Phenotypic and Genotypic Characterization of Klebsiella Pneumoniae Obtained from Egyptian Vultures and Steppes Eagles from India

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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 123 Klebsiella pneumoniae Egyptian vultures and Steppes eagles
INTRODUCTION
Emerging antibiotic resistance of infectious diseases among
domestic animals, wildlife and human being draw attrac-
tion toward increased interface between humans, domestic
animals and free-ranging wildlife. Te role of wild animals
including bird of prey as reservoirs and disseminators of
pathogens of potential zoonotic importance has major im-
plications for public health (1, 2). Since the migratory scav-
engers feed at refuse dumps, abattoirs and other sources of
domestic refuse and ofal from various and diferent places of
world they may act as disseminators of bacterial pathogens
associated with those food sources (3). Raptors feeding on
carcass dumps are exposed to a variety of pathogenic or-
ganisms. Hundreds of endangered Egyptian vultures N. p.
percnopterus and Steppe eagles (Aquila nipalensis), henceforth
referred to as ‘the raptors’, overwinter at Jorbeer carcass dump
(4) situated 10 km from Bikaner city, in state of Rajasthan.
Klebsiella pneumoniae is a gram negative opportunistic
pathogen associated with many enteric infections of animals,
mastitis in cattle (5), bacteremia in calves (6), metritis in
mares (7), pneumonia and urinary tract infections in dogs,
and pneumonia, septicemia in foals (8). Epidemic and en-
demic nosocomial infections caused by Klebsiella pneumoniae
are also the leading cause of morbidity and mortality in hu-
man beings (9).
At present, strains expressing capsule serotypes K1 and
Phenotypic and Genotypic Characterization of Klebsiella Pneumoniae
Obtained from Egyptian Vultures and Steppes Eagles from India
Sharma, S.K.,
1
* Sharma, P
2
. and Shringi, B.N.
1
1
Department of Veterinary Microbiology and Biotechnology. College of Veterinary and Animal Sciences, Bikaner. Rajasthan
University of Veterinary and Animal science, Bikaner-334001 (Rajasthan), India.
2
Veterinary ofcer. Department of Animal Husbandry, Government of Rajasthan, India.
* Corresponding Author: Dr. Sharma, S.K., Department of Veterinary Microbiology and Biotechnology. Tel: 09414775879, Email: drsharmask01@hotmail.com
ABSTRACT
Wild migrating Egyptian vultures (Neophron percnopterus percnopterus) and Steppes eagles (Aquila nipalensis)
can potentially harbor and spread pathogenic microorganisms and may associated with antibiotic resistance
genes over long distances. In the present study, we reported the prevalence of Klebsiella pneumoniae from
feces of wild Egyptian vultures and Steppes eagles, which were sampled for two consecutive winter seasons,
at a carcass dump in the state of Rajasthan India, and to the best knowledge of the authors as a frst
record from the Indian subcontinent. After phenotypic characterization and genotypic confrmation based
on 16S-23S rDNA internal transcribed spacer (ITS) region showing a prevalence of 11% (n=27), and
these isolates displayed resistance to multiple antibiotics including beta-lactams, glycopeptides, macrolides,
polypeptides and sulfonamides groups. All isolates were screened for virulence associated serotypes K1, K2
and K5 with serotype specifc primers and the potentially pathogenic K5 serotype was confrmed in one
isolate obtained from an Egyptian vulture. Occurrence of multiple antibiotic resistance and K5 serotype
of K. pneumoniae in endangered Egyptian vultures, and steppe eagles requires further investigations in the
context of epidemiology and pathogenicity of K. pneumoniae including concerning efects on population
health and conservation context of these scavenging raptors.
Keywords: Steppe Eagle, Egyptian Vulture, Klebsiella pneumoniae, K5 serotype.
