Presence of bla_TEM-116 Gene in Environmental Isolates of Aeromonas Hydrophila and Aeromonas Jandaei from Brazil

Abstract

It is known that Aeromonas spp. possess different chromosomal β-lactamase genes. Presence and phenotypic expression of bla_TEM, bla_SHV, and bla_CTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of bla_SHV and bla_CTX-M genes was not observed, and bla_TEM gene was verified in 91% of the isolates. Sequencing of 10 fragments showed the occurrence of bla_TEM-116.

Members of the genus Aeromonas have been associated with a wide range of illnesses in humans, including gastrointestinal disorders and systemic infections in both immunocompromised and healthy hosts (4). Several studies have shown the presence of Aeromonas spp. in food and drinking water samples, suggesting that these sources may act as dissemination vehicles of the human pathogen, with implications in the public health. Furthermore, it is known that Aeromonas spp. are among the few microorganisms harboring different chromosomal β-lactamase genes, including cphA (also named imiH), cepH and ampH, encoding class B, C and D β-lactamases, respectively (1).

Antibiotic resistance has been classified by the World Health Organization as one of the three major public health threats of the 21st century (6). The rapid emergence of antibiotic resistance among bacteria is, to a great extent, due to the dissemination of antibiotic resistance genes by horizontal transfer mediated by plasmids, transposons and integrons (5). Among the clinical populations of Gram-negative microorganisms, bla_TEM-1 is the most frequently detected antimicrobial resistance gene and, although its expression results in penicillin resistance, diverse point mutations in the bla_TEM-1 gene have contributed to the emergence of TEM-type extended-spectrum β-lactamases (ESBLs), resulting in simultaneous resistance to penicillins and broad-spectrum cephalosporins (8). Although, almost all previous studies and efforts to control the dissemination of these genes have been based on isolates from clinical samples, antibiotic resistance genes can also occur in nonpathogenic bacteria, which can then be transferred via lateral gene transfer (6).

The aim of this study was to investigate the presence and phenotypic expression of bla_TEM, bla_SHV, and bla_CTX-M ESBL-encoding genes in 87 environmental water isolates of Aeromonas hydrophila (n=41) and Aeromonas jandaei (n=46). The identification of Aeromonas species was performed as previously described (1). All isolates were screened for ESBL production by a double-disc synergy test using clavulanic acid (amoxicillin-clavulanate disk, 20/10 µg) and cloxacillin (200 µg/ml-containing Mueller-Hinton agar plates) as ESBL and AmpC inhibitors, respectively, and ceftazidime, cefpodoxime and cefotaxime-containing disks as ESBL substrates (9). Presence of ESBL-encoding genes was evaluated by PCR (2), and plasmid extraction was carried out using the commercial kit Wizard Plus SV Miniprep (Promega-USA). Also the search for class 1 integron was carried out according to previous work (7).

Although production of ESBL was not confirmed by phenotypic methods, and no amplification of the bla_SHV and bla_CTX-M genes was observed, the presence of the bla_TEM gene was verified in 97.6% (40/41) and 85% (39/46) of A. hydrophila and A. jandaei isolates, respectively. Nucleotide sequencing showed 100% sequence identity with the bla_TEM-116 gene (GenBank accession numbers FJ767900 to FJ767909). Plasmids were found in 24.4% (10/41) of A. hydrophila and in 34.9% (16/46) of A. jandaei isolates, suggesting no association between plasmid occurrence and presence of bla_TEM genes. Also the association of bla_TEM genes with the occurrence of class 1 integrons was not observed.

The present work raises a question concerning the possible origin of bla_TEM genes and their dissemination among environmental isolates. According to the data shown herein, the presence of these genes could not be associated to the occurrence of plasmids, suggesting a chromosomal location in A. hydrophila and A. jandaei isolates in Brazil. On the other hand, the bla_TEM-116 gene variant is closely related to the bla_TEM-1 gene, which does not possess ESBL activity. In this work, the Aeromonas species harboring the bla_TEM-116 gene did not showed ESBL activity. In fact, this absence of ESBL activity was also observed in a bla_TEM-116 gene-carrying Klebsiella pneumoniae strain isolated in a teaching hospital in São Paulo, Brazil (3).

In conclusion, these data suggest that presence of bla_TEM-like genes in Aeromonas species recovered from natural water reservoirs could be intrinsic. Thus, risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view. Finally, additional investigation about plasmidial or chromosomal occurrence of bla_TEM-116 in environmental isolates of Aeromonas is worthy of evaluation.