野生动物(野鸟)对全球耐药基因传播的影响

1Over several decades, antimicrobial resistance has become a global clinical and public health threat against the effective treatment of common infections caused by resistant pathogens, resulting in treatment failure and increased mortality. The development of bacterial resistance is a natural evolution of microorganisms, but the widespread use and misuse of antibacterial agents in humans and animals has accelerated this process. Furthermore, the increasing frequency of global travel and trade has also contributed to the rapid worldwide spread of antimicrobial resistance. Some resistant clones, such as Escherichia coli ST131, Klebsiella pneumoniae ST258 and ST11, and methicillin-resistant Staphylococcus aureus (MRSA) USA 300, which are involved in the spread of resistance to crucially significant antibiotics in human medicine, have been widely disseminated. Antimicrobial resistance is a complex and multifaceted problem involving humans, animals, and the environment. However, the role of wildlife in the emergence of antibacterial resistance might be underestimated. The first report of antibacterial resistance in wildlife revealed chloramphenicol resistance in E. coli isolates obtained from Japanese wild birds. Since then, the occurrence of resistant bacteria in wild animals has been increasingly reported within diverse animal species across different geographical areas. In addition, several important antimicrobial resistant pathogens, such as MRSA, vancomycin-resistant enterococci, Salmonella spp., Vibrio cholerae, and Campylobacter spp., have been described in wild animals, highlighting the importance and complexity of wildlife, not normally exposed to antibiotics directly, in the transmission of resistant bacteria.

2This review gives a brief overview of the emergence and prevalence of expanded-spectrum β-lactamase (ESBL), AmpC β-lactamase, carbapenemase, and colistin resistance genes in Enterobacteriaceae strains from wild animals, all of which have significant public health impact. Furthermore, this review aims to better understand the role of wildlife in the transmission of clinically significant antimicrobial resistance in Enterobacteriaceae.

3The global dissemination of ESBL-producing Enterobacteriaceae in human clinics is an urgent problem that poses a serious challenge to the treatment of infectious diseases, particularly the worldwide emergence of CTX-M-15-producing ST131 E. coli. ESBL-producing Enterobacteriaceae have also been increasingly reported in livestock, companion animals, and food (;;;; Michael et al., 2016). The CTX-M-type β-lactamases are the most common ESBLs among Enterobacteriaceae isolates of human and veterinary origin worldwide.

4Since the first report on ESBL-producing E. coli isolates from wild animals in Portugal in 2006, ESBL-producing Enterobacteriaceae of wildlife origin have so far been reported in Europe, Africa, Asia, South America, North America, and Australia (Table 1). Although ESBLs have been found in various Enterobacteriaceae, most ESBL-producing bacterial pathogens in wild animals are E. coli, followed by K. pneumoniae (Table 1). To date, at least 80 wildlife species have been found to be carriers of ESBL-producing Enterobacteriaceae, most being wild birds (Table 1). Similar to that among isolates from human and veterinary medicine, the CTX-M family is the most prevalent type of ESBL-producing Enterobacteriaceae found in wild animals (Table 1). Both blaCTX-M-1 and blaCTX-M-15 are commonly reported in wild animals and are the most prevalent ESBL genes, followed by blaCTX-M-14, blaCTX-M-32, blaCTX-M-9, blaCTX-M-3, blaCTX-M-2, and blaCTX-M-22. Other ESBL genes, such as blaCTX-M-27, blaCTX-M-55, blaCTX-M-8, blaCTX-M-24, blaCTX-M-25, blaCTX-M-28, blaCTX-M-29, and blaCTX-M-124, have also been detected, though infrequently (Table 1). Significant geographical differences have been observed in the occurrence of CTX-M enzymes. As summarized in Table 1, CTX-M-15 is the only reported CTX-M-type β-lactamase in Africa to date, and is the most common CTX-M-type enzyme reported in Bangladesh. In Canada and the US, CTX-M-14 is dominant, followed by CTX-M-15. Diversity in CTX-M β-lactamases has been reported in European countries, with the predominance of CTX-M-1 and CTX-M-15. Interestingly, CTX-M-15 is also reported to be the most common CTX-M enzyme in Franklin's gulls (Leucophaeus pipixcan) in northern Chile, although CTX-M-1 was previously reported to be dominant in the same gull species in central Chile. hypothesized that, based on their migratory habits, Franklin's gulls from the north acquired resistant CTX-M-15-producing ST131 and ST10 E. coli clones, which are highly prevalent in humans in the US and Canada but scarce in Chile. However, this hypothesis, though possible, needs further investigation.

