Journal of Global Antimicrobial Resistance 22 (2020) 515–518

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Journal of Global Antimicrobial Resistance

journal homepage: www.elsevier.com/locate/jgar

Short Communication

Genomic analysis of a multidrug-resistant Klebsiella pneumoniae

ST11 strain recovered from Barbary deer (Cervus elaphus barbarus)
in Akfadou Forest, Algeria
Assia Mairia , Olivier Barraudb , Anaelle Muggeoc,d , Christophe de Champsc,d ,
Abdelaziz Touatia,*
a
Laboratoire d’Ecologie Microbienne, FSNV, Université de Bejaia, Béjaïa, Algeria
b
INSERM, CHU Limoges, UMR 1092, Université de Limoges, Limoges, France
c
INSERM UMR-S 1250 P3Cell, SFR CAP-Santé, Université de Reims-Champagne-Ardenne, Reims, France
d
Laboratoire de Bactériologie-Virologie-Hygiène Hospitalière-Parasitologie-Mycologie, CHU Reims, Hôpital Robert Debré, Reims, France

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: The emergence and worldwide spread of carbapenemase-producing Enterobacterales (CPE) is
Received 2 December 2019 a great public-health concern. This study aimed to screen for the presence of CPE isolates from Barbary
Received in revised form 9 April 2020 deer in Akfadou Forest, Béjaïa (Algeria).
Accepted 20 April 2020
Methods: Faecal samples (n = 39) were obtained from Barbary deer in Akfadou Forest between March–
Available online 4 May 2020
June 2018. Whole-genome sequencing (WGS) was performed to characterise one representative strain of
Klebsiella pneumoniae. Data analysis was performed using online tools.
Keywords:
Results: A total of 13 carbapenem-resistantK. pneumoniae isolates were obtained. The isolates showed an
Klebsiella pneumonia
Antimicrobial resistance genes
identical antimicrobial resistance pattern and were susceptible to colistin and fosfomycin. WGS analysis
Wild animal revealed the complete resistome of K. pneumoniae strain CF21, including blaNDM-1, blaCTX-M-15, blaSHV-182,
WGS blaDHA-1, blaOXA-1, aac(3)-IIa, aac(3)-IId, aac(6ʹ)-Ib-cr, rmtC, sul1, qnrB9, fosA, tetA, dfrA14, catA2, catB3 and
Algeria mphA. Multilocus sequence typing (MLST) analysis assigned this strain to the international clone ST11.
Plasmid analysis showed that this K. pneumoniae strain possesses five different plasmids including
IncA/C2, IncFIA(HI1), IncFIB(K), IncFII(K) and ColRNAI.
Conclusion: This study reports a multidrug-resistantK. pneumoniae strain recovered from Barbary deer in
Algeria and confirms that wild animals could serve as a reservoir of antimicrobial resistance genes.
© 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial
Chemotherapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).

1. Introduction resistant K. pneumoniae (CRKP) are the most isolated strains in

Carbapenemase-producing Enterobacterales (CPE) are now a
serious public-health problem. The most widespread carbapene-
mases found among Enterobacterales in the world include
Klebsiella pneumoniae carbapenemase (KPC), metallo-β-lactamases
(MBLs) such as imipenemase (IMP), Verona imipenemase (VIM)
and New Delhi metallo-β-lactamase (NDM) types, and the class D
oxacillinases (OXA-48-like) [1]. NDM-1 carbapenemase was first
described in an Enterobacterales strain isolated in 2008 in a
Swedish patient previously hospitalised in New Delhi, India. More
than 20 variants of NDM have been described, with NDM-1 and
NDM-5 being particularly widespread [2]. In Algeria, carbapenem-
* Corresponding author.
E-mail address: ziz1999@yahoo.fr (A. Touati).
human (78.5%) and extra-human samples (51.1%). However, NDM-
producing K. pneumoniae strains are only sporadically reported
(5% of all CPE) compared with OXA-48 carbapenemase [3].
Only a few reports of CRKP in wild animals have been published,
including blaIMP-4 and blaIMP-26 from silver gulls in Australia [4] and
blaOXA-48 from wild boars [5], bat guano [6], wild birds and wild fish
from Algeria [7]. The aim of the present study was to investigate by
whole-genome sequencing (WGS) a CRKP isolate recovered from
faeces of Barbary deer (Cervus elaphus barbarus) because, to our
knowledge, no CRKP have been reported in this species and the
living conditions for these animals are particular in Algeria, where
they live freely in an enclosure in Akfadou Forest, Béjaïa, without
contact with human beings.
Barbary deer (or Atlas deer), a subspecies of European red deer,
disappeared from North Africa more than 200 years ago. In Algeria,
this species has been reintroduced and kept in semi-captivity in

