GENOMIC CHARACTERIZATION OF MULTIDRUG-RESISTANT EXTENDED-SPECTRUM β-LACTAMASE–PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE FROM CHIMPANZEES (PAN TROGLODYTES) FROM WILD AND SANCTUARY LOCATIONS IN UGANDA

Extended-spectrum β-lactamases (ESBLs) confer resistance toward oxyimino-cephalosporins (cefotaxime, ceftazidime, ceftriaxone, cefuroxime, and cefepime) and monobactams (aztreonam), but not to carbapenems (imipenem, meropenem, doripenem, and ertapenem) or to cephamycins (cefoxitin, cefotetan), although these are inhibited by β-lactamase inhibitors such as clavulanic acid (Livermore 2008). They were initially restricted to the clinical environment with high selective pressure but have since emerged in pathogens in the community (Gandolfi-Decristophoris et al. 2013). Mainly, ESBLs are found within the Enterobacteriaceae family with Escherichia coli as the primary ESBL-carrying pathogen involved in community-acquired infections (Pitout 2010). The most clinically relevant ESBLs are the CTX-M, TEM, and SHV types, of which CTX-M-15 is the most prevalent type in both humans and animals worldwide (Ghafourian et al. 2015).

Increasingly, ESBL-producing bacteria are observed within animal populations, including healthy wild animals worldwide (Smet et al. 2010; Gandolfi-Decristophoris et al. 2013). Several studies have identified ESBLs in migratory bird species and in rodents, suggesting these species act as carriers and contribute to dissemination of ESBL genes (Stedt et al. 2015; Mohsin et al. 2017; Schaufler et al. 2018). Studies performed in Uganda suggest that transmission of E. coli between humans, mountain gorillas (Gorilla beringei beringei), and livestock is influenced by habitat overlap (Rwego et al. 2008a, b).

Chimpanzees (Pan troglodytes) live in the wild, but in several places, including Uganda, sanctuaries have been set up to rescue animals that for various reasons have been left or taken from their community. It is currently unknown to which extent such chimpanzees are at risk for being carriers of ESBL genes that could be passed on to pathogenic strains causing resistant infections. Our aims were to determine the occurrence of Enterobacteriaceae with resistance to extended spectrum β-lactams in chimpanzees living in sanctuaries or in forest reserves and to characterize putative ESBL isolates obtained from these animals.

A total of 86 fecal samples were collected from chimpanzees from two different locations in Uganda, Budongo Forest (n=56) and Ngamba Island Chimpanzee Sanctuary (n=30), in January and February 2018. Samples were collected in 30-mL screw cap containers with spoons (109117, Globe Scientific, Paramus, New Jersey, USA) containing Stuart Transport Medium (M306-500G, HiMedia®, Mumbai, India). Fresh fecal matter was collected after the chimpanzee had defecated on the ground and was picked up with the spoon from the sample container without touching the ground, to avoid environmental contamination. A single sample per animal was collected. Samples were kept in a cool box until arrival at the laboratory at Makerere University, Kampala, Uganda. The Uganda Wildlife Authority, the Chimpanzee Sanctuary & Wildlife Conservation Trust, and Uganda National Council for Science and Technology granted approvals for sample collection in Ngamba and Budongo.

For each fecal sample, 1 g was diluted in 9 mL of phosphate-buffered saline (AMRESCO®, Solon, Ohio, USA); 100 µL of the mixture was spread on MacConkey agar (MCA; Oxoid, Basing-stoke, UK) containing 2 µg/mL cefotaxime (Sigma-Aldrich, St. Louis, Missouri, USA). Plates were incubated overnight at 37 C. If plates showed no growth or insignificant growth, 1 g of feces was mixed with 9 mL of tryptic soy broth (Bacto®, Liverpool, New South Wales, Australia) and incubated overnight at 37 C, then restreaked on MCA with 2 µg/mL cefotaxime.