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Sharma, S.K. 124
K2 of K. pneumoniae are considered especially likely to be
virulent, although only a few of the 77 diferent K antigens
have been systematically studied in this regard (10). Also
serotypes K1, K2 and K5 are often associated with severe
infections in humans, animals and are highly virulent in ex-
perimental infection in mice (11, 12).
Extensive survey of the literature including peer-reviewed
journals revealed a dearth of information on occurrence of
Klebsiella spp. from Egyptian vultures, and no report of the
organism from Steppe eagles. No information is available on
the role of scavenging raptors in epidemiology of the poten-
tially pathogenic serotypes including K1, K2 and K5 of K.
pneumoniae. Information on multiple antibiotic resistance in
fecal bacteria of scavenging raptors is generally lacking and
no information could be accessed on Klebsiella specifcally
from the Indian subcontinent.
Te objectives of this investigation were to (i) study preva-
lence of Klebsiella pneumoniae in fecal matter of the migratory
scavenging raptors (ii) employ phenotypic characterization
of the K. pneumoniae, (iv) genotypic confrmation of the iso-
lates from the scavenging raptors employing 16S-23S rDNA
internal transcribed spacer (ITS) region specifc primers (v)
confrmation of virulent capsular serotype K1, K2 and K5
with serotype specifc primers; and (vi) study the occurrence
of multiple antibiotic resistance in these isolates.
MATERIAL AND METHODS
Study site
Jorbeer carcass dump, with coordinates: N 27°57.958’ E
73°22.598’ (± 3m), is a dumping site for livestock carcass-
es near Bikaner in Rajasthan state of India. Te site is in
the Tar Desert at 235 m above sea level, with a landscape
dominated by arid and sparse desert vegetation composed
of scattered trees and shrubs. Carcasses were mainly of cat-
tle but included camels, dogs, equines and poultry waste.
Approximately 15–20 carcasses were being dumped each day
at the site providing a regular and ample source of food to the
scavenging raptors. Te probability of carcasses containing
diferent type of veterianry drugs considered high as some of
these originate from veterinary university clinics, and other
veterinary clinical setting.
Te carcass dump hosts fve other species of vultures
(White-rumped Gyps bengalensis, Long-billed G. indicus,
Eurasian Grifon G. fulvus, Cinereous G. monachus, and Red-
headed vultures Sarcogyps calvus), Black kite (Milvus milvus),
and few other breeding as well as migrating eagles belonging
to the genus Aquila (4). Many avian species, migratory as well
as resident, also inundate the dump site along with hundreds
of resident stray dogs.
Sample collection, isolation and identifcation
Fresh fecal samples were collected from roosting sites of
the raptors by noninvasive means through sterile cotton
swabs (Himedia Laboratories Pvt. Ltd., Mumbai, India).
Te birds were identifed and monitored using binoculars
(Nikon Monarch 10x50, Nikon Inc., USA) and fecal sam-
ples collected aseptically from inside of lump of drooping by
HiCulture™ Transport Swabs (Himedia Laboratories Pvt.
Ltd., Mumbai, India) as soon as the raptors defecated. 27
fresh fecal samples of the raptors in two consecutive win-
ter seasons (includes 25 Egyptian vultures and two steppe
eagles) were examined for presence of Klebsiella pneumoniae.
Te raptors always existed in excess of 500 in numbers with
in the sampling period and thus probability of duplicate
sampling was statistically insignifcant (P<0.05). Te pure
Table 1: Phenotypic characterization of K. pneumoniae isolated from
Steppe eagles and Egyptian vultures.
