Transmissible fosfomycin resistance markers in urinary isolates and imported foodstuffs in the UK during 1994 and 1995.

Sir, There has been great interest in the transfer of antibiotic resistance markers between clinically important bacteria and bacteria present in foodstuffs. We carried out two small studies where fosfomycin resistance was detected in clinical isolates and isolates from foodstuffs during 1994 and 1995. Fosfomycin is a broad-spectrum antibiotic that has been used extensively in European countries since 1973. It is a phosphoenol pyruvate analogue that inhibits phosphoenol pyruvate transferase, an enzyme that catalyses the early stages of peptidoglycan synthesis. Fosfomycin was introduced into the UK market in February 1994 but was withdrawn in 1996 for commercial reasons. Routine screening of clinically significant urinary isolates against fosfomycin during the period September to December 1994 was carried out at the Leeds General Infirmary. Surprisingly, 15 resistant Enterobacteriaceae (six isolates of Morganella morganii, five of Klebsiella pneumoniae, two of Enterobacter spp., one of Providencia rettgeri and one of Klebsiella oxytoca) were identified, despite the previous lack of use of fosfomycin in this region. Fosfomycin resistance in Gramnegative Enterobacteriaceae has been associated with acquisition of the plasmid-mediated fosA gene. Resistance encoded by fosA results from the addition of fosfomycin and the tripeptide glutathione in a reaction catalysed by a dimeric 16 kDa protein FosA. The protein was recently characterized as a metalloenzyme (metalloglutathione transferase). This mechanism of resistance has not been reported previously in the UK. Two of the 15 fosfomycinresistant isolates (both Enterobacter spp.) were found by PCR to carry the fosA gene using the oligonucleotide primers 5 -GCTGACGCTGCACGC-3 and 5 -AGGG CTTCTCGCACAC-3 , designed to hybridize with an internal sequence of fosA, generating a 311 bp product. The reactions were carried out in 25 L volumes containing 0.5 M each primer, 1.5 mM magnesium chloride, 200 M dATP, dCTP, dGTP and dTTP, 1 U Taq DNA polymerase (Promega, Madison, WI, USA) and cell lysate. The reactions were heated at 94 C for 2 min followed by 30 cycles of 93 C for 40 s, 57 C for 40 s and 72 C for 40 s using an ERICOMP thermal cycler (ERICOMP, San Diego, CA, USA). This finding led us to believe that the vehicle for carriage of fosfomycin resistance into West Yorkshire may have been food imported from countries where fosfomycin is used extensively. Thirty-five food samples (17 from Spain, six from Italy, four from Germany, five from France, one from Hungary, one from Austria and one from Portugal)— a mixture of cured meats (20 samples) and vegetables (15 samples)—were bought from delicatessens, market stalls and shops in Leeds during November and December 1995. A 10 g sample of each purchase was emulsified in 100 mL of nutrient broth (Oxoid, Basingstoke, UK) in a stomacher (Lab Blender 80; Merck, Dorset, UK). Pour plates were made using serial dilutions of the food in IsoSensitest agar (Oxoid) supplemented with 10 mg/L vancomycin, 187 mg/L fosfomycin trometamol and 25 mg/L glucose-6-phosphate. Thirteen fosfomycin-resistant isolates (seven Enterobacter spp., two Serratia spp., three K. pneumoniae and one Rahnella acquatilis) were recovered from seven food samples (five vegetable and two cured meat samples). Amplification of the fosA gene was shown in two isolates (Enterobacter sakazakii and K. pneumoniae). Both of these samples originated in Spain and isolates were recovered from the skin of a courgette and a tomato, respectively. Transfer of the fosfomycin resistance markers was attempted for all the fosfomycin-resistant food isolates and the two clinical urinary isolates that carried fosA. Escherichia coli strains UB1637 and UB5201 were used as recipients in conjugation experiments using a plate-mating method. Transconjugants were selected on IsoSensitest agar containing 25 mg/L nalidixic acid and 187.6 mg/L fosfomycin trometamol potentiated with 25 mg/L glucose6-phosphate. We were unable to demonstrate transfer of fosfomycin resistance from the clinical urinary isolates. However, four of the 13 fosfomycin-resistant isolates from Correspondence Journal of Antimicrobial Chemotherapy (2001) 48, 735–748