaceae can develop resistance to antibiotics.Nowadays, a significant increase in antimicrobial resistance of Enterobacteriaceae is observed. 2Moreover, the emergence and spread of MDR (multiple drug resistance) and XDR (extensively drug resistance) Enterobacteriaceae, in the community and hospitals, is increasing in the world. 3Due to the high rate of antibiotic resistance, selection of antibiotics against MDR bacteria has been limited. 4MDR bacteria can transfer the gene to other clinical strains, so the detection of this strain is important.In an era of MDR and XDR, emphasis should be given not only to the development of new antibiotics but also to the reassessment of older and forgotten antimicrobial agents. 5Recently, there has been renewed interest in the use of fosfomycin for the management of infections caused by MDR Gram-negative bacteria, mainly Enterobacteriaceae that are resistant to usually used agents. 6Fosfomycin is a natural, forgotten antibiotic, known for nearly four decades, broad spectrum and a bactericidal antibacterial agent that inhibits cell wall synthesis in bacteria by inactivating the UDP-N-acetyl-glucosamine-3-o-enolpyruvultransferase. 6,7There are a few studies on the in vitro activity of fosfomycin against commonly encountered bacteria, except for Escherichia coli and Enterococcus faecalis. 8In this regard, the aim of this study was to evaluate the antimicrobial activity of fosfomycin against MDR and XDR Enterobacteriaceae that are resistant to traditional antibiotics.

Bacterial isolates
A total of 303 non-duplicates Enterobacteriaceae isolates were collected in tertiary care hospitals during February 2014 through August 2015 from 3 cities of Iran; Tabriz, Khoy, and Uremia.Identification of isolates was done by using biochemical tests in the Department of Microbiology, Tabriz University of Medical Sciences, Iran.

Statistical analyses
The results were analyzed using SPSS software for Windows (version 17SPSS Inc., Chicago, IL, USA).In this study, P≤0.05 was regarded statistically significant.

Results
The mean age of patients was 52 years old, ranging from 1 to 90 years old, including 125 males and 178 females.The median hospitalization of patients was 6 days.most frequently isolated bacteria (Table 1).
According to the results, the highest rate of resistance was in the penicillin group (ampicillin) with 87.5%, followed by the cehems group (cefazolin) with 80.5%, and the folate pathway inhibitors with 64.4% (Table 2).In contrast, the highest sensitivity rates were discovered in fosfomycin with 98% and the carbapnem group (imipenem) with 94.7% (Figure 1).Out of 6 fosfomycin resistant isolates, 5 isolates were isolated from males and 1 was from a female.None of the isolates were sensitive to all antibiotics.The frequency of ESBL, AmpC, KPC and MBL-producing isolates were 112 (36.9%), 28 (9.2%), 4 (1.3%) and 9 (2.9%) (

Discussion
In the present study, we investigated the susceptibility profile of MDR Enterobacteriaceae isolates to fosfomycin.The main finding of this study is that fosfomycin showed a high in vitro susceptibility to MDR, XDR, CRE, KPC, OXA48 and ESBL producing isolates.In total, 2% of the MDR and the XDR isolates were resistant to fosfomycin.Numerous studies have distinguished good activity of fosfomycin against MDR Enterobacteriaceae. 5,10,12Fairly few studies have assessed the antibacterial activity of fosfomycin against Enterobacteriaceae isolates with XDR, and have provided favorable findings concerning the potential worth of fosfomycin in this regard. 5,11,13n comparison to E. coli (0.9%), K. pneumonia (1.8%) showed higher rates of resistance to fosfomycin.The low level of resistance to fosfomycin probably is due to limited use of fosfomycin for the treatment of infections in this area.The susceptibility to fosfomycin has not been widely studied for other Enterobacteriaceae.In our study, the majority of isolates were susceptible to fosfomycin; susceptibility rates for P. mirabilis, C. freundii, K. oxytoca and P. vulgaris were 100%, except for E. cloacae and M. morganii which was detected in 92.9% and zero, respectively.These results are partly in concordance with previous reports, which indicated that low rate of E. coli, P. mirabilis and P. vulgaris were non-susceptible to fosfomycin. 3,14Unfortunately, the spread of ESBL significantly limits the treatment options.The gene encoding the ESBL is located in plasmids and is transmitted among bacteria.These plasmids can carry MDR genes against cotrimoxazole, quinolones and aminoglycosides at the same time. 15In the previous studies, 90% or more of the isolates of Enterobacteriaceae with advanced resistance