S. No. Biochemical Test Reaction
1 Catalase test Positive
2 Oxidase test Negative
3 Capsule staining Capsulated
4 Oxidation- fermentation test Fermentative
5 IMViC Pattern -ve-ve+ve+ve
6 Colony Muco-viscosity Positive
7 Growth on 10°C Negative
8 Growth on 44.5°C Positive
9 Growth on MacConkey agar Mucoid pink colonies
10 Growth on SCAI Agar Yellow mucoid dome shaped colonies
11 Growth on EMB Agar Dark mucoid non-metallic colonies
12 Growth on BCP Agar Yellow mucoid colonies
13 Growth on TSI Agar
Acid/Acid/No H2
S
14 Gelatin Liquefaction Negative
15 Aesculin Hydrolysis Positive
16 Nitrate Reduction Positive
17 Malonate Utilization Positive
18 Arginine Hydrolysis Negative
19 Lysine Decarboxylation Positive
20 Phenylalanine Deamination Negative
21 ONPG (o-Nitrophenyl-β-d
Galactopyranoside)
Positive
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 125 Klebsiella pneumoniae Egyptian vultures and Steppes eagles
colonies of organisms were phenotypically confrmed and
characterized by standard biochemical tests and colony char-
acters according to the techniques described by Edwards and
Ewing (13) (Table 1). All of the confrmed K. pneumoniae
isolates were also examined for sugar fermentation reaction
of 15 sugars (Table 2).
Te genotypic confrmation was based on 16S-23S
rDNA internal transcribed spacer (ITS) region as method
described by Liu et al., (14). PCR reactions were carried out
in 50 µl volumes with fnal concentrations of 3 µl of extracted
template DNA, as template DNA obtained through tech-
nique described by Chen and Kuo (15), 10 µl 5x PCR assay
bufer (with 1.5 mM MgCl2), 1 µl dNTP (10 mM), 1 µl each
forward and reverse primer set, 0.25 µl Taq DNA polymerase
(5 U/ µl) and 33.75 µl MiliQ water. Te cycling conditions
were 10 min at 94 °C followed by 35 cycles of 30 s at 94 °C,
20 s at 57 °C, and 20 s at 72 °C then 10 minutes hold at
72 °C. PCR products (130 bp) were separated by 8% native
PAGE (Hoefer SE 600 Hoefer Inc., Massachusetts , USA).
Detection of serotype (K1, K2 and K5) and antibiotic
susceptibility testing
Genotypically confrmed K. pneumoniae were evaluated for
presence of serotype K1, K2 and K5 using serotype specifc
primers with genomic DNA as template and PCR condi-
tions as described by Turton et al., (16) and the PCR prod-
ucts were separated by 8% native PAGE (17).
Each confrmed K. pneumoniae isolate was examined for
susceptibility to 24 antibiotics of diferent chemical classes
(Table 3) and resistance pattern was determined by disc dif-
fusion method of Bauer et al., (18) on Mueller Hinton agar
(Himedia Laboratories Pvt. Ltd., Mumbai, India).
RESULTS AND DISCUSSION
In the present study, Klebsiella pneumoniae showed a preva-
lence of 11.11% (n=27) as phenotypic (Table. 1) and geno-
typic characterization confrmed three K. pneumoniae isolates
(Figure 1); two from Egyptian vultures and one from a steppe
eagle. Furthermore out of three isolates, one isolate from the
Egyptian vulture was detected as K5 serotype (280bp) and
the K1 and K2 serotypes were not found in any of the isolates
Table 2: Diferentiation of K. pneumoniae isolates based on sugar
fermentation and gas production
Isolate ID
Name of Sugar EV1 SE1 EV2
Xylose +G +G +G
Dulcitol +G -- --
Arabinose +G -- +G
Lactose +- +G --
Mannose +G +G +G
Inositol +G +G +G
Rafnose +G +G +G
Rhamnose +G +G +G
Maltose +G +G +G
Sucrose +G +G +G
Sorbitol +G +G +G
Dextrose +G +G +G
Fructose +G +G +-
Mannitol +G +G +G
Trehalose +G +G +G
+ Positive for Fermentation; G Positive for gas production; - Negative
for fermentation or gas production
Table 3: Multiple Antibiotic resistance pattern of K. pneumoniae
isolates from Egyptian vultures (EV) and Steppe eagles (SE)
Isolate ID
Antibiotics EV1 SE1 EV2
Ampicillin R R R
Bacitracin R R R
Ciprofoxacin S S S
Ceftazidime S S S
Cephlothin R I I
Norfoxacin S S S
Oxacillin R R R
Erythromycin R R R
Imipenem S S S
Vancomycin R R R
Gentamicin S S S
Tetracyclin I I I
Rifampicin R R R
Trimethoprim S S S
Kanamycin I S S
Amoxycillin+Clavulanic Acid R R R
Ampicillin + Sulbactam S S S
Amoxycillin R R R
Cefepime S S S
Cefotaxime S S S
Clinadmycin R R R
Faropenem R I R
Polymyxin R R R
Sulfadiazine R R R
R: Resistant; S: Sensitive; I: Intermediate
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Sharma, S.K. 126
(Figure 2). Except for three sugars dulcitol (33.3%), arabi-
nose (66.6%) and lactose (66.6%), all sugars were fermented
by all the isolates (Table 2). All isolates showed multiple anti-
biotic resistance and all of the isolates were resistant to 11 an-
tibiotics used in the study and remaining antibiotics showed
variable patterns of efcacy against the isolates (Table. 3).