http://dx.doi.org/10.1016/j.jgar.2020.04.027
2213-7165/© 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
516 A. Mairi et al. / Journal of Global Antimicrobial Resistance 22 (2020) 515–518
animal enclosures. It is the only representative of the Cervidae
family in Africa. It has been listed since 1968 in the protected
species category A of the African Convention for the Conservation
of Nature and Natural Resources, is currently in Appendix III of the
Convention in International Trade of Endangered Species (CITES),
and its status by the International Union for Conservation of Nature
(IUCN) has recently been changed from ‘Vulnerable’ to ‘Lower Risk
(Near Threatened)’ [8].
2. Methods
2.1. Study area
Akfadou Forest, located 160 km east of Algiers and 20 km from the
sea, is under the jurisdiction of Tizi-Ouzou and Béjaïa departments
(Algeria) and is one of the most important forests in North Africa. The
forest massif extends over an area of approximately 16 000 hectares
(ha) and is mainly composed of different species of oak. The
reintroduction site of the Barbary deer consists of two enclosures:
the main enclosure (180 ha) in which deer are released; and a
quarantine enclosure (65 ha) in the event of forest fires (Supple-
mentary Fig. S1). Barbary deer from Tunisia were reintroduced in
Akfadou Forest on three occasions: in December 2005, with three
deer (one male and two females); in February 2006, with three deer
(one male and two females); and in November 2006, with two deer
(two females). An enumeration operation was carried out in January
2019 and at least 38 deer were inventoried.
2.2. Sample collection and microbiological testing
Collection of faecal samples took place within the Barbary deer
enclosure of Akfadou Forest during the period March–June 2018. A
total of 39 fresh faeces samples were collected using sterile swabs.
All samples were transported under refrigeration (4  C) to the
laboratory and were analysed within a maximum of 4 h.
For CPE screening, each swab was introduced into 1 mL of the
in-house Carba MTL-broth containing ertapenem (0.5 mg/L),
cloxacillin (250 mg/L), vancomycin (64 mg/L) and amphotericin B
(2 mg/L). Following incubation for 12 h at 37  C, tubes of Carba
MTL-broth showing a colour change from green to yellow were
considered as positive for CPE. A volume of 200 mL of positive
culture was streaked on MacConkey agar supplemented with
crystal violet, sodium chloride and 0.15% bile salts (Fluka, St Louis,
MO, USA) and was incubated at 37  C for 24 h [9]. Suspected
colonies (up to five) were subcultured onto MacConkey plates.
Species identification was performed using an API 20E
identification system (bioMérieux, Marcy-l’Étoile, France) and
was confirmed using matrix-assisted laser desorption/ionisation
time-of-flight mass spectrometry (MALDI-TOF/MS) (microflexTM;
Bruker Daltonik GmbH, Bremen, Germany) with the MALDI
Biotyper software package database version 4.2.90 (Bruker
Daltonik GmbH). Identification was made on whole cells and a
score of 2.000 was required for an accurate species-level
identification as recommended by the manufacturer.
2.3. Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by the disk
diffusion method on Mueller–Hinton agar according to the
recommendations of the Comité de l’ántibiogramme de la Société
Française de Microbiologie (CA-SFM) [10]. The following anti-
biotics were tested: ampicillin (10 mg); ticarcillin (75 mg); ticar-
cillin/clavulanic acid (TCC) (75/10 mg); piperacillin/tazobactam
(TZP) (30/6 mg); amoxicillin/clavulanic acid (AMC) (20/10 mg);
ceftazidime (10 mg); cefotaxime (5 mg); cefoxitin (30 mg); aztreo-
nam (30 mg); cefepime (30 mg); imipenem (10 mg); ertapenem
(10 mg); amikacin (30 mg); tobramycin (10 mg); gentamicin
(10 mg); norfloxacin (10 mg); ofloxacin (5 mg); ciprofloxacin
(5 mg); trimethoprim/sulfamethoxazole (SXT) (1.25/23.7 mg);
trimethoprim (5 mg); fosfomycin (200 mg); and chloramphenicol
(30 mg) (Bio-Rad, Marnes-la-Coquette, France). The minimum
inhibitory concentrations (MICs) of carbapenems (ertapenem,
imipenem and meropenem) were determined by Etest (bioMérieux)
and the MIC of colistin was determined by microbroth dilution
(UMIC1 ; Biocentric, Bandol, France). Susceptibility patterns were
interpreted according to the CA-SFM v.2.0 (2019) guidelines [10].
Escherichia coli ATCC 25922 was used as a quality control strain.
2.4. Genome sequencing
One K. pneumoniae strain (CF21) was further characterised by
WGS using Ion ProtonTM technology (Thermo Fisher Scientific,
Waltham, MA, USA) according to the manufacturer’s instructions.
Quality check of raw reads was done using FastQC v.0.11.5 and
MultiQC v.0.9, and no trimming was performed. Reads are available
under Sequence Read Archive (SRA) accession no. PRJNA604085.
Mean coverage of 40 was determined after alignment against the
K. pneumoniae subsp. pneumoniae HS11286 reference genome
(GenBank CP003200.1). Reads were then assembled using
Mimicking Intelligent Read Assembly (MIRA) software. Contigs
were analysed using Geneious software (Biomatters Ltd., Auckland,
New Zealand). Average nucleotide identity (ANI) was estimated
using ANI calculator from Kostas lab (http://enve-omics.ce.gatech.
edu/ani) using K. pneumoniae HS11286 reference strain and
contigs: 99.85% identity was obtained.
Antimicrobial resistance genes were identified using ResFinder
3.2 (https://cge.cbs.dtu.dk/services/ResFinder/). Genes with a 60%
minimum length and a percentage of identity >98% were
considered. Plasmid replicon types were determined using the
PlasmidFinder 2.1 database (https://cge.cbs.dtu.dk/services/Plas-
midFinder/). Sequence types (STs) were determined using the
Klebsiella multilocus sequence typing (MLST) database (https://
cge.cbs.dtu.dk/services/MLST/). Mutations in the quinolone resis-
tance-determining regions (QRDRs) of gyrA and parC were
analysed.
3. Results
From the 39 faecal samples, 13 CRKP (33.3%) were obtained on
selective medium. These CRKP strains were isolated at two
different times, including eight strains obtained during March
2018 and five strains obtained during June 2018.
All 13 K. pneumoniae strains showed the same antimicrobial
resistance profile and were resistant to ertapenem, imipenem,
ampicillin, ticarcillin, TCC, TZP, AMC, ceftazidime, cefotaxime,
cefoxitin, cefepime, aztreonam, amikacin, tobramycin, gentamicin,
chloramphenicol, trimethoprim, norfloxacin, ofloxacin, ciproflox-
acin and SXT (Table 1). All isolates were susceptible to colistin and
fosfomycin.
WGS of K. pneumoniae CF21 strain showed the presence of 17
antimicrobial resistance genes (Table 1), including: blaNDM-1,
blaCTX-M-15, blaSHV-182, blaDHA-1 and blaOXA-1 encoding β-lactam
resistance; aac(3)-IIa, aac(3)-IId, aac(6ʹ)-Ib-cr and rmtC encoding
aminoglycoside resistance; sul1 encoding sulfonamide resistance;
the plasmid-mediated quinolone resistance gene qnrB9; fosA
encoding fosfomycin resistance; tetA encoding tetracycline resis-
tance; dfrA14 encoding trimethoprim resistance; catA2 and catB3
encoding chloramphenicol resistance; and mphA encoding macro-
lide resistance. Mutations were detected in GyrA (Ser83Phe;
Asp87Ala) and ParC (Ser80Ile) topoisomerase proteins that are
responsible for resistance to quinolones. MLST analysis assigned
this isolate to the international clone ST11.
 A. Mairi et al. / Journal of Global Antimicrobial Resistance 22 (2020) 515–518 517

