In vitro activity of fosfomycin trometamol and other oral antibiotics against multidrug-resistant uropathogens

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Short Communication

In vitro activity of fosfomycin trometamol and other oral antibiotics

against multidrug-resistant uropathogens

Maria Lina Mezzatesta

a,

_*

_{, Giulia La Rosa}

a

_{, Gaetano Maugeri}

a

_{, Tiziana Zingali}

a

_,

Carla Caio

a

_{, Andrea Novelli}

b

_{, Stefania Stefani}

a

a_{Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania, Catania, Italy} b_{Department of Health Sciences, Clinical Pharmacology and Oncology Section, University of Florence, Florence, Italy}

A R T I C L E I N F O Article history: Received 28 November 2016 Accepted 14 January 2017 Keywords: Fosfomycin trometamol Uropathogens Urinary tract infections ESBL

A B S T R A C T

Clinical midstream and urinary catheter isolates (n= 106) of extended-spectrum β-lactamase (ESBL)-positive Escherichia coli, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae, Proteus

mirabilis and meticillin-resistant Staphylococcus saprophyticus were tested against fosfomycin using the

agar dilution method, the broth microdilution method and the gradient test described by the Clinical and Laboratory Standards Institute. Nitrofurantoin, co-trimoxazole, amoxicillin/clavulanic acid, cefuroxime, levofloxacin and ciprofloxacin were tested using the gradient test alone. Breakpoints from the Europe-an Committee on Antimicrobial Susceptibility Testing 2015 guidelines were used. Fosfomycin inhibited all of the ESBL-positive E. coli, P. mirabilis and meticillin-resistant S. saprophyticus strains isolated from urine, as well as 82% of KPC-producing K. pneumoniae isolates. Substantial agreement for fosfomycin ac-tivity was found for the three test methods, particularly for Enterobacteriaceae. This study confirmed that fosfomycin has good in vitro activity against more common multidrug-resistant uropathogens. Fosfomycin could be a reliable empirical therapeutic option for uncomplicated urinary tract infections caused by these organisms, and a valid option for sparing parenteral antibiotics, such as carbapenems. © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

1. Introduction

Urinary tract infections (UTIs) are the most common infections observed in the community setting, and one of the main causes of increasing morbidity and the increasing economic burden. Uncom-plicated UTIs, such as cystitis, are mainly caused by Escherichia coli, representing 70–90% of uropathogens according to various epide-miological studies. In complicated and nosocomial UTIs, other Enterobacteriaceae (e.g. Proteus mirabilis and Klebsiella pneumoniae) and Gram-positive strains (e.g. Enterococcus faecalis and Staphylo-coccus saprophyticus) can be isolated as well as E. coli[1,2].

Over the last few years, the emerging presence of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae has reduced the number of antibiotics that are active against uropathogens[3,4]. In Italy in 2014, according to the European Antimicrobial Resistance Surveillance Network, the rates of antibiotic resistance to aminopenicillins, aminoglycosides and ﬂuoroquinolones among E. coli were 65%, 19% and 44%, respectively,

and the rates of antibiotic resistance to aminoglycosides and ﬂuoroquinolones among K. pneumoniae were 49% and 56%, respec-tively. ESBL-positive E. coli and K. pneumoniae accounted for 28.7% and 56.5% of isolated strains, respectively[5].

The current epidemiological situation and the spread of multidrug-resistant (MDR) clones in hospitals and community set-tings, resistant to the most important classes of oral antibiotics, are inducing clinicians to reconsider older antibiotics, such as fosfomycin trometamol, for the treatment of UTIs caused by MDR uropathogens. Fosfomycin trometamol, a phosphonic acid derivative that inacti-vates UDP-N-acetylglucosamine-3-enolpyruvyltransferase, the enzyme responsible for ligating phosphoenolpyruvate to the 3= -hydroxyl group of UDP-N-acetylglucosamine in the ﬁrst step of peptidoglycan synthesis, is approved in several countries for the treatment of uncomplicated UTIs and prophylaxis of urological ma-noeuvres. Fosfomycin is very active in vitro against common uropathogens, including ESBL-producing strains; in addition, the sus-ceptibility of these pathogens to fosfomycin remained relatively stable over time[6].