All morphologically different colonies on MCA plates with cefotaxime were preserved. To exclude Pseudomonadaceae, non–lactose-fermenting colonies that tested positive with oxidase test strips (Oxoid) were discarded. All lactose-fermenting and non–lactose-fermenting oxidase-negative colonies were purified on MCA and incubated overnight at 37 C. Purified colonies were stored in Luria Bertani media (BD Difco™, Franklin Lakes, New Jersey, USA) with 15% glycerol at –80 C until further analysis.

Species identification was obtained with the use of fresh colony material from bacteria grown on Luria Broth (BD Difco™, Franklin Lakes, New Jersey, USA) overnight at 37 C by matrix assisted laser desorption ionization time-of-flight (MAL-DI-TOF) mass spectrometry (MS) (VITEK® MS, bioMérieux, Marcy-l'Étoile, France). Strains of E. coli and K. pneumoniae were preserved for detailed analysis, whereas strains that were not identified as one of these two species by MALDITOF MS were not analyzed further.

A disk diffusion test for antimicrobial sensitivity was performed on Mueller-Hinton agar (Oxoid) according to Clinical and Laboratory Standards Institute guidelines (Papich et al. 2015). Testing was carried out with disks of the following antimicrobials: aztreonam 30 µg, cefpodoxime 10 µg, ceftazidime 30 µg, ceftriaxone 30 µg, cefotaxime 30 µg, and imipenem 10 µg (all Oxoid). An ESBL confirmatory test was performed with Etest strips containing cefotaxime and clavulanic acid (bioMérieux) according to the package insert. Briefly, an isolate was interpreted as an ESBL producer if the minimum inhibition concentration (MIC) for cefotaxime (CT) was ≥0.5 and the MIC ratio of CT/cefotaxime + clavulanic acid (CTL) was ≥8, or by presence of a phantom zone (an additional zone of inhibition between the ellipses of the CT and the CTL, or a deformation of the CT inhibition zone, irrespective of the ratios or MICs). Strains from the same chimpanzee with different phenotypic resistance profiles, or of different species, were considered as different strains and included separately for further analysis.

Selected ESBL strains were reincubated on Luria broth and incubated overnight at 37 C. One colony was picked and transferred to 5 mL Luria Bertani medium and incubated overnight at 37 C with shaking. Genomic DNA was isolated with the Maxwell® RSC Cultured Cells DNA kit (Promega, Madison, Wisconsin, USA) on the Maxwell® RSC instrument (Promega) according to the supplier's instructions. An agarose gel with the isolated DNA and GeneRuler 100-base pair Plus DNA Ladder (0.5 µg/mL, Thermo Scientific, Waltham, Massachusetts, USA) was run for 30 min at 100 V on a 1% agarose gel to ensure the quality of the genomic DNA. Whole-genome sequencing was carried out at a 250-base pair paired-end read format with the Nextera XT Library Preparation Kit and the MiSeq instrument (Illumina, San Diego, California, USA). Raw reads of whole-genome–sequenced isolates were submitted to European Nucleotide Archive and are available for download under study accession number PRJEB43637.

We assembled and quality checked E. coli genomes with programs implemented in EnteroBase (Alikhan et al. 2018). Genomes were assembled by SPAdes 3.10.1 (Bankevich et al. 2012), and the assembly quality was evaluated by QUAST 2.3 (Gurevich et al. 2013). All genomes were annotated by the Prokka command-line software tool (Seeman 2014). Species were identified by KmerFinder 2.5 (Hasman et al. 2014; Larsen et al. 2014). Sequence type (ST) determination was done by MLST 2.0 (Larsen et al. 2012). Antimicrobial resistance genes were identified by ResFinder 3.0 (Zankari et al. 2012). Virulence genes were identified by VirulenceFinder 2.0 (Joensen et al. 2014). We constructed single-nucleotide polymorphism (SNP)–based maximum likelihood phylogenetic trees by CSI Phylogeny (Kaas et al. 2014) and reference isolates of E. coli MG1655 (accession no. NC_000913) and of K. pneumoniae HS11286 (accession no. NC_016845) retrieved from Gen-Bank. Phylogenetic trees were imported to the Interactive Tree of Life (Letunic and Bork 2016), where bootstrap values, sequence type information, and heatmaps showing antibiotic resistance genes were added (Letunic and Bork 2016).