Table 1
Antimicrobial resistance characteristics of carbapenem-resistant Klebsiella pneumoniae strain CF21 detected from Barbary deer (Cervus elaphus barbarus) in Akfadou Forest,
Algeria.

Resistance phenotype MIC (mg/L) [interpretation] Associated ARGs Plasmids MLST

ETP IPM MEM COL

TIC, AMC, TCC, CTX, CAZ, FEP, 8 (R) 6 (R) 6 (S) <0.25 (S) blaNDM-1, blaCTX-M-15, blaSHV-182, IncA/C2, IncFIA ST11
ATM, IPM, ETP, FOX, TZP, GEN, blaDHA-1, blaOXA-1, aac(3)-IIa, (HI1), IncFIB(K),
TOB, AMK, CIP, CHL, TMP, aac(3)-IId, aac(6')-Ib-cr, rmtC, IncFII(K), ColRNAI
OFX, NOR, SXT, AMP qnrB9, fosA, sul1, tetA, dfrA14,
catA2, catB3, mphA

MIC, minimum inhibitory concentration; ARG, antimicrobial resistance gene; MLST, multilocus sequence typing; TIC, ticarcillin; AMC, amoxicillin/clavulanic acid; TCC,
ticarcillin/clavulanic acid; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam; IPM, imipenem; ETP, ertapenem; FOX, cefoxitin; TZP, piperacillin/tazobactam;
GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; CHL, chloramphenicol; TMP, trimethoprim; OFX, ofloxacin; NOR, norfloxacin; SXT, trimethoprim/
sulfamethoxazole; AMP, ampicillin; MEM, meropenem; COL, colistin; R, resistant; S, susceptible.

Table 2
Results of plasmid replicon typing determined by PlasmidFinder 2.1.