Figure 1. Susceptibility rates (%) of multiple drug resistance, extensively drug resistance, extended spectrum beta-lactamase, class C cephalosporinases (AmpC), OXA48, mannanbinding lectin and Klebsiella pneumoniae carbapenemase (KPC)-producing
Enterobacteriaceae to fosfomycin.  to antimicrobial drugs were susceptible to fosfomycin. 5,16By contrast, in two studies, 10,14 fewer than 60% of the isolates (which were isolates of E. aerogenes and K. pneumoniae) were susceptible to fosfomycin.E. coli seem to be the most susceptible to fosfomycin of the Enterobacteriaceae that create ESBL that in concordance with previous reports. 10,14,16owever, in the current study, ESBL producing strains showed 98.2% sensitivity to fosfomycin which concur with reports of previous studies. 17,18However fosfomycin is chemically unrelated to other anti-bacterial agents, due to the unique mechanism of action it may provide a synergistic effect with other antibiotics including beta-lactams, aminoglycosides and fluoroquinolone. 7hile ESBLs are the chief reason of resistance to cephalosporins between Enterobacteriaceae in the world, AmpC betalactamases are emerging as a probable risk to the activity of cephalosporins in many regions. 19Cefepime is believed a fourth-generation and frequently effective to AmpC betalactamases.A new worldwide review of 23,918 isolates from ICUs demonstrated that 74-100% of isolates were sensitive to cefepime. 20In the current study, the most effective antibiotics against AmpC producer Enterobacteriaceae were fosfomycin and imipenem (92.9%), followed cefepime (85.7%).Furthermore, fosfomycin and carbapenems are considered extremely effective treatment for AmpC producing infections. 21n comparison to tested antibiotics, apart from fosfomycin, imipenem (94.7%) showed high activity against bacterial isolates.Fosfomycin seems to have retained antibacterial activity against Enterobacteriaceae with progressive resistance patterns, even carbapnem-resistant K. pneumoniae.However, CRE is the main threat, and CRE is increasingly common in various parts of the world. 14hough, among beta-lactamases, KPC and OXA48 are all carried on plasmids and with no trouble transferred to other isolates.OXA48 enzymes are rising predominantly in the Middle Eastern and European regions and management options are limited. 14In the present study, all KPC, AmpC-porin loss, and OXA48 strains were sensitive to fosfomycin.One of the MBL strains was resistant, and 8 were susceptible to fosfomycin.According to results, fosfomycin seems to be a choice antibacterial agent for the management of such difficult to treat infections.Several studies have reported excellent in vitro activity of fosfomycin against CRE isolates. 6ne important consideration for the clinical use of fosfomycin is the possible for the emergence of resistance during management and for the choice of resistant mutants. 6While the natural mutation rate of fosfomycin resistance in Enterobacteriaceae appears to be reasonably high in vitro, this has not commonly been related to the increase of clinically very important fosfomycin resistance in clinical practice. 11Nowadays, the oral form of fosfomycin has mainly been used in the management of uncomplicated urinary tract infections in the United States, the United Kingdom, and other countries.However, the intravenous form has been used for indications beyond urinary tract infections in some countries such as Germany, France, Spain and Japan. 5The resistance rate to fosfomycin was zero in Enterobacteriaceae isolated from blood, CSF, peritoneal fluid, and burn samples.However, the frequency of resistance to fosfomycin was high (58.3%) in Enterobacteriaceae isolated from respiratory samples.Current data recommend that fosfomycin might be considered as an option in the management of MDR bacterial infections other than of UTIs, except respiratory infections.The activity of fosfomycin seems to be prone by the site from which the pathogen is isolated.The absence of cross-resistance to fosfomycin with other antibiotics may be attributed to the exclusive mechanism of action of this antimicrobial agent, which includes inhibition of a primary step in bacterial cell wall synthesis.Furthermore, fosfomycin does not seem to be a substrate for common resistance mechanisms of XDR and MDR such as efflux pumps.Moreover, the main type of fosfomycin resistance seems to be chromosomal rather than via plasmid, which reduces the likelihood of co-transmission of resistance to fosfomycin along with resistance to other antibiotics. 11n the current study, the disk diffusion assay was used for fosfomycin susceptibility testing.The agar dilution assay is an appropriate method for fosfomycin susceptibility testing, whereas broth dilution tests might give conflicting findings. 5Considering that MIC is a time-consuming assay, that several automated systems have not yet included fosfomycin, and that the E-test has shown incompatible results, disc diffusion appears to be the most useful option in routine laboratories to assess susceptibility to fosfomycin. 22

Conclusions
In this study, we find high levels of resistance to commonly used antibiotics.The most effective antibiotic is fosfomycin.So, fosfomycin can potentially be considered in the management of infections caused by MDR and XDR Enterobacteriaceae if recognized therapeutic options are not obtainable.Furthermore, fos-fomycin can be included in the routine panel of antibiotics for susceptibility testing by disc diffusion to provide fast and reliable information for the selection of treatment alternatives for MDR and XDR strains.