Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil

Abstract

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the bla _CTX-M was the most prevalent (n = 12 positive isolates), followed by bla _TEM (n = 5) and bla _SHV (n = 4). bla _CTX-M-15 (n = 5), bla _CTX-M-14 (n = 1) and bla _CTX-M-2 (n = 1) variants were detected in conserved genomic contexts: bla _CTX-M-15 flanked by ISEcp1 and Orf477; bla _CTX-M-14 flanked by ISEcp1 and IS903; and bla _CTX-M-2 associated to an ISCR element. For 4 strains the transfer of bla _CTX-M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. bla _CTX-M-15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., bla _OXA-208 and bla _ADC-like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.

Keywords: Acinetobacter baumannii; Escherichia coli; antibiotic resistance; blaCTX–M; whole genome analysis.

FIGURE 1

Map of the Utinga State Park (dark gray area). Sampling points are identified as S1, S2, S3, S4, S5, and S6. The urban area of Belém is represented by the gray area in the upper left half of the map. Therefore, the city is closer to the sampling points S1, S2, S5, and S6.

FIGURE 2

Synteny analysis of ISEcp1 element carrying bla_CTX–M–15 gene in Escherichia coli APC43A. Conserved genes are connected by lines. The pink arrow represents the ISEcp1 element that is described in detail at the bottom of the figure. E. coli APC43A contains two CDSs identified as MATE efflux family proteins flanking the IS element. These CDSs are complementary and probably are part of the same gene that was fragmented during the insertion of the IS element.

FIGURE 3

Comparative genomic ring designed in Gegenees program. The innermost ring to the outermost is presented in this figure, as follows: GC skew and the GC content of E. coli APC43A; the genome sequence of E. coli APC43A and E. coli K-12; the Resistance Islands (RIs) detected by GIPSy; the Pathogenicity Islands (PAIs) detected by GIPSy; CDSs identified in the genome of E. coli APC43A. The location of the resistance genes detected by CARD and ResFinder are shown and identified by their respective names in red.

FIGURE 4

Comparative genomic ring designed in Gegenees program. The innermost ring to the outermost is presented in this figure, as follows: GC skew and the GC content of Acinetobacter baumannii APC25; the genome sequence of A. baumannii APC25 and A. calcoacetius CA16; the Resistance Islands (RIs) detected by GIPSy; the Pathogenicity Islands (PAIs) detected by GIPSy; CDSs identified in the genome of A. baumannii APC25. The location of the resistance genes detected by CARD and ResFinder are shown and identified by their respective names in red.