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RASOOL, MUHAMMAD HIDAYAT

ALMATROUDI, AHMAD

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KHURSHID, MOHSIN

Polish Journal of Microbiology
2020, Vol. 69, No 2, 231–234
https://doi.org/10.33073/pjm-2020-017
SHORT COMMUNICATION
* Corresponding author: M. Khurshid, Department of Microbiology, Government College University Faisalabad, Pakistan;
 e-mails: mohsin.mic@gmail.com, mohsinkhurshid@gcuf.edu.pk
© 2020 Zeeshan Taj et al.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons.
org/licenses/by-nc-nd/4.0/).
Acinetobacter baumannii is the most prevalent spe-
cies of genus Acinetobacter that caused various nosoco-
mial infections in clinical settings. A. baumannii is quite 
ubiquitous and has been found in water, air, and soil. 
Although the studies related to the animal infections 
caused by A. baumannii are limited, the reports have 
highlighted the involvement of Acinetobacter species in 
respiratory, urinary, bloodstream, and wound infections 
with an attributable mortality of 47% in pets (Pomba 
et al. 2017). The therapeutic management of carbape-
nem-resistant A. baumannii (CRAB) is challenging in 
clinical medicine (Sohail et al. 2016; Khurshid et al. 
2017). The emergence of multidrug-resistant CRAB 
isolates has been increasingly reported and is mainly 
associated with the acquisition of the blaNDM gene and 
overexpression of the blaOXA-23 gene in bovines and 
equines (Poirel et al. 2012; Smet et al. 2012; Zhang et al. 
2013). However, the majority of carbapenem-resistant 
phenotypes in A. baumannii isolates from the pets are 
mainly linked with the increased expression of the 
intrinsic genes (Ewers et al. 2017).
The data regarding the mechanisms underlying the 
antimicrobial resistance and molecular epidemio logy of 
Acinetobacter species from the veterinary origin are lim-
ited compared to the A. baumannii strains from humans. 
However, the studies have revealed that the A. bau - 
mannii isolates from veterinary sources may harbor 
identical antimicrobial resistant determinants as well 
as share the identical clonal lineages as human strains 
suggesting a common source of infection (Zordan et al. 
2011; Puntener-Simmen et al. 2019). Here, we have 
described a CRAB isolate harboring the blaOXA-23 gene 
from a pet cat suffering from urinary tract infection.
A two-years-old pet cat was brought to our pet clinic 
with dysuria and hematuria. The urine sample was 
aseptically collected, which showed significant bacteri-
uria, and A. baumannii was solely obtained. The cat was 
having a history of persistent fever, pyuria, anorexia, 
weight loss, postural changes, and mood disorders from 
the last three months, which were previously attempted 
to treat with multiple courses of antimicrobial agents 
empirically. Initially, the oral amoxicillin-clavulanate 
Extensively Drug-resistant Acinetobacter baumannii Belonging
to International Clone II from A Pet Cat with Urinary Tract Infection;
The First Report from Pakistan
ZEESHAN TAJ1, 2, MUHAMMAD HIDAYAT RASOOL1, AHMAD ALMATROUDI3,
MUHAMMAD SAQALEIN1, 2 and MOHSIN KHURSHID1* 
1 Department of Microbiology, Government College University Faisalabad, Pakistan
2 Pet Care Clinic, Faisalabad, Pakistan
3 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Qassim, Saudi Arabia
Submitted 30 December 2019, revised 26 March 2020, accepted 29 March 2020
A b s t r a c t
The carbapenem-resistant Acinetobacter baumannii (CRAB) has got global attention as a notorious nosocomial pathogen. This study 
describes a case of urinary tract infection in a 2-years old pet female cat infected with A. baumannii. The susceptibility profiling, screening 
for the resistance determinants, and the multilocus sequence typing was performed. The A. baumannii isolate was found to harbor the 
blaOXA23-like gene and corresponded to International clone II that has been widely reported to be involved in human infections. The 
study proposes that the pets may contribute towards the spread of clinically relevant antimicrobial-resistant pathogens.