We obtained 18 putative ESBL E. coli and K. pneumoniae isolates from the 86 chimpanzees sampled. Of these, 17 strains were obtained from chimpanzees from the Ngamba sanctuary, where animals were in close contact with humans; only one was from a chimpanzee living in the wild in Budongo National Park. In 11 E. coli and seven K. pneumoniae isolates, we confirmed resistance to cefpodoxime, ceftazidime, aztreonam, cefotaxime, and ceftriaxone, but sensitivity to imipenem. Potential ESBL production was supported by the confirmatory test represented by the presence of a phantom zone or a MIC of CT≥0.5 and MIC ratio of CT/CTL≥8 for all isolates (Table 1). The isolates were obtained from 14 different chimpanzees; in chimpanzee N003 two E. coli strains (isolates 1 and 20) were obtained, and in chimpanzee N017 one K. pneumoniae (isolate 7) and two E. coli (isolates 8 and 22) were obtained (Table 1). These isolates were further characterized by whole-genome analysis. Metadata for these strains, details on sequences obtained and accession numbers are listed in Table 2.

Of the seven K. pneumoniae ESBL isolates sequenced, two isolates were excluded because of low coverage of sequence (<25), failed identification with KmerFinder, and failure of MLST call. Among the remaining strains, three strains belonged to ST1540 and two to ST597. Overall, 24 different antibiotic resistance genes conferring resistance to nine different antibiotic classes were detected. All strains harbored the bla_CTX-M-15, strA, strB, oxqA, oxqB, qnrS1, fosA, mph(A), catA2, sul1, and sul2 genes conferring resistance to βlactams, aminoglycosides, fluoroquinolones, fosfomycins, macrolide-lincosamide-streptogramin B, phenicols, and sulfonamides and could be classified as multidrug resistant on the basis of the definition given by Magiorakos et al. (2012). Additional resistance genes were observed in all strains: β-lactamases, in addition to bla_CTX-M-15, included bla_TEM-1B, bla_TEM-1C, bla_SHV-11, bla_SHV-12, bla_OXA-1, and bla_LEN24, of which bla_CTX-M-15 and bla_SHV-11 are classified as ESBL genes (Fig. 1). Core genome SNP analysis clustered strains by sequence type. Strain 5 of ST1540 resembled the two strains of ST597 on the basis of resistance profile, showing that resistance was not correlated with phylogeny (Fig. 1).

Of the nine ESBL E. coli isolates sequenced, four isolates were found to be ST5204, two ST2852, and one each ST215, ST405, and ST315. Twenty different antibiotic resistance genes were identified among the strains conferring resistance to seven different antibiotic classes (Fig. 2). The ESBL phenotype was found to be caused by bla_CTX-M-15 in all nine strains, and additional SHV-12 genes were demonstrated in all four ST5204 strains. Some genes such as strA, strB, and sul2, including the bla_CTX-M-15, were found in all isolates, whereas bla_TEM and tetA were more frequently observed, and qnrS1 and dfrA14 were less frequent. All isolates were found to be multidrug resistant. Isolates clustered according to sequence type by SNP core genome analysis, and isolates with the same sequence type showed the same resistance gene profile. Out of the ESBL-producing E. coli strains in this study, isolate 70 (ST315) from Budongo was the isolate harboring the greatest number (n=15) of resistance genes. A search for virulence-associated genes revealed that the ESBL-producing strains encoded between 0 and 13 known E. coli virulence genes. The most commonly found virulence genes were lnfA and iss. One E. coli isolate (70) had more virulence genes than others (Table 3).