5In addition to CTX-M enzymes, SHV and TEM enzymes have also been reported in wildlife, especially SHV-12 and TEM-52, which accords with that found in ESBL-producing isolates from humans, livestock, and companion animals (Table 1) (;;; Michael et al., 2016; ). For example, SHV-12 has been frequently detected in wildlife in Spain, and is highly prevalent in ESBL-producing E. coli obtained from 8-to 16-month-old healthy children in northern Spain and in raw poultry meat from southern Spain, as well as from hospitals. Other SHV-type enzymes, such as SHV-102, SHV-1, SHV-2, and SHV-5, and TEM-type enzymes, such as TEM-19, TEM-40, TEM-176, and TEM-20, have also been sporadically reported in wild animals (Table 1).

6More than 170 different sequence types (STs) have been identified in ESBL-producing E. coli isolates of wildlife origin (Table 1). Among them, ST131 is the most commonly detected clone. The dominant ST131 clone identified in wild animals, which has been frequently described in humans, companion animals, food products, and the environment, is involved in the international dissemination of blaCTX-M-15 and blaCTX-M-14. Additionally, other STs described in wild animals, such as ST10, ST69, ST405, ST410, and ST648, have also been reported in various sources and are responsible for the intercontinental distribution of CTX-M. However, some STs found in wildlife, such as ST1340, ST1646, ST2687, ST3018, and ST3056, have been identified as new types and have not yet been reported in human or veterinary isolates.

7As for ESBL-producing K. pneumoniae, limited studies are currently available on the clonal group of K. pneumoniae from wildlife (Table 1). found an SHV-11-encoding K. pneumoniae strain from mouflon (Ovis orientalis musimon) in Austria belonging to the epidemic clone ST11, which is associated with carbapenemase and ESBL in humans worldwide, and previously described in companion animals and Eurasian beaver (Castor fiber). In Algeria, all 17 blaCTX-M-15-bearing K. pneumoniae isolates found in wild boars and Barbary macaques belong to ST584, which has also been detected in silver gulls as carriers of carbapenemase IMP-4 in Australia as well as in human in Brazil (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html).

8Successful clones found in humans and domestic and wild animals indicate possible interspecies transmission of ESBL-producing isolates. However, horizontal transfer mediated by mobile elements, such as insertion sequences and plasmids, is also one of the main methods for ESBL dissemination worldwide. Only limited (mostly European) studies are available on ESBL-encoding plasmids in wild animals (Table 1). For example, blaCTX-M-15 is reportedly associated with IncF plasmids (mostly multiple replicons containing IncFIA and IncFIB) and IncI1, which agrees with previous research involving CTX-M-15-producing Enterobacteriaceae obtained from the environment, healthy cattle, and humans. IncHI2 plasmids have also been reported as carriers of blaCTX-M-15 in humans, companion animals, pigs, and wild birds. Though rare, IncI2 plasmid has also been described with blaCTX-M-15 in the lesser black-backed gull from the Netherlands and identified in a chicken E. coli strain in China. In Enterobacteriaceae of human and veterinary origin, blaCTX-M-1 has been frequently found associated with IncN and IncI1 plasmids. Interestingly, blaCTX-M-1 has been mainly located on IncI1 plasmids in wildlife in Europe as well. However, IncN, IncF, and IncHI1 plasmids have also been reported as carriers of blaCTX-M-1. Furthermore, IncI1 plasmids are also carriers of other ESBL genes in wild animals, such as blaCTX-M-3, blaSHV-12, and blaTEM-52. Similarly, IncF plasmids are also reported to be associated with blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-27, blaCTX-M-32, and blaSHV-12 in wildlife. The narrow-host-range plasmid IncX has been found to carry several ESBL genes, namely blaTEM-135, blaTEM-52b, blaTEM-176, and blaSHV-12, in E. coli isolated from diverse sources in Australia, Czech Republic, Spain, and Poland. Notably, plasmid replicon typing was performed on ESBL-producing isolates with multiple plasmids in several studies, thus the replicons of ESBL-carrying plasmids could not be confirmed.