The aim of this study was: (1) to evaluate the in vitro activity of fosfomycin against recently isolated MDR uropathogens; (2) to compare the in vitro activity of fosfomycin with that of other com-monly used oral antibiotics for UTIs; and (3) to evaluate susceptibility to fosfomycin using the agar dilution method, the broth microdilution method and the gradient test.

* Corresponding author. Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania, Via Santa Soﬁa n.97, 95123 Catania, Italy.

E-mail address:mezzate@unict.it(M.L. Mezzatesta).

http://dx.doi.org/10.1016/j.ijantimicag.2017.01.020

International Journal of Antimicrobial Agents 49 (2017) 763–766

Contents lists available atScienceDirect

International Journal of Antimicrobial Agents

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2. Materials and methods

In total, 106 clinical midstream and urinary catheter isolates of ESBL-positive E. coli (n= 24), Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (n= 56), P. mirabilis (n = 10) and meticillin-resistant S. saprophyticus (n= 16), collected in 2014, were tested at the Department of Microbiology, University of Catania, Italy. All isolates were identiﬁed by conventional methods.

Phenotypic screening for the presence of ESBLs and carbapenemases in E. coli and K. pneumoniae strains, respectively, was performed according to the criteria of the Clinical Laboratory Standards Institute (CLSI) using a commercial synergy test (Rosco Diagnostica, Taastrup, Denmark). In order to characterize the re-sistance proﬁles of these strains fully, ampliﬁcation and sequencing for detection of ESBLs and carbapenemases was performed using previously described primers[7,8]. Meticillin resistance in staph-ylococci was evaluated by the cefoxitin disc diffusion method and correlated with the presence of the mecA gene.

Fosfomycin antimicrobial susceptibility testing was performed according to the agar dilution method, the broth microdilution method and the gradient test described by CLSI. The inoculated plates were incubated in ambient air at 35 °C for 16–18 h. Mueller Hinton agar and broth supplemented with 25 μg/mL of glucose-6-phosphate (Sigma Aldrich Co, Milan, Italy) were used for susceptibility testing by the agar dilution and broth microdilution methods, respective-ly. The gradient test was performed on Mueller Hinton agar with strips containing fosfomycin and glucose-6-phosphate (Lioﬁlchem, Roseto degli Abruzzi, Italy).

All other antibacterial agents (nitrofurantoin, co-trimoxazole, amoxicillin/clavulanic acid, cefuroxime, levofloxacin and ciprofloxacin) were tested using the gradient test alone (Liofilchem). Fosfomycin was supplied by Zambon SpA, Bresso (Milan, Italy). Com-mercial products were used for all other antibiotics.

E. coli ATCC 25922 and S. aureus ATCC 25923 were used as the quality control strains.

Breakpoints of fosfomycin and other antibiotics for the inter-pretative criteria for clinical isolates were used according to the European Committee on Antimicrobial Susceptibility Testing Guide-lines Version 5.0 for isolates of Enterobacteriaceae. The fosfomycin

intravenous breakpoint, established by EUCAST 2015 for Staphylo-coccus spp. (32 mg/L), was applied to S. saprophyticus. Susceptibility results for fosfomycin, evaluated by the gradient test, were com-pared with those for the other antibiotics tested. The susceptibility results for fosfomycin obtained with the broth microdilution and gradient test methods were compared with those obtained from the agar dilution method (the latter was used as the reference method). Agreements and discrepancies between the evaluated and refer-ence methods were classiﬁed as described previously[9].

3. Results

In vitro activity of fosfomycin and comparative oral antimicro-bial agents, measured by the gradient test, against the 106 clinical urinary isolates of uropathogens, is shown inTable 1. Fosfomycin minimum inhibitory concentration (MIC) distribution data and sus-ceptibility rates of fosfomycin using the agar dilution method for all isolates are shown inTable 2.

According to the selected breakpoints, fosfomycin inhibited all of the ESBL-positive E. coli, P. mirabilis and meticillin-resistant S. saprophyticus strains isolated from urine (Table 1).

