High-Level Multidrug-Resistant Escherichia coli Isolates from Wild Birds in a Large Urban Environment

Introduction

Since the 2000s, Escherichia coli isolates producing extended-spectrum beta-lactamase (ESBL) of the CTX-M-type have been reported to be associated with clonal E. coli lineages causing human and animal infections.^1–3 ESBL-producing isolates frequently carry co-resistance to other beta-lactam and several non-beta-lactam drugs and pose a major challenge to the management of human and veterinary infections.^4,5

The intensive use of antibiotics in humans and in food animals, together with lack of sanitation, favors the dissemination of ESBL genes in the community, especially in cities with limited public health and sanitation infrastructure resources.^6–8

The proximity of wild birds with an environment contaminated with fecal matter may lead these birds to become colonized with ESBL-producing E. coli.^9,10 Drug-resistant isolates contribute to the dissemination of resistance genes globally via contaminated food products or human travel, whereas colonized nonmigratory birds may disseminate such genes locally. ¹¹ We hypothesized that wild birds living in unsanitary and untreated environments in Brazilian cities are likely to harbor high levels of multidrug-resistant (MDR) bacteria. The aim of this study was to assess the presence of ESBL- or carbapenemase-producing E. coli isolates, or other MDR E. coli isolates, in the intestinal microflora of wild birds rescued in Rio de Janeiro city.

Materials and Methods

Results

We isolated 353 E. coli isolates from 112 (56%) of 199 rescued birds. At least one MDR isolate was obtained from 43 of these birds. The frequency of MDR isolation was 38% considering the subgroup of 112 birds with E. coli isolation or 22% if we took into account the whole collection of 199 birds (Fig. 1). Resistance frequency was 68% among isolates from roadside hawks and black vultures compared with 15% in isolates from blue-fronted parrots, striped owls, and other birds (p < 0.001). Resistance frequencies among the 353 E. coli isolates were 42% to ampicillin, 31% to cefuroxime, 28% to ceftriaxone, 23% to cefotaxime, 20% to cefepime, 14% to trimethoprim–sulfamethoxazole, 13% to ceftazidime, 12% to aztreonam and ciprofloxacin, 8% to nitrofurantoin, 7% to gentamicin, 5% to cefoxitin, 4% to amoxicillin–clavulanate, and 3% to amikacin, and none was resistant to imipenem or ertapenem. In total, 109 isolates were MDR. Table 1 shows resistance profiles of 87 MDR isolates after exclusion of ESBL producers with exclusive beta-lactam resistance. We obtained 41 ESBL-producing isolates from 14 (13%) birds. ESBL-producing E. coli was most frequently found in black vultures (Fig. 1). All ESBL-encoding genes were bla_CTX-M type, as shown in Table 2. Only two birds carried the bla_CMY-2 gene. Additional resistance genes included aac(6′)-lb-cr and qnrB found in three and four different bird species, respectively. No isolate carried the mcr-1 or qepA genes (Table 2).

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FIG. 1.

Distribution of multidrug-resistant Escherichia coli isolates among wild birds. ESBL, extended-spectrum beta-lactamase; MDR, multidrug-resistant non-ESBL; not resistant, isolates susceptible.

Most isolates belonged to E. coli phylogroups B2 and F (N = 58, 53% of the 109 MDR isolates). ERIC-PCR gave a clear profile with at least seven bands for all isolates typed. MLST was performed on 35 (32%) of the 109 MDR isolates; 23 belonged to known clonal complexes (CC) and 12 were singletons. ERIC band profiles of isolates included in the same sequence types had various percent similarities. For example, 13 isolates belonging to ST648 were typed from three birds; only isolates of the same bird belonged to same ERIC type (100% similarity). Clusters of isolates from different birds shared <85% similarities. Two CC predominated: CC648 and CC155, each found in three birds, all MDR isolates. As shown in Table 2, a diversity of clonal lineages and beta-lactamases was observed in a single bird.

Discussion

The present study shows the occurrence of clinically relevant drug-resistance genes in E. coli from wild birds from Rio de Janeiro, the second largest city in Brazil, with an estimated population of 16.6 million in 2016. ²⁴ We found several instances of single birds with E. coli strains belonging to different CC carrying different beta-lactamase genes. In addition, several isolates belonged to phylogroup B2, known as highly pathogenic for humans, and F, which is closely related to B2. These findings support the hypothesis that urban wild birds are heavily colonized with drug-resistant bacteria and may indeed serve as sentinel markers of environment pollution with drug-resistance genes. ¹¹

Two other studies investigated antimicrobial drug resistance among birds in Brazil^25,26; however, neither investigated ESBL production with a phenotypic test, and one of them that looked for ESBL-encoding genes did not find any. ²⁶ However, it is unclear from this study how many birds in total had E. coli or were screened for ESBL genes, making any comparison impossible.

Here, we detected E. coli CC648 from three species of birds, including vultures, hawks, and caracaras. CC648 isolates carried bla_CTX-M15, bla_CTX-M2, and bla_CTX-M8 (including one with bla_CMY-2) and other quinolone and aminoglycoside resistance genes. CC648 represents a novel pandemic clonal lineage found in humans, horses, and wild birds, including a high frequency of MDR isolates.^27–29 CC648 isolates encoding bla_CMY-2 have been frequently described from human sources, but there are reports from companion animals in South Korea and Japan.^30,31 We detected E. coli CC155 in five isolates from three birds, all of them carrying bla_CTX-M. Three isolates were resistant to ciprofloxacin, two carrying qnrB gene, as described by others in Europe. ³² CC10, found in four ESBL-producing isolates from four distinct species of birds, is a CC commonly found among clinical isolates obtained from humans, in addition to chicken meat, poultry, food animals, and wild birds.^5,32,33 CC10 was described as ESBL-producing E. coli in Italy, Brazil, the Netherlands, and Canada.^28,29,33,34 This lineage has successfully entered a variety of ecological niches.

CC156 and CC446, both with bla_CTX-M and CC446 carrying qnr genes, although infrequently reported, have been isolated by others from avian sources.^28,29 Some of these lineages could be considered as extraintestinal pathogenic E. coli because they have been isolated from human bloodstream infections or urinary tract infections.^3,5,31,35,36

Several singleton isolates carried resistance markers, such as ST1844 with qnrB, ST2064 with bla_CMY-2 and qnrB, and ST212 with bla_CTX-M8. Such isolates may evolve to represent a public health threat as they show a capacity to acquire important resistance genes.

The urban wild birds studied here carried E. coli clonal lineages with a diversity of bla_CTX-M types, plasmid-mediated quinolone resistance, and ampC-type beta-lactamase genes. These findings indicate a relatively common dissemination of resistance genes in the urban environment. Such E. coli isolates from wild birds in Rio de Janeiro city may pose a potential zoonotic risk.

The limitation of this study is that since we did not simultaneously examine E. coli isolates from human infections, we cannot determine the human clinical implication of finding E. coli isolates from wild birds. More detailed analyses, such as whole-genome sequencing, may be needed to assess the direction of transmission of such E. coli organisms. Nevertheless, our observation suggests that the distribution of MDR E. coli in wild birds is reflective of a degree of environmental pollution with these drug-resistant human pathogens.