Ceftazidime resistance in a Salmonella enterica serotype Bareilly in Ireland N.DeLappe, D Morris, C.O’Hare, E.Costello, G.Corbett-Feeney and M.Cormican. 1 Department of Bacteriology, NUI, Galway 2 Interim National Salmonella Reference Laboratory, UCHG, Galway

Abstract Antimicrobial resistance in Salmonella enterica is a cause of increasing concern. In many cases resistance to multiple agents is observed, with resistance to five antimicrobials (ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline) or more, now frequently observed in Salmonella Typhimurium. More recently, reduced susceptibility to flouroquinolones and resistance to the third generation cephalosporins have been observed in a number of European countries and in the USA. Resistance to third generation cephalosporins in Salmonella has not been previously reported from Ireland. On 13th March 2000, an isolate of Salmonella enterica serotype Bareilly (O6,7 H-y,5) was isolated from a sample of coastal water (E.coli count 11/100ml). The isolate was resistant to ceftazidime, cefuroxime, cephalothin, cefpodoxime, ampicillin, intermediate to ceftriaxone, and susceptible to aztreonam, imipenem and cefoxitin. The pattern of resistance to  -Lactam antibiotics is most consistent with the production of an Extended Spectrum  -Lactamase and this is supported by the results of the confirmatory broth microdilution test of the National Committee for Clinical Standards (NCCLS). The isolate was sensitive to all other classes of antibiotic tested (chloramphenicol, streptomycin, sulphonamides, tetracyline, trimethoprim, nalidixic acid, kanamycin,

ciprofloxacin, nitrofurantoin, gentamicin, minocycline and spectinomycin). This is the first report of resistance to extended spectrum cephalosporins in Salmonella enterica from Ireland. The detection of the isolate in coastal waters suggests that the organism is capable of survival and expression of antimicrobial resistance after exposure to the environment for extended periods. There may be potential for transfer of such strains to humans in the food chain through contamination of shellfish, or through ingestion of bathing water during recreational pursuits. Conjugation studies showed that the resistance determinant is transferable to other strains of salmonella.

Introduction Salmonella enterica Bareilly was first isolated in India in 1928 and is mainly associated with the Indian sub-continent where it is the third most prevalent serotype. It is an uncommon isolate in Ireland and the U.K., of 66 isolates identified in the PHLS laboratory in Colindale since January 1999 the vast majority were associated with foreign travel, especially to India. It has also been isolated from various imported foodstuffs including fish from Bangladesh, chilli powder and corianders. Since 1991, Salmonella species resistant to extended-spectrum cephalosporins have been reported in several countries, but not previously in Ireland. Since these are the drug of choice for invasive Salmonella disease in persons under 18 years, the presence of ESBL-producing Salmonellas pose a serious therapeutic problem. Objective : To determine the mechanism of this isolate’s resistance to ceftazadime and whether or not it is located on a transferable plasmid.

Materials and Methods Source: – Isolated from 1L of filtered bathing seawater Antibiotic Sensitivity Testing performed by –NCCLS disk-diffusion –E-test from AB Biodisk –Double-disk diffusion synergy test –NCCLS broth microdilution Plasmid Analysis : Performed by the method of Kado and Liu. Iso-electric Focussing : Performed using clarified extracts of bacterial suspensions on polyacrylamide gels containing ampholytes. Conjugation : A liquid conjugation using E.coli J53Nal and S.Enteritidis PT 1 (wild strain-Nal res) recipients was carried out. Transconjugants were selected for using MacConkey agar supplemented with ceftazadime (30ug/ml) and Naladixic acid (30ug/ml).

Isolate CTX CTX+Clav CTX+Clav CTX+Clav 4ug/ml 2ug/ml 1ug/ml 871>128 >128 >128 >128 ATCC < 0.25 < 0.25 < >128 < cork-1 64 < S.Bareilly 8 < Table 2: Cefotaxime broth microdilution CTX = Cefotaxime 871 = po 332 = ESBL producer (TEM) Clav = Clavulanate ATCC = ESBL producer 377 = AmpC producer

Isolate CAZ CAZ+Clav CAZ+Clav CAZ+Clav 4ug/ml 2ug/ml 1ug/ml 871>128 >128 >128 >128 ATCC < 0.25 < > cork-1 32 < S.Bareilly 32 < Table 1: Ceftazadime broth microdilution CAZ = Ceftazadime Clav = Clavulanate 871 = po 332 = ESBL producer (TEM) ATCC = ESBL producer 377 = AmpC producer

Results 1. Antibiotic sensitivity testing : Disk diffusion : Caz zone size 13mm Resistant < 15mm E-test MIC : Caz = 32ug/ml Resistant >= 32 Ctx = 4ug/ml Resistant >= 64 Double-disk diffusion synergy test : Synergy not demonstrated. NCCLS broth microdilution : > 4-fold increase in Caz and Ctx MIC with 4ug/ml clavulanate. (Table 1,2) 2. Iso-electric focussing : An ESBL with a PI of 8.1, consistent with SHV-! like enzyme was detected. (Fig.1) 3. Plasmid analysis : A single plasmid > 30 kb was detected. (Fig.2) 4. Conjugation : Liquid mating with both E.coli and S.Enteritidis proved unsucessful. However, solid mating with S.Enteritidis was achieved with a conjugation frequency of 9.5 x 10 . The transconjugants exhibited resistance to ceftazadime.

Conclusion Resistance of this isolate to Ceftazadime is consistent with production of an ESBL. The isolate produces a  -lactamase of pI 8.1 (similar to SHV-1) The strain contains a large (>30kb) plasmid.. Resistance to ceftazadime is transferable by conjugation.

Bibliography 1. Bridges Rf, Scott WM. A new organism causing paratyphoid fever in India, Salmonella type ‘ Bareilly’. J R Army Medical Corps 1931 ;