9Insertion sequences also play an important role in facilitating the spread of ESBL genes. Though few studies are available on horizontal transfer mediated by insertion sequences, associations of insertion sequence ISEcp1 and blaCTX-M, including blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-32, have been observed in E. coli from wild animals.

10In summary, the ESBL gene types identified in wild animals are the same as those in human and veterinary medicine. Thus, interspecies transmission mediated by successful pandemic ESBL-producing clones and plasmids in humans, domestic animals, and wildlife might occur.

11Plasmid-mediated AmpC β-lactamases among Enterobacteriaceae in human and veterinary medicine are of considerable global concern because they confer resistance to clinically important cephalosporin antibiotics and β-lactamase inhibitors. CMY-2 is the most prevalent AmpC β-lactamase and has been globally disseminated among Enterobacteriaceae in humans, companion animals, food-producing animals, and retail meat. As shown in Table 2, AmpC β-lactamases have been reported in E. coli, K. pneumoniae, and Enterobacter cloacae isolates of wildlife origin in Europe, North America, and Asia, and particularly in central Europe, similar to ESBL-producing Enterobacteriaceae (Table 2). To date, 20 different wild animal species, mostly birds, have been identified as blaCMY-2 carriers (Table 2). Like that in human and veterinary medicine, blaCMY-2 is the most commonly detected AmpC-type β-lactamase among wild animals, though its identification has been limited to E. coli isolates.

12The blaCMY-2 gene is mainly located on IncA/C and IncI1 plasmids from Enterobacteriaceae isolates of human and veterinary origin. Among wild animals, blaCMY-2 has been reported to be mainly associated with IncI1 plasmids in E. coli from Dutch wild birds and wild seagulls in the US. Other plasmid types, such as IncB/O, IncK, and IncF, have also been identified as carriers of blaCMY-2 in wildlife, as previously reported in humans, companion animals, broiler chickens, and retail meat. Thus far, the IncA/C plasmid, a major carrier of blaCMY-2, has not yet been identified in wild animals. The absence of blaCMY-2-bearing IncA/C plasmids could simply reflect the limited studies on the characterization of blaCMY-2-carrying plasmids in wildlife, or might indicate that blaCMY-2-harbouring IncI1 plasmids are more successful among wildlife.

13In addition to CMY-2, DHA-type AmpC β-lactamase genes have also been detected in K. pneumoniae from mouflons (Ovis orientalis musimon) in Austria, E. coli from hill mynah (Gracula religiosa) in Saudi Arabia, and K. pneumoniae ST11 isolates from Eurasian beaver (Castor fiber) in Switzerland. FOX-5 encoded by an IncA/C plasmid has been obtained from K. pneumoniae isolates in the US. Furthermore, a novel variant of the ACT AmpC β-lactamase gene has been identified in an Enterobacter cloacae strain originating from glaucous gull (Larus hyperboreus) in Arctic Svalbard, Norway.

14Carbapenemase-producing Enterobacteriaceae isolates pose an urgent public health threat. New Delhi metallo-β-lactamase (NDM), as one of the most widespread carbapenemases, has been increasingly reported in human clinics, foods, domestic animals, and the environment worldwide.

15The first reported carbapenemase-producing bacteria in wild animals were isolated from black kites (Milvus migrans) in Germany. Among 184 cefotaxime-resistant Salmonella spp. isolates, only one Salmonella Corvallis isolate belonging to ST1541 has shown reduced susceptibility to carbapenem, and carries the carbapenemase gene blaNDM-1 located on~180 kb IncA/C conjugative plasmid pRH-1738. The broad-host-range IncA/C plasmids are among the most predominant plasmids associated with blaNDM-1 in humans. supposed that the blaNDM-1-bearing Salmonella Corvallis isolate might have originated from non-European countries and was transferred to Germany through the black kite migratory route, since Salmonella Corvallis was prevalent in South-East Asia and was emerging in North Africa and Nigeria, rather than in European countries. The complete sequence of plasmid pRH-1738 further confirms this hypothesis. Plasmid pRH-1738 exhibited high relatedness with plasmid pMR0211 obtained from human Providencia stuartii isolate in Afghanistan, but showed distinct differences from other sequenced NDM-1-IncA/C2 plasmids from Western countries. In addition, fosfomycin resistance gene fosA3, which has been rarely detected in Europe but is prevalent among CTX-M-encoding E. coli and K. pneumoniae isolates in Asia (i.e., China, Japan, and South Korea), has also been identified on NDM-1-producing plasmid pRH-1738. blaNDM-1 transferred with fosA3 on IncA/C plasmid has only been described in clinical E. coli and Citrobacter freundii isolates in China. Taken together, these findings suggest that the origin of this plasmid might be in the Asiatic region.