Fosfomycin was slightly less active against KPC-producing K. pneumoniae (82% susceptibility rate by gradient test method). Ni-trofurantoin showed good activity against ESBL-positive E. coli and meticillin-resistant S. saprophyticus strains (92% and 100% suscep-tibility rates, respectively), but was not effective against other tested Enterobacteriaceae (P. mirabilis and KPC-producing K. pneumoniae). On the contrary, co-trimoxazole was active against P. mirabilis and meticillin-resistant S. saprophyticus (80% and 100% susceptibility rates, respectively), but its antibacterial action against the other two MDR Enterobacteriaceae was lower. Finally, P. mirabilis alone was sus-ceptible to beta-lactams (amoxicillin/clavulanic acid, cefuroxime) and fluoroquinolones (levofloxacin, ciprofloxacin).

Table 3shows the in vitro antibacterial activity of fosfomycin against urinary isolates according to the three test methods. Looking at susceptibility rates, substantial agreement was observed between the three methods, particularly for Enterobacteriaceae. A higher vari-ability of results was detected for meticillin-resistant S. saprophyticus

Table 1

In vitro antibacterial activity of antimicrobial agents by gradient test method against 106 urinary isolates.

Species Susceptibility rate (%)

n Fosfomycin Nitrofurantoin Cotrimoxazole Amoxicillin/ clavulanic acid

Cefuroxime Levoﬂoxacin Ciproﬂoxacin

MIC90 %S MIC90 %S MIC90 %S MIC90 %S MIC90 %S MIC90 %S MIC90 %S

ESBL Escherichia coli 24 4 100 32 92 >32 50 >256 50 >256 0 >32 0 >32 0

KPC Klebsiella pneumoniae 56 48 82 >512 28 >32 32 >256 0 >256 0 >32 0 >32 0

Proteus mirabilis 10 1 100 >512 0 0.094 80 1 100 1.5 100 0.125 80 0.02 100

Meticillin-resistant Staphlococcus saprophyticus

16 12 100 12 100 1 100 4 NA 16 NA >32 50 >32 50

ESBL, extended-spectrum β-lactamase-producing; MIC90, minimum inhibitory concentration required to inhibit the growth of 90% of organisms; %S, percentage susceptibility.

Table 2

Minimum inhibitory concentration (MIC) distribution and susceptibility rates of fosfomycin by the agar dilution method.

Species No. of isolates No. of isolates with MIC (mg/L) MIC50 MIC90 %S

0.50 1 2 4 8 16 32 64 128

ESBL Escherichia coli 24 16 6 2 0.50 1 100

KPC Klebsiella pneumoniae 56 2 8 14 24 2 6 32 128 78

Proteus mirabilis 10 6 2 2 0.50 2 100

Meticillin-resistant Staphlococcus saprophyticus 16 2 4 4 2 4 16 128 75

ESBL, extended-spectrum β-lactamase-producing; MIC50, minimum inhibitory concentration required to inhibit the growth of 50% of organisms; MIC90, minimum inhibi-tory concentration required to inhibit the growth of 90% of organisms;%S, percentage susceptibility.

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isolates, where the susceptibility rate ranged from 75% with the agar dilution method to 100% with the gradient test. The results of the gradient test and broth microdilution method were compared with those obtained from the agar dilution method (reference method) to evaluate their feasibility to characterize fosfomycin susceptibil-ity. In cases of ESBL-positive E. coli and P. mirabilis, no evaluation errors were observed; on the contrary, some errors were found with respect to the reference method for KPC-producing K. pneumoniae and meticillin-resistant S. saprophyticus. In particular, the very major error (VME) rate for the broth microdilution method and the gra-dient test for KPC-producing K. pneumoniae was 14.3%: eight strains showed resistance to fosfomycin by the agar dilution method (MIC values 64–128 mg/L) and susceptibility to fosfomycin by the broth microdilution method and the gradient test (MIC values 16–32 mg/ L). Two strains of S. saprophyticus had MIC values of 128 mg/L by the agar dilution method and MIC values of 32 mg/L by the broth microdilution method (VME 12.5%), and four strains had MIC values of 128 mg/L by the agar dilution method and 16–32 mg/L by the broth microdilution method (VME 25%).

