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First detection of the AmpC beta-lactamase ACC-1 in a Klebsiella pneumoniae isolate in Bulgaria

Plasmid-encoded AmpC enzymes were first described in 1988. Since then, they have been disseminated all over the world. During the last years reports on extended spectrum beta-lactamase (ESBL)-producing isolates in Bulgaria appeared, mostly showing increasing rates of CTX-M-type ESBL producers; however, so far, no plasmidic AmpC enzymes have been described for Bulgaria. During a survey on ESBL-producing Escherichia coli and Klebsiella pneumoniae conducted between 2006 and 2009, the K. pneumoniae isolate AH11-390 drew our attention due to the absence of the inhibitory effect of clavulanic acid. The isolate was recovered in December 2008 from blood of a 70 years old male patient, hospitalized in the surgery ward of the Alexandrovska University Hospital in Sofia, Bulgaria. Species identification was performed by routine biochemical tests. MICs obtained by broth microdilution technique according to the CLSI guidelines are listed in Table 1. In addition, susceptibility to trimethoprim/sulfamethoxazole and chloramphenicol was shown by the diskdiffusion method according to CLSI guidelines. The combination of cefotaxime or ceftazidime with clavulanic acid or ceftazidime with sulbactam showed no effect of the beta-lactamase inhibitors (Table 1). The beta-lactam resistance determinant was not transferable in conjugation experiments with E. coli K12:W3110 lac 2 resistant to rifampicin. Analytical isoelectric focusing indicated two isoelectric points (pIs) of 5.4 and 7.7. The hydrolytic activity of individual beta-lactamase bands was assessed by a bioassay and showed ceftazidime hydrolysis by the beta-lactamase with pI 7.7. The ceftazidime nonhydrolysing enzyme with pI 5.4 was assumed to be TEM-1. The enzyme with pI 7.7 was suspected to be an AmpC beta-lactamase due to the lack of an inhibitory effect of clavulanic acid. However, AmpC betalactamases usually confer resistance to cefoxitin, except for the enzymes of the ACC-group. As our isolate was susceptible to cefoxitin we applied a PCR with ACCtype specific primers (Kiel10, 59-TACCTGTCTGGCAGC-39 and Kiel9, 59-TTCGGCAGAGAAACC-39) which yielded a PCR product of the expected size of 884 bp. For sequencing of the entire gene primers binding outside the coding region (KUS-EcoRI-V, 59CGGAATTCGAGTGTTGCTCTGTG-39and Kiel1V, 59-ATAAGCAAACCCACTAC-39) were used and the PCR-product was subjected to sequencing at Eurofins MWG Operon (Ebersberg, Germany). Analyses of sequences and multiple alignments were performed using Chromas Lite 2.01 (Technelysium Pty Ltd, Brisbane, Australia) and DNAMAN 4.11 Software (Lynnon BioSoft, Vaudreuil-Dorion, Canada). Sequencing revealed 100% identity of the gene with blaACC-1 (AJ133121). Additional 223 bp upstream and 99 bp downstream of the blaACC-1 gene were identical to the environmental sequence of other blaACC genes (AY856832, EF504260, AJ270942) except for one nucleotide exchange. Further PCRs using primers derived from environmental sequences of blaACC-1 genes 5 revealed the presence of the gdha gene encoding a glutamate dehydrogenase downstream from blaACC-1 of K. pneumoniae AH11-390. Upstream from blaACC-1 the insertion sequence ISEcp1 was found. This structure is common to all the blaACC-1 environments analysed by Doloy et al. For K. pneumoniae isolates from France and Tunisia Doloy et al. showed an ISEcp1 truncated by 1433 bp due to the insertion of an IS26. However, this structure could not be found in our isolate. A PCR based replicon typing scheme described by Carattoli et al. was applied and showed the presence of a plasmid of the IncHI1 incompatibility group. As conjugational plasmid transfer was not successful, it remains unproven whether the blaACC-1 gene resides on that IncHI1-type plasmid. Up to now blaACC-1 has been associated with IncI1 replicon type plasmids only. ACC-1 was first described in Germany in a K. pneumoniae isolate, and subsequently it has been shown that this enzyme originated from an AmpC beta-lactamase intrinsic to the species Hafnia alvei. ACC-1 has been found repeatedly in France, where multiresistant ACC-1-producing K. pneumoniae isolates caused an outbreak, as well as in Spain and Tunisia. In the Netherlands, ACC-1 producers have been found from patients, however, also from poultry