16Large-scale transmission of IMP-producing bacteria into wildlife was first reported in 2015. In total, 120 carbapenemase-producing Enterobacteriaceae of 10 species were obtained from silver gulls in Australia, mainly E. coli (n=85), carrying blaIMP-4, blaIMP-38, or blaIMP-26. The blaIMP-4 gene has been found in 116 isolates, and is the most commonly detected gene among carbapenemase-producing Enterobacteriaceae isolates in human clinics in Australia. blaIMP-4 in gulls is carried by various conjugative plasmids, mostly IncHI2-N plasmid type, followed by IncA/C plasmids, as well as IncL/M and IncI1, and is associated with a class 1 integron-containing blaIMP-4-qacG-aacA4-catB3 array in most positive strains. The same array carried by IncA/C and IncL/M plasmids is also reportedly responsible for the dissemination of blaIMP-4 in clinical isolates in Australia, and by the IncHI2 plasmid in Salmonella Typhimurium from a cat in Australia. Furthermore, 19 different STs have been detected in IMP-4-producing E. coli isolates, including five prevalent lineages (ST216, ST58, ST354, ST167, and ST224), in which ST58, ST354, and ST167 are clinically relevant clone lineages.

17Although carbapenem resistance reported in wild animals is rare, the emergence of NDM-1 and IMP carbapenemases in wild birds is of concern.

18Colistin is widely applied in food-producing animals and is currently used as the last resort for treating infections caused by multi-resistant gram-negative bacteria. Since the first identification of the plasmid-mediated colistin resistance gene mcr-1 in China in 2015, it has been identified in Enterobacteriaceae isolates from food-producing animals, companion animals, food products, the environment, and humans worldwide.

19The role of wild birds as reservoirs and vectors for the global distribution of mcr-1 should be considered. Recently, mcr-1 was described in an E. coli strain isolated from European herring gull (Larus argentatus) feces collected from the Kaunas (Lithuania) city dump. However, the emergence of mcr-1 in wildlife could be traced back to E. coli strains isolated in 2012. Five extended-spectrum cephalosporin-resistant E. coli isolates obtained from kelp gulls in Ushuaia, Argentina in 2012 were found to carry mcr-1 and blaCTX-M-2 (n=1) and blaCTX-M-14 (n=4) and exhibited elevated colistin MICs (4-8 mg/L). The mcr-1 gene was located on a~57 kb IncI2 plasmid without blaCTX-M in all five isolates. IncI2 plasmids, which have been detected in E. coli and Salmonella isolates from food, food-producing animals, and humans in China, Great Britain, the US, Venezuela, and Denmark, have been reported to be associated with the transmission of mcr-1. Notably, four mcr-1-carrying isolates, which belong to ST744, have been previously described in Denmark and carry the mcr-1-bearing IncI2 plasmid.

20Clinically relevant resistance, such as ESBL, AmpC cephalosporinase, carbapenemase, and colistin resistance, has been detected in wild animals, particularly wild birds, from distinct geographical areas. Thus, wild animals could serve as important reservoirs of resistant bacteria. Although the origin of bacterial resistance genes in wild animals remains unclear, as wildlife are not exposed to antibiotics directly, contact with sewage or animal manure might be one possibility. Additionally, the potential of wild animals as vectors of resistant bacteria or genetic determinants should not be underestimated. Wildlife, especially migratory birds with their instinctive mobility, can carry resistant bacteria over long distances, even between continents; thus, this might be a new transmission route and partly responsible for the global dissemination of bacterial resistance. Contamination of food or water by wildlife is recognized as an important risk factor for the transmission of antimicrobial resistance or pathogens to food animals and humans.

21Wild animals might play a vital role in the worldwide spread of clinically relevant pathogens or resistance genes. Pandemic ESBL-producing E. coli clones or plasmids shared by humans, domestic animals, and wildlife further strengthen this hypothesis. Thus, continued surveillance of multi-resistant bacteria in wild animals is warranted.