Our study showed the presence of E. coli and K. pneumoniae harboring ESBL genes in chimpanzees in both Budongo National Park and Ngamba Island Chimpanzee Sanctuary in Uganda. Consistent with studies from a community setting in Cameroon, in outpatients in urban and rural districts in Uganda, and in companion and domestic farm animals in Tanzania, E. coli was the most commonly isolated ESBL producer (Lonchel et al. 2012; Najjuka et al. 2016; Seni et al. 2016). The likelihood of observing colonies growing on MacConkey plates with 2 mg/mL cefotaxime was highest from chimpanzees living in close contact with humans, and all but one ESBL-producing E. coli and K. pneumoniae characterized were identified in the chimpanzees residing in a sanctuary. Sanctuary chimpanzees are orphaned and rescued animals from the illegal trade that live in a safe seminatural environment. Visitors are allowed to observe the chimpanzees from a distance as they feed, play, rest, and prepare their nests. However, the facility caregivers, which include international visitors, come into close direct and indirect contact with the chimpanzees when preparing food, feeding, cleaning night-time holding facilities, performing medical procedures, and creating behavioral enrichment structures (Chimpanzee Sanctuary Wildlife Conservation Trust 2020), which may explain the higher number of ESBL-producing strains, confirming previous observation of higher risks of transmission associated with close contact between humans and livestock, mountain gorillas, and wild chimpanzees (Goldberg et al. 2007; Rwego et al. 2008a, b). In Taï National Park, Côte d'Ivoire, no ESBL-producing bacteria were identified from chimpanzees or other wildlife tested, despite a prevalence of ESBL-producing E. coli in the villages nearby of 27% in humans and 32% in dogs, and the majority of the assistants working in the park live in those villages (Albrechtova et al. 2014). This does not rule out that chimpanzees, who often leave the forest and enter other habitats such as human settlements to travel and raid (Kortlandt 1983), may come into contact with environments contaminated by human bacteria, as pointed out by Goldberg et al. (2007), which may account for the observation of ESBL even in the protected environment.

The most commonly identified ESBL gene was bla_CTX-M-15, detected in all strains. This correlates well with this gene being the most widely distributed in the world (Ghafourian et al. 2015) and what other studies have observed in countries such as Tanzania, France, Iran, and Venezuela (Arpin et al. 2009; Seni et al. 2016; Moghanni et al. 2018; Araque and Labrador 2018). The global distribution of bla_CTX-M has been referred to as the CTX-M pandemic and is of certain concern to public health (Cantón and Coque 2006) because most CTX-M producers display coresistance to aminoglycosides, tetracycline, sulfonamides, and fluoroquinolones, which limits therapeutic options significantly (Morosini et al. 2006). In accordance with this notion, the ESBL-producing E. coli and K. pneumoniae in the current study were all classified as multidrug resistant. The ESBL genes bla_SHV-11 in K. pneumoniae and bla_SHV-12 in E. coli were also identified among the sequenced isolates; bla_TEM-1B, bla_TEM-1C, bla_OXA-1, and bla_LEN24 were also detected in this study, but they are not considered ESBLs because they are not capable of hydrolyzing third generation cephalosporins (Ghafourian et al. 2015).

None of the chimpanzees in this study harbored the globally disseminated E. coli clone ST131, which has been identified in Australia, Canada, India, Spain, UK, New Zealand (Petty et al. 2014), and, more recently, in hospitalized patients in Nigeria and Tanzania and in farm animals in Tanzania. However, in common with our study, other studies identified E. coli ST405 and ST2852 in Tanzania and Nigeria (Seni et al. 2016, 2018; Sonda et al. 2018a). The E. coli ST315 clone identified in Budongo has been found in countries such as India, Canada, and Switzerland in humans and in hospital effluent (Peirano et al. 2012; Seiffert et al. 2013; Beg and Khan 2018).

Clinically relevant ESBL-producing K. pneumoniae ST307 have been detected in a rat population in Guinea (Schaufler et al. 2018), and ST14 and ST48 have been involved in outbreaks in neonatal units in Tanzania (Mshana et al. 2013). Although ST17 was the most prevalent type causing community-onset infections in China (Zhang et al. 2016) and in hospitalized patients in Kilimanjaro, Tanzania (Sonda et al. 2018b), none of these studies identified ST597or ST1540, which we detected. There has been a high focus on the widespread carbapenemase-producing ST258 and ST307 K. pneumoniae clones (Baraniak et al. 2009; Samuelsen et al. 2009; Woodford et al. 2011; Villa et al. 2017). Carbapenemaseproducing Klebsiella, however, was not detected in our study, which correlates well with a study on outpatients from rural and urban districts of Uganda (Najjuka et al. 2016). Use of drugs in the carbapenem class in the community is less likely because they have to be administered parenterally and are quite expensive.

No specific β-lactamases are affiliated with animals; therefor, it is believed that the most probable origin of ESBL producers is humans (Guenther et al. 2011). Both E. coli and K. pneumoniae can be transmitted from humans to animals through the fecal-oral route, probably through indirect contact with contaminated environmental sources rather than by direct contact (Goldberg et al. 2007; Rwego et al. 2008a; Smet et al. 2010). Spread of human pathogens through reintroduction of chimpanzees to the wild is known to pose a threat to the wild population (Schaumburg et al. 2012), and our study shows that antimicrobial-resistant bacteria may also be transmitted. Several isolates from our study were genetically similar, and some were isolated from different chimpanzees. Identical K. pneumoniae isolates were isolated (7 and 3) from two different chimpanzees (N017 and N029, respectively), and a second pair of isolates (15 and 11) were also identical and isolated from chimpanzees N004 and N021, respectively. Similarly, identical isolates of E. coli (strains 1, 21, 22, and 30) were isolated from chimpanzees N003, N022, N017, and N030, respectively; these belonged to ST5204 and had similar resistance profiles. Some chimpanzees, however, yielded multiple E. coli strains of different STs, such as chimpanzee N003, which harbored two E. coli strains (strain 1 and 20) belonging to different STs (ST5204 and ST405, respectively), whereas chimpanzee N017 harbored one K. pneumoniae (7) and two E. coli (8 and 22 of ST2852 and ST5204, respectively), and chimpanzee N030 had two E. coli (29 and 30 of ST117 and ST5204, respectively). The harboring of unrelated sequence type resistant bacteria is not unexpected and has been demonstrated in this study, has been reported previously (Goldberg et al. 2007; Rwego et al. 2008a; Smet et al. 2010), and is a major concern for transmission of resistance.

The establishment of strict hygiene guidelines while working with chimpanzee in sanctuaries should be instituted to limit the spread of antimicrobial-resistant bacteria. Further studies with paired samples between chimpanzees and caretakers are needed to show whether caretakers may play a role in transmission of ESBL to the chimpanzee.

In conclusion, ESBL-producing K. pneumoniae and E. coli were isolated more frequently from chimpanzees in close contact with humans; however, they could also be obtained from free-living chimpanzees without known human contact. A diverse pool of STs of these Enterobacteriaceae was identified. The most commonly identified ESBL gene was the worldwide-disseminated gene identified in all strains, bla_CTX-M-15 followed by bla_SHV-11 in K. pneumoniae and bla_SHV-12 in E. coli. All ESBL strains were multidrug resistant. These findings underline the importance of limiting further spread of these resistant bacteria, because treatment alternatives for infections with multidrug-resistant ESBL-producing E. coli and K. pneumoniae are becoming scarce. The findings underpin the existential threat of antimicrobial-resistant bacteria to chimpanzee health management and conservation and needs urgent attention.

The authors express the warmest thanks to the laboratory staff at Makerere University and University of Copenhagen for assistance with practical laboratory work.