K e y  w o r d s: MLST, sequence types, carbapenemases, Acinetobacter baumannii, companion animals
Taj Z. et al. 2232
suspension was administered at a dose rate of 62.5 mg/
cat PO twice daily for 14 days, followed by ciprofloxacin 
at a dose rate of 6 mg/kg PO q12h for 10 days.
The A. baumannii isolate was identified by amplifi-
cation of the recA gene and ITS region in a multiplex 
PCR as described previously, as well as the amplifi-
cation of the blaOXA-51 gene (Khurshid et al. 2017; 
Khurshid et al. 2020). The broth microdilution method 
was used to determine the minimum inhibitory con-
centrations (MICs) according to the CLSI guidelines 
(CLSI 2015). The genes encoding the carbapenem 
resistance and the presence of insertion element i.e., 
ISAba1, were detected using PCR as described previ-
ously using specific primers (Khurshid et al. 2017). 
The PCR was performed to detect the presence of 16S 
rRNA methyltransferase genes (armA, rmtA, rmtB, 
rmtC, rmtD, and rmtE) and aminoglycoside modifying 
enzymes (AMEs) i.e., aphA1, aphA6, aadB, aadA1, and 
aacC1 and tetracycline and sulfonamide resistant genes 
including tetA, tetB, sul1, sul2, and sul3 genes (Khur-
shid et al. 2019). The isolates were also screened for 
plasmid-mediated quinolone resistance genes (qnrA, 
qnrB, and qnrS) as well as mutations in the quinolone 
resistance-determining region by sequencing gyrA and 
parC gene (Gu et al. 2015). The multi-locus sequence 
typing (MLST) was performed using primers recom-
mended by the MLST database for A. baumannii fol-
lowing the Pasteur scheme.
The strain was susceptible only to colistin (MIC 
0.5 µg/ml), and tigecycline (MIC 1 µg/ml). The higher 
MICs of imipenem (MIC 16 µg/ml), meropenem (MIC 
32 µg/ml), ceftazidime, cefotaxime, ceftriaxone (MIC 
64 µg/ml), cefepime (MIC 32 µg/ml), piperacillin-tazo-
bactam (MIC 128/4 µg/ml), and ampicillin-sulbactam 
(MIC 64/32 µg/ml) were linked with the production of 
blaOXA-23 (Opazo et al. 2012; Khurshid et al. 2017). 
The resistance to aminoglycoside i.e., MICs of amikacin 
(MIC 1024 µg/ml), gentamicin, and tobramycin (MIC 
512 µg/ml) was attributed to the presence of 16S rRNA 
methyltransferase genes i.e., the armA gene as well as 
AMEs i.e., aphA6, aadB, and aacC1. Moreover, the MIC 
of trimethoprim-sulfamethoxazole was 16/304 µg/ml 
attributed to the presence of the sul2 gene. The A. bau-
mannii isolates showed resistance to tetracycline/doxy-
cycline with a doxycycline MIC equal to 128 µg/ml, and 
it was related to the presence of the tetB gene. The strain 
was found resistant to ciprofloxacin (MIC 16 μg/ml), 
which was attributed to the mutation (Ser83Leu) in the 
gyrA gene. The genes conferring resistance to different 
antimicrobial agents that were found in the A. baumannii 
strain are summarized in Table I. The ISAba1 was found 
upstream to the blaOXA-51 and blaOXA-23 genes.
The concerns related to the possible threats of the 
blaOXA-23 harboring CRAB among the pets and other 
farm animals have been increasing (Ewers et al. 2017). 
The information on A. baumannii in veterinary settings 
is, however, limited, and data related to the compari-
son of strains isolated from the humans and veterinary 
sources are quite inadequate (van der Kolk et al. 2019). 
From Pakistan, this is the very first report of exten-
sively drug-resistant (XDR) CRAB isolates harboring 
the acquired the bla-OXA-23 and armA genes from an 
infected pet cat, which drives the attention towards the 
possible transmission of these XDR pathogens from 
the companion animals to humans.
The blaOXA-23 gene is a major cause of carbap-
enem resistance throughout the world; therefore, it can 
be considered a virulence marker and is located on the 
chromosome as well as on the plasmids. Moreover, 
the studies have found a strong correlation between 
the occurrence of the blaOXA-23 gene and multidrug-
resistant phenotypes (Pomba et al. 2014; Zowawi et al. 
2015; Khurshid et al. 2017).
The MLST has shown that the A. baumannii strain 
belonged to the sequence type 2 (ST2), and the eBURST 
analysis has revealed that it corresponded to the inter-
Aