Colonization with Enterobacteriaceae-Producing ESBLs,AmpCs,and OXA-48 in Wild Avian Species,Spain 2015–2016

Introduction

Antibiotic resistance is a global threat of complex and changeable epidemiology. Antibiotic-resistant bacteria occur in humans, animals, food, and the environment, and require a broad and integrated One Health approach. ¹

While the increase of antibiotic resistance in livestock is mainly driven by the direct antibiotic usage, the emergence of resistant bacteria in wildlife may be related to the passage of antibiotic-resistant genes to the environment. ² This naturally occurring phenomenon is exacerbated by the influx to the environment of large amounts of antibiotic residues and antibiotic-resistant genes from intensive livestock, human waste, the pharmaceutical industry, and hospitals.^2,3 In addition, the increasing frequency of global travel has also contributed to the rapid worldwide spread of antimicrobial resistance. ³ However, the role of wildlife in the dissemination of antibacterial resistance might be underestimated.

The Group for Rehabilitation of the Autochthonous Fauna and its Habitat (GREFA) (www.grefa.org) is a Spanish nongovernmental organization for the study and conservation of nature; one of its main objectives is the maintenance of a hospital for acute care of the wild animals. The Study Group on the Medicine and Conservation of Wild Animals (GEMAS) is a multidisciplinary project, led by GREFA, aimed at improving the knowledge of the health status of native wildlife in Spain; in addition, due to the evident interrelationships between some of the diseases of wild animals, domestic animals, and people, GEMAS may play an important role in public health issues.

The aims of this study were to know the prevalence of colonization by Enterobacteriaceae isolates resistant to third-generation cephalosporins in wild birds, and to characterize the acquired antibiotic resistance genes implicated in this resistance. In addition, we tested if isolates resistant to third-generation cephalosporins also produced carbapenemases and/or harbored mcr-1 genes.

Materials and Methods

Results

After overnight incubation at 37°C, bacterial growth was observed on the screening plates obtained from 95 (14.2%) birds; seven of them had nonfermentative gram-negative rods, and the remaining 88 (13.2%) had cefotaxime-resistant enterobacterial isolates. These 88 specimens were isolated from 28 different species of wild birds as follows: diurnal bird of prey (28 specimens and 11 species), storks and jackdaws (17 specimens and two species), seagulls (14 specimens and two species), scavenger birds (12 specimens and two species), nocturnal bird of prey (6 specimens and three species), and others (11 specimens and 8 species) (Table 1). After bacterial identification, we obtained 94 different isolates of the following Enterobacteriaceae species: 60 E. coli, 10 Klebsiella pneumoniae, 10 Hafnia alvei, 8 Enterobacter spp., 4 Proteus mirabilis, 1 Citrobacter freundii, and 1 Morganella morganii. Six birds were colonized by two Enterobacteriaceae strains nonsusceptible to cefotaxime: three had two different strains of E. coli, two had E. coli plus K. pneumoniae, and one had E. coli plus M. morganii.

Sixty-one of the 94 (64.9%) isolates produced ESBLs. The prevalence of colonization by ESBL-producing Enterobacteriaceae was 8.7% (58 of 668 wild birds); three birds had two different ESBL-producing strains. The ESBL types detected belonged to the CTX-M-1 group (39, 63.9%), of which 27 (44.3%) were CTX-M-15; CTX-M-9 group (14, 23%), of which 8 were CTX-M-9 and 6 CTX-M-14; and SHV-group (8, 13.1%), of which 6 were SHV-12 and 2 SHV-2 (Table 2). The remaining 33 isolates displayed a resistance phenotype consistent with AmpC production on the basis of their resistance to amoxicillin/clavulanic acid and cefoxitin, and inhibition with phenyl boronic acid and cloxacillin ⁴ ; but only 15 of them (12 E. coli and 3 P. mirabilis; 2.5% of the 668 wild birds) were positive for pAmpC β-lactamases of the bla_CIT family. The remaining 18 isolates presenting the AmpC phenotype but negative for pAmpC were 10 H. alvei, 5 Enterobacter spp., and one isolate each of M. morganii, C. freundii, and E. coli; this is most probably due to the expression of the intrinsic chromosomal AmpC (cAmpC) present in these species (Table 2).

Carbapenemase detection showed that five of the 94 cefotaxime-resistant isolates were also positive for bla_OXA-48: three K. pneumoniae (isolated from Eurasian eagle-owl, lesser kestrel, and common buzzard), one E. coli (common buzzard), and one E. cloacae (cattle egret), all five also carried the bla_CTX-M-15 gene. In addition, an E. coli isolate producing a plasmid mediated AmpC of CIT family also carrying mcr-1 colonized a black vulture.

By MLST, the three OXA-48-producing K. pneumoniae isolates belonged to the high-risk clones ST11 (two) and ST15 (one) frequently identified in humans; the OXA-48-producing E. coli belonged to ST23, and the CIT-producing and mcr-1-positive E. coli belonged to ST162.

PFGE analysis of the E. coli isolates revealed a high degree of genetic diversity because 44 different PFGE patterns were observed among the 54 cefotaxime-resistant E. coli isolates studied (Fig. 1); in six isolates, digestion with XbaI failed. However, two well-defined clusters, with more than two isolates each, were detected with a genetic linkage >90% (Fig. 1). Cluster 1 (C1) included nine CTX-M-15-producing isolates belonging to ST23; these isolates came from eight different species of birds (Eurasian eagle-owl (2), common buzzard, booted eagle, black vulture, barn owl, griffon vulture, and mallard), and one of them was the OXA-48-producing E. coli from the common buzzard previously mentioned. Cluster 2 (C2, Fig. 1) included four CIT-producing isolates belonging to ST744 from three different species of birds (white stork (2), northern goshawk, and common kestrel). Four additional clusters of two isolates each were also detected (Fig. 1).

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FIG. 1.

Dendrogram illustrating the PFGE profiles of 54 cefotaxime-resistant Escherichia coli from wild birds. PFGE, pulsed field gel electrophoresis.

Discussion

Remarkably, our study identified a high diversity of wild bird species (n = 28) from very diverse ecosystems and with different life and eating habits harboring ESBL-producing bacteria (Table 1). This finding suggests that ESBLs are widely distributed in the environment. Previously, several studies have described the occurrence of ESBL-producing enterobacteria in wild birds, but not in such a wide and diverse number of species. A previous Spanish study detected 15 wild birds colonized by ESBL-producing E. coli among 100 specimens studied, a total of nine wild avian species were represented. ¹⁴ In another study, up to 37% of samples from Alaskan gulls carried E. coli and/or K. pneumoniae harboring ESBLs. ¹⁵

In comparison with the 8.7% of wild birds colonized by ESBLs in the present study, a recent meta-analysis, ¹⁶ including 28,909 healthy human individuals, showed that the pooled prevalence of colonization by ESBL-producing Enterobacteriaceae was 14%. ¹⁶ However, this percentage varied among continents (6% in southern Europe and 2% in the Americas). The most important ESBL types detected in wild birds in our study (CTX-M-15, CTX-M-9, CTX-M-14, and SHV-12) are also the predominant ESBL types circulating in humans in Spain.^17,18

In relation to the production of carbapenemases in this study, while production of VIM, NDM, KPC, and IMP classes would had been fully detected using the agar plates with 1 mg/L of cefotaxime because all of them generate coresistance to cefotaxime, production of OXA-48 alone could had been underestimated because OXA-48 does not generate coresistance to third-generation cephalosporins by itself. Accordingly, OXA-48-producing isolates would only grow on plates containing cefotaxime if the isolates had an additional cefotaxime resistance mechanism such as ESBL or AmpC, a fact that often occurs in K. pneumoniae. ¹⁹

Acquired carbapenemases currently pose one of the most worrying public health threats. Carbapenemase-producing bacteria have been detected from some rivers, sewage plants, pets, and food animals. ¹ However, there is very little information available on carbapenemase-producing Enterobacteriaceae isolated from wild birds,^20–23 and as far as we know OXA-48 has not been previously reported. Recently, a case of OXA-48 in wild boars in Algeria has been reported. ²⁴ Also, NDM-1-producing Salmonella spp., VIM-1-producing E. coli, and IMP-4-producing Enterobacteriaceae were isolated from one black kite, ²¹ yellow-legged gulls, ²² and silver gulls, ²³ respectively.

The high-risk clones ST11 and ST15, detected in OXA-48-producing K. pneumoniae in this study in wild birds, are also two of the most prevalent clones detected in humans. In a multicenter study carried out in Spain, ¹⁹ ST11 and ST15 were identified in 42.6% of 221 carbapenemase-producing K. pneumoniae from humans. The presence, in wild birds, of OXA-48-producing Enterobacteria belonging to some of the most prevalent clones circulating in humans is very remarkable and worrying.

Detection of mcr-1 in a CIT-producing E. coli is also a remarkable finding because few cases of mcr-1 have been described in wild birds. Recently, mcr-1 was reported in E. coli isolated from a European herring gull in Lithuania, ²⁰ and it was also retrospectively detected in ESBL-producing E. coli isolated from kelp gulls in 2012 in Argentina. ²⁰

Clonal complex ST23 has been previously described as one of the most frequent clonal complexes found among ESBL-producing E. coli from humans, ²⁵ also it has been often detected in isolates from wild birds.^26,27

Diversity of eating patterns and migratory habits of the multiple avian species capable of carrying multiresistant bacteria may contribute to their global dissemination from human sources. In addition, the presence of different species of birds, with different ecological guilds, colonized by multidrug-resistant bacteria from human origin, suggests that these bacteria are more widespread in the environment than previously thought. Any initiative for global control of antibiotic resistance based on the One-Heath approach must take into account the undeniable pathways of communication between the human being, livestock, environmental, and wild animal ecosystems.