4. Discussion

The antibiotics currently recommended by national and inter-national guidelines for the treatment of uncomplicated UTIs are fosfomycin trometamol, pivmecillinam and nitrofurantoin mono-hydrate. Co-trimoxazole, ﬂuoroquinolones and beta-lactams can be prescribed according to local epidemiology of resistance[2,10]. Mecillinam was not tested because it is not available in Italy; however, a representative oral penicillin, amoxicillin/clavulanic acid, was tested in this study. A very recent European survey on antimi-crobial resistance of E. coli causing uncomplicated UTIs showed that the susceptibility rates of this pathogen against mecillinam and amoxicillin/clavulanic acid are equivalent[11]. However, the current epidemiological situation in Italy and in most European countries, where the spread of MDR Enterobacteriaceae is increasing[12], dis-courages the empirical use of co-trimoxazole, ﬂuoroquinolones and beta-lactams for the treatment of these infections[5]. For this reason, fosfomycin trometamol has been recommended for the treatment of uncomplicated UTIs caused by ESBL-producing E. coli and other MDR uropathogens[6,13].

In the authors’ experience, fosfomycin was the only tested an-tibiotic found to be fully active against the most important MDR Enterobacteriaceae responsible for UTIs. In fact, 100% of recently (2014) obtained urinary isolates of ESBL-positive E. coli and P. mi-rabilis were susceptible to fosfomycin; fosfomycin also demonstrated

marked activity against carbapenemase-producing K. pneumoniae (78% by agar dilution reference method). The present results dem-onstrate that fosfomycin is very active against meticillin-resistant S. saprophyticus (75%)[13], although intrinsically resistant (Table 4, v3.1 – EUCAST 2016). On the contrary, nitrofurantoin showed good activity against ESBL-positive E. coli and meticillin-resistant S. saprophyticus strains alone. Other than antibacterial activity, fosfomycin and nitrofurantoin also differ in terms of pharmacoki-netics: a single dose of fosfomycin trometamol is able to reach a very high urinary concentration[6], whereas the urinary levels of nitrofurantoin are lower[13,14]. Finally, these in vitro data confirm that co-trimoxazole, fluoroquinolones and beta-lactams can no longer be used empirically for the treatment of UTIs caused by MDR uropathogens in Italy. These findings are consistent with those pre-sented previously. The very recent systematic review by Vardakas et al. evaluated the susceptibility of contemporary isolates (2010– 2015) to fosfomycin[14]. The results showed that fosfomycin susceptibility ranged from 81% to 100% [frequency 95.1%, 95% con-fidence interval (CI) 94.3–95.9%] for ESBL-producing E. coli, from 15% to 100% (frequency 83.8%, 95% CI 78.7–89.4%) for ESBL-producing K. pneumoniae, and from 39.2% to 100% (frequency 73.5%, 95% CI 66.4–81.4%) for carbapenemase-producing K. pneumoniae[14,15]. Other recent reports confirmed the in vitro activity of fosfomycin against ESBL-producing uropathogens, as well as carbapenemase-producing Enterobacteriaceae, suggesting that, guided by local susceptibility data, fosfomycin could be considered for the treat-ment of patients with infections due to problematic MDR bacteria

[15,16].

In terms of susceptibility testing of fosfomycin, discrepancies have been reported between broth and agar dilution MICs for fosfomycin, and, to date, agar dilution is the only method approved for testing fosfomycin MIC susceptibility (CLSI 2014). The study results showed that the three methods tested (agar dilution method, broth microdilution method and the gradient test) are suitable for testing the susceptibility of fosfomycin against ESBL-positive E. coli and P. mirabilis. When carbapenemase-producing K. pneumoniae and meticillin-resistant S. saprophyticus are susceptible to fosfomycin, with MICs of 16–32 mg/L obtained by the broth microdilution method and the gradient test, the results for resistance should be conﬁrmed by the reference method (agar dilution).

Other studies evaluated different susceptibility methods for evalu-ating the in vitro activity of fosfomycin against MDR pathogens. Lu et al. evaluated the antimicrobial activity of fosfomycin against 960 strains of common uropathogens (Enterobacteriaceae, Pseudomo-nas aeruginosa, Acinetobacter baumannii, StenotrophomoPseudomo-nas

Table 3

In vitro antibacterial activity of fosfomycin against 106 urinary isolates using three test methods, and agreement of the broth microdilution method and the gradient test with the reference (agar dilution) method.

Species Fosfomycin

n Agar dilution Broth microdilution Gradient test Agreement

MIC90 %S MIC90 MBC90 %S MIC90 %S

Gram-negative bacteria 90 No. (%) of isolates

ESBL Escherichia coli 24 1 100 8 8 100 4 100 Methods EA A VME ME

Broth microdilution 2 (8.3) 6 (25) 0 0

Gradient test 14 (58.3) 6 (25) 0 0

KPC Klebsiella pneumoniae 56 128 78 32 64 89 48 82 Broth microdilution 32 (57.1) 16 (28.6) 8 (14.3) 0

Gradient test 34 (60.7) 12 (21.4) 8 (14.3) 6 (10.7)

Proteus mirabilis 10 1 100 4 4 100 1 100 Broth microdilution 6 (60) 0 0 0

Gradient test 8 (80) 0 0 0 Gram-positive bacteria 16 Meticillin-resistant Staphylococcus saprophyticus 16 128 75 32 32 88 12 100 Broth microdilution 12 (75) 4 (25) 2 (12.5) 0 Gradient test 0 2 (12.5) 4 (25) 0

ESBL, extended-spectrum β-lactamase-producing; MIC90, minimum inhibitory concentration required to inhibit the growth of 90% of organisms; %S, percentage suscepti-bility; EA, essential agreement; A, agreement; VME, very major error; ME, major error.

765 M.L. Mezzatesta et al. / International Journal of Antimicrobial Agents 49 (2017) 763–766

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maltophilia, meticillin-susceptible and -resistant Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium) using stan-dard agar dilution and disc diffusion methods. E. coli was uniformly susceptible to fosfomycin, as were most strains of K. pneumoniae and Enterobacter cloacae. Comparison of the two methods showed that the disc diffusion test can be considered an alternative test to evaluate the susceptibility of these species to fosfomycin [17]. Another in vitro study compared the agar dilution, broth microdilution and disc diffusion methods to establish the suscep-tibility of 428 ESBL-positive E. coli and K. pneumoniae to fosfomycin. The results demonstrated the very high activity of fosfomycin against all ESBL-producing strains, and showed excellent agree-ment between the three susceptibility methods for E. coli. On the contrary, marked discrepancies were observed for K. pneumoniae; in fact, the results of susceptibility testing showed poor correla-tion for both the broth microdilucorrela-tion method and the disc diffusion method with the agar dilution method, reporting greater resis-tance to fosfomycin. When the broth microdilution or disc diffusion methods are used[18,19], it has been suggested that the results should be conﬁrmed using the reference method (agar dilution) for resistance and intermediate susceptibility of K. pneumoniae to fosfomycin.

Finally, Endimiani et al. conﬁrmed that the result of fosfomycin susceptibility testing is dependent on the method used and the micro-organisms tested, and that the gradient test and the broth microdilution method are not reliable ways to test fosfomycin MICs against some MDR strains, such as K. pneumoniae[20].

Despite the well-known problem of rapid development of fosfomycin resistance under experimental conditions, resistance in clinical isolates is still rare. Many authors have addressed this feature and many hypotheses have been proposed[13]. One of most accredited ideas is that fosfomycin-resistant mutants are unable to establish themselves in the bladder due to their lowered ﬁtness, as demonstrated by experimental data and mathematical models

[21].

In conclusion, the study data suggest that fosfomycin has good in vitro activity against more frequently isolated MDR uropathogens, namely ESBL-positive E. coli, KPC-producing K. pneumoniae, P. mi-rabilis and meticillin-resistant S. saprophyticus, suggesting that fosfomycin trometamol may be a reliable empirical therapeutic option for uncomplicated UTIs caused by these organisms, and a valid option for sparing parenteral antibiotics, such as carbapenems. Furthermore, the broth microdilution method and the gradient test can be considered alternative methods to determine fosfomycin sus-ceptibility of ESBL-positive E. coli and P. mirabilis alone, whereas the agar diffusion method is the most reliable test against all uropathogens.

Acknowledgements

The authors wish to thank the Scientiﬁc Bureau of the Univer-sity of Catania for language support.

Funding: This study was supported by a research grant from Zambon SpA, Bresso, Italy.

Competing interests: None declared. Ethical approval: Not required.

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