| Popis: |
BACKGROUND AND AIM The patient was a 30 year old male, who inhaled sewage during work due to the rupture of the sewage pipeline. He developed bacillary dysenthery with frequent stools mixed with blood and mucus and was hospitalized in Sestre Milosrdnice Hospital in Zagreb, Croatia. He was released after 1 day without antibiotic therapy. Shigella sonnei was isolated from the stool specimen. MATERIAL AND METHODS The antibiotic susceptibility testing to a wide range of antibiotics was performed by disk- diffusion and broth microdilution method. Double disk synergy test and combined disk test with clavulanic acid were done to determine the production of an extended-spectrum beta-lactamase (ESBL). The transferability of cefotaxime and ciprofloxacin resistance was done by broth mating method at 35°C employing E. coli J65 recipient strain resistant to sodium-azide. The transconjugants were selected on MacConkey agar containing either ampicillin (0.5 mg/L) or ciprofloxacin (0.5 mg/L) and sodium azide (100 mg PCR was applied to detect genes encoding ESBLs(blaSHV, blaTEM, blaCTX-M, blaOXA-9, blaOXA-1 and blaPER-1) and fluoroquinolone resistance (qnrA, qnrB, qnrS). The CTX-M β-lactamase cluster was detected with multiplex PCR including five primer pairs: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. The genetic context of blaCTX-M genes was determined by PCR mapping with forward primer for insertion sequences ISEcp1 and IS26 combined with primer MA-3 (universal reverse primer for blaCTX-M genes) Amplicons were visualised after electrophoresis in 1 % agarose by staining it with ethidium bromide. WGS was done using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) according to the manufacturer's instructions. Plasmids were extracted from ampicillin resistant donor strains and their respective transconjugants with Qiagen plasmid mini kit according to the manufacturer’s instructions (Hilden, Germany) PCR-based replicon typing (PBRT) was applied to determine the plasmid content of the tested strains RESULTS The strain exhibited resistance to amoxicillin, cephalexin, cefuroxime, azythromycin, tetracycline, cefotaxime, ceftriaxone, and ciprofloxacin but remained susceptible to ceftazidime, cefepime, amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem and gentamicin. Cotrimoxazole and cefoxitin tested resistant and susceptibile in disk-difusion test with the inhibition zones of 6 and 25 mm, respectively. Phenotypic tests confirmed production of an ESBL. PCR identified blaCTX-M gene belonging to the cluster 1. WGS demonstrated a variety of aminoglycoside (aadA, aph(6), aph(3), β-lactam (blaCTX-M-27), macrolide (Erm(B, ) Mph(A)), sulphonamide (sul 1), trimethoprime (dfrA17, dfrA) and tetracycline (tet A). Four different plasmids were found: IncFIB, IncFII, IncI1 and IncCol. The isolate was found to belong to the widespread ST152 which is dominant in Europe and North America and usually associate with MSM population. CONCLUSIONS To our best knowledge, this is the first report of an ESBL in Shigella spp from Croatia. This allelic variant was recently described in S. sonnei in UK and Australia. Plasmid-mediated resistance determinants to fluoroquinolones were not found and thus it is very likely that ciprofloxacin resistance was associated with mutations of gyrA and parC genes. This report emphasizes the ability of S. sonnei belonging to the widespread ST 152 to accumulate various resistance determinants. |