Te availability of literature on Klebsiella pneumoniae iso-
lated from Egyptian vultures and steppes eagles is very scarce
but Klebsiella spp. have been reported from diferent avian
genera such as red kites (Milvus milvus), Egyptian vultures,
and Antarctic skua (Catharacta spp.) showing a prevalence of
7.96% (n=113) (19), 8.82% (n=68) (20) and 4.54 % (n=22)
(21) respectively. Prevalence of fecal Klebsiella spp. from Red-
billed choughs (Pyrrhocorax pyrrhocorax) was 15.0%, 12.8%
and 15.6% for three diferent locations in Spain (22). K.
pneumoniae was isolated from wild turkey vultures (Cathartes
aura) (23) and peregrine falcon (Falco peregrinus) (24) in
USA showed prevalence of 5% (n=20) and 42.85% (n=14)
respectively. In the context of phenotypic characterization
and sugar fermentation patterns of K. pneumoniae, similar
result were obtained in present study as described in previous
literature in which K. pneumoniae was obtained from various
animals and humans (25, 26 and 27).
To the best knowledge of the authors, the presence of K5
serotype of K. pneumoniae from feces of the wild Egyptian
vulture is a frst report although particular serotypes of K.
pneumoniae have been incriminated in epidemics in animals
(28), community-acquired liver abscesses, pneumonia, endo-
ophthalmitis, and as an opportunistic nosocomial pathogen
in humans worldwide, especially Asia and the USA (10,
29). Serotypes K2, K5 and K7 were found to be the most
pathogenic in equines (30), whereas serotypes K1, K2, K5
caused metritis epidemics in mares (31). K5 strain caused
pneumonia in humans (32) and outbreak of enteritis and
septicemia in canines (33). Apart from isolated case reports
describing K. pneumoniae as a cause of pathogenic lesions in
the infra-trochlear area and brainstem in Barn owl (Tyto alba)
(34) the ability of the bacteria to cause disease at diferent
spatial scale in raptorial birds including endangered Egyptian
vultures, largely remains unknown requiring further studies
in reference to the population health of the species.
Te presence of Klebsiella spp. from substantially diferent
spatial and biological sources suggests a widespread distribu-
tion, adaptation and pathogenicity in diverse host species.
Tis study frstly documents Egyptian vultures, which can
migrate thousands of kilometers (35), as host and carrier of
Figure 1: Genotypic confrmation of K. pneumoniae based on 16S-23S
rDNA internal transcribed spacer (ITS) region.
Figure 2: Genotypic detection of serotype K5 of K. pneumoniae using
serotype specifc primers.
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 127 Klebsiella pneumoniae Egyptian vultures and Steppes eagles
serotype K5. Tis fnding is epidemiologically important as
bacteria from vultures can potentially spread to other wildlife
or human populations passively, possibly causing diseases
(3, 1 and 36). Te raptors studied were apparently healthy
as no visible clinical symptoms could be observed suggest-
ing K. pneumoniae can exist as commensals in their gastro-
intestinal tract. Reported prevalence of 7.29% (n=96) of the
K5 serotype, associated with pneumonia in dromedary cam-
els (Camelus dromedarius) in and around Bikaner city (37),
proposes additional novel host species, which can contribute
to this microbial ecological niche of carcass dumps receiving
such pneumonic camel carcasses.
Strains of Klebsiella spp. are naturally resistant to amino-
penicillins (ampicillin and amoxicillin) and other penicil-
lins due to chromosomal class-A β-lactamase production,
but susceptible to most other β-lactam antibiotics (38). Te
observed bacterial resistance to antibiotics belonging to mac-
rolide, cyclic polypeptides, β-lactams (including penem sub-
group), rifamycin, sulfonamide, and specially combinations of
β-lactam/β-lactamase inhibitor classes is signifcant since the
sampled migratory raptors were wild, and in all probability,
were without any prophylactic history concerning antibiot-
ics. Te probability of ingestion of residual antimicrobials
by the raptors, in carcasses of livestock, administered prior
to their death was high as many carcasses originated from
veterinary clinics. Practically it is possible that such regu-
lar and long exposure of residual antibiotics to the raptors
could be attributed to the development of resistance in the
K. pneumoniae isolates. A second alternative however impor-
tant explanation is that the transfer of resistance genes from
resistant bacteria to susceptible ones was possible as many of
the bacterial resistance genes are plasmid borne. Escherichia
coli strains are potentially harbored by wild birds of diverse
species and spread conjugative resistance plasmids (R plas-
mids) (39, 40). However Klebsiella spp. are poorly studied in
this context even if the bacteria harbor widely distributed R
plasmids, which can be transferred to other bacterial strains
and species (41) conferring resistance to quinolones and
β-lactams antibiotics (42). Such transmission of R plasmids
to distant geographical locations may possibly be facilitated
by diferent dynamically interacting populations of migrat-
ing scavenging raptors, which although breed in diferent
geographic locations still winter in similar ecological niches
of carcass dumps such as Jorbeer. Te third reason for the ob-
served resistance to antimicrobials could be the ingestion of
already resistant K. pneumoniae originating from the carcasses
of livestock species including camels, which are susceptible
hosts of K. pneumoniae, as food sources are known to afect
the gastro-intestinal micro-fora. It is also possible that all
the three proposed mechanisms responsible for the observed
antimicrobial resistance, as mentioned above, were not mutu-
ally exclusive and probably worked simultaneously. Further
expansions of this study to investigate these mechanisms may
illuminate exactly which processes govern the occurrence of
the observed resistance in these wild raptors in this study. Te
ability of K. pneumoniae to spread intercontinentally (43), the
presence of New Delhi metallo-β-lactamase-1 (NDM-1)
enzymes in K. pneumoniae strains isolated from India con-
ferring resistance to carbapenem antibiotics (44), and wide-
spread distribution of such strains (45) are rational reasons to
study the role of these migratory raptors in epidemiological
perspectives of K. pneumoniae and its serotypes.
Te populations of birds under study, which are declining,
thrive in similar ecological niches of carcass dumps sharing
almost the same food sources, which may facilitate potential
spread of pathogenic bacteria such as K. pneumoniae among
the birds’ populations. Also since the species under study are
wild and migratory any potential disease or outbreak caused
by the virulent strains of K5 and resistance to multiple an-
tibiotics may probably go unnoticed and spread to distant
geographic locations. Tus focused molecular and clinical
studies are needed to ascertain pathogenicity, adaptability
and virulence of the K5 serotypes to ascertain if these avian
genera are biological hosts or mere mechanical vectors. More
exhaustive epidemiological studies are needed to study the
prevalence and pathogenicity of other potentially pathogenic
serotypes of Klebsiella among these raptors.
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