Plasmid Identity (%) Query/template length Contig Position in contig GenBank accession no.
ColRNAI 100 130/130 mid42-urc2_c68 cov = 65.50 len = 2294 1641..1770 DQ298019
gc = 47.21 nseq=773
IncA/C2 100 417/417 mid42-urc2_c66 cov = 48.91 len = 10584 8213..8629 JN157804
gc = 50.73 nseq=2568
IncFIA(HI1) 98.45 387/388 mid42-urc2_c65 cov = 46.20 len = 4755 3306..3692 AF250878
gc = 51.19 nseq=1099
IncFIB(K) 100 560/560 mid42-urc2_c33 cov = 35.24 len = 17060 3334..3893 JN233704
gc = 45.86 nseq=3014
IncFII(K) 97.32 149/148 mid42-urc2_c44 cov = 34.73 len = 23064 2171..2318 CP000648
gc = 46.78 nseq=3990

Plasmid analysis showed that K. pneumoniae CF21 possesses
five different plasmids including IncA/C2, IncFIA(HI1), IncFIB(K),
IncFII(K) and ColRNAI, with high identity to the previously
described pNDM-KN (GenBank JN157804), pTR2 (GenBank
KJ187752), pKPN-IT (GenBank JN233704), pKPN3 (GenBank
CP000648] and pIGMS32 (GenBank DQ298019), respectively
(Table 2).
4. Discussion
The global spread of acquired CPE has become a serious
public-health problem. In addition to humans, pets and
livestock, antimicrobial resistance has also been reported in
wildlife that has not been exposed to antibiotics [11]. In this
respect, Fischer et al. reported for the first time the isolation of
NDM-1-producing Salmonella from a wild bird in Germany [12].
Also, a few reports of CRKP isolates in wildlife have been
published, including blaIMP in silver gulls from Australia [4],
blaOXA-48 in wild boars [5], wild birds and wild fish [7], and blaOXA-
48 and blaKPC-3 in bat guano from Algeria [6]. This report confirms
that wildlife in Algeria could serve as reservoir of multidrug-
resistant (MDR) K. pneumoniae.
It is interesting to note that the Barbary deer from which
these CRKP have been isolated are living in an enclosed area
with rare contact with anthropogenic sources in the surround-
ing area. This raises the question of the origin of these CRKP
isolates. We could hypothesise contamination from other wild
animals such as birds and small rodents that can cross the
enclosed area easily.
CRKP strain CF21 was assigned to ST11 clone. This successful
clone has been reported worldwide in wildlife, humans and
companion animals. ST11 has also been reported in a clinical NDM-
1-producing K. pneumoniae strain in Tunisia [13]. It is interesting to
highlight that the Barbary deer reintroduced into Akfadou Forest
originated from Tunisia. Besides, this clone has now spread
globally.
In this study, it was observed that a gene encoding
resistance to fosfomycin was detected in K. pneumoniae strain
CF21, however phenotypically this strain was categorised as
susceptible. Thus, it is important to test in vitro the susceptibility
of strains to ensure the concordance of the genotype (presence of
antimicrobial resistance gene) with the antimicrobial susceptibili-
ty phenotype.
Current data indicate that MDR bacteria can spillover from their
anthropogenic sources into natural ecosystems, possibly creating
secondary reservoirs in the environment where clinically impor-
tant resistance can be maintained and from where it can spread
further [14]. Reports describing the emergence of plasmid-
associated resistance genes in wildlife could indicate the possible
exchange of mobile genetic elements between human, livestock
and environmental niches [15]. The current study may suggest that
the dynamics of MDR plasmids could be maintained in antibiotic-
free environments such as wildlife.
In conclusion, these findings strongly suggest that Barbary deer
may serve as a reservoir of MDR K. pneumoniae strains that are
characterised by resistance to various antimicrobial agents in
clinical use. Algeria is one of the countries with the highest rate of
CPE reported. Indeed, further research should be conducted and
proper measures applied to limit the transmission of these MDR
pathogens in the human population, animals and the environment.
Competing interests
None declared.
Ethical approval
Not required.
518 A. Mairi et al. / Journal of Global Antimicrobial Resistance 22 (2020) 515–518
Acknowledgments
The authors would like to thank Mr Lahelal Abane (Béjaïa forest
conservation, Algeria) for his kind help. The authors also thank
Dr Valentin Tilloy (UF9481 Bioinformatique, CHU Limoges,
Limoges, France) for his kind help in the bioinformatics analysis
and the ‘Direction Générale de la Recherche Scientifique et du
développement technologique (DGRSDT)’ of the Algerian Ministry
of Higher Education and Scientific Research.
Appendix A. Supplementary data
Supplementary material related to this article can be
found, in the online version, at doi:https://doi.org/10.1016/j.
jgar.2020.04.027.
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