Determination of susceptibility to fosfomycin and tigecycline of Enterobacteriaceae , particularly Escherichia coli isolates , producing extended-spectrum β-lactamases from multiple regional Canadian hospitals

BACKGROUND
The worldwide spread of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, particularly Escherichia coli, has significantly limited therapeutic options, especially for urinary tract infections. Although limited in their indications, fosfomycin and tigecycline are potential agents to treat infections due to ESBL-producing organisms. Although not routinely performed, susceptibility testing to both is necessary to ensure there is not an increase in resistance.


METHODS
A total of 160 isolates of ESBL-producing E coli were isolated from patients at multiple regional hospitals in the Interior Health Region of British Columbia from June 2009 to January 2012. Isolates were obtained from various body fluids and sites including urine (78.2%), wounds, blood, gall bladder drain and respiratory specimens. All isolates were tested using the E-test method (Etest, bioMérieux, France) for tigecycline and Kirby Bauer disk diffusion method for fosfomycin using European Committee of Antimicrobial Susceptibility Testing breakpoints for tigecycline and Clinical and Laboratory Standards Institute zone sizes for fosfomycin.


RESULTS
All 160 isolates were found to be susceptible to tigecycline, while five isolates (3.1%) were resistant to fosfomycin (four resistant, one intermediate).


CONCLUSION
Although resistance to these antibiotics has previously been reported, the present study confirmed that isolates of ESBL-producing E coli from the Interior Health Region of British Columbia remain highly susceptible to both tigecycline and fosfomycin.


Susceptibility of Enterobacteriaceae to fosfomycin and tigecycline
Can J Infect Dis Med Microbiol Vol 24 No 3 Autumn 2013 e81 potential option for intra-abdominal or wound infections involving ESBL-producing Escherichia coli.The use of these agents is further complicated because automated susceptibility panels in Canada typically do not test for resistance to these antibiotics and, hence, offline manual testing is required.Susceptibility testing is not typically performed because of cost and reports indicating that ESBL-producing Enterobacteriaceae are susceptible to these two antibiotics, fosfomycin and tigecycline (3).Therefore, the need to understand susceptibility patterns to these agents for more resistant organisms (ie, ESBLs) in the Interior Health Region of British Columbia (BC) is vital.Due to the emerging threat of Enterobacteriaceae with ESBLs causing infections in hospitals worldwide, it is a benefit to regional hospitals in the BC interior and across Canada to monitor the presence of ESBL-producing Enterobacteriaceae from infected patients.Specifically, results of the present study may help determine the effectiveness of using fosfomycin or tigecycline as an alternative choice to treat infections caused by ESBL-producing bacteria.

MEtHoDS
A total of 160 isolates of ESBL-producing E coli were collected from multiple regional hospitals in the Interior Health region of BC (Royal Inland Hospital, Kelowna General Hospital, Vernon Jubilee Hospital and Penticton Regional Hospital) from June 2009 to January 2012.Isolates were obtained from various body fluids and sites including urine (78.2%), wounds, blood, gall bladder drain and respiratory specimens.All isolates were tested using the E-test method (Etest, bioMérieux, France) for tigecycline and Kirby Bauer disk diffusion method for fosfomycin using European Committee of Antimicrobial Susceptibility Testing (EUCAST) breakpoints for tigecycline and Clinical and Laboratory Standards Institute (CLSI) zone sizes for fosfomycin.
All isolates were identified as ESBL-producing Enterobacteriaceae using Vitek Gram Negative identification (AST N96 panels, bioMérieux, France).Confirmatory testing for ESBL-producing E coli was performed using MAST discs (MASTDISCS D68C, United Kingdom).
For the susceptibility testing of fosfomycin and tigecycline, all isolates were subcultured on Mueller-Hinton plates and incubated at 35°C; the density of the culture was equalized to a 0.5 McFarland standard.All isolates were tested using the E-test method for tigecycline and Kirby Bauer disk diffusion method for fosfomycin using EUCAST breakpoints for tigecycline and CLSI zone sizes for fosfomycin (4)(5)(6).Quality control testing was performed using E coli (American Type Culture Collection 25923) for the E-test and disk diffusion test.

rESultS
All 160 isolates of ESBL-producing E coli were susceptible to tigecycline, while five isolates demonstrated resistance to fosfomycin (four resistant and one intermediate).These results demonstrated that overall, in Interior Health, there was 3.1% resistance to fosfomycin.

DISCuSSIoN
In the past 10 years, there has been a steady increase in the number of ESBL-producing Enterobacteriaceae resistant to antibiotics typically prescribed to treat UTIs (1)(2)(3)7).This has been an alarming problem because resistance is increasing at a faster rate than the development of antibiotics.More importantly, this is of great concern to clinicians because new antibiotics are needed to overcome and avoid β-lactamase activity.A study conducted in Spain in 2005 determined that of 428 E coli isolates producing ESBL, many from UTIs, only 0.3% of E coli were resistant to fosfomycin (3).ESBL-producing Enterobacteriaceae have been shown to be susceptible to tigecycline.In a study by Fritsche et al (7), 95.7% of ESBL-producing Enterobacteriaceae isolates isolated from the urinary tract, bloodstream infections, respiratory tract, skin tissue and gastrointestinal tract were susceptible to tigecycline at ≤2 μg/mL.This study included isolates from Europe, North American and Latin America.Unlike tetracyclines, tigecycline is able to overcome drug-specific efflux pump acquisition and ribosomal modification (7).Our finding correlates with a number of studies published in that there is no resistance to tigecycline observed in ESBL-producing Enterobacteriaceae.There has been little resistance observed throughout the world, with the highest resistance rate observed in Spain (97.5% susceptibility) (8).There has also been little resistance to fosfomycin observed in ESBLproducing Enterobacteriaceae (8).Our data supports these reports, with 96.9% susceptibility to fosfomycin.The highest resistance rate was observed in Japan, with 73% susceptibility (8).This is a high resistance rate compared with the literature, with most studies having a recorded susceptibility rate of 90% to 100%.Our observed resistance rate fits into this range.
The increase in the prevalence of ESBL-producing Enterobacteriaceae has necessitated need for alternative therapeutic agents.Figure 1 illustrates the resistance patterns of tigecycline and fosfomycin observed in countries around the world (8)(9)(10)(11)(12).The susceptibility rates ranged from 73% to 100% for fosfomycin, and 97.5% to 100% for tigecycline.

CoNCluSIoN
ESBL-producing E coli remain highly susceptible to fosfomycin and tigecycline.These agents may be considered in the management of infections due to these organisms.Microbiology laboratories should consider performing susceptibility testing of these agents for multiresistant isolates for which therapeutic options are limited.

aCKNoWlEDgEMENtS:
The authors sincerely thank the staff in the microbiology laboratory at Royal Inland Hospital, Kelowna General Hospital, Vernon Jubilee Hospital and Penticton Regional Hospital for collecting and storing isolates.
Thanks to the Department of Biological Sciences and Faculty of Science at Thompson Rivers University for ongoing support in research.Many thanks to the Interior Health Authority for funding our research and the Comprehensive University Enhancement Fund Travel Fund for funding to attend the Association of Medical Microbiology and Infectious Disease Annual Meeting in Vancouver, British Columbia, May 3 to 5, 2012.They appreciate the help received from Carolynne Fardy in the microbiology laboratory at Thompson Rivers University.This work was also accepted and presented at the 112th American Society of Microbiology (ASM) General Meeting and the 2012 AMMI Annual Meeting student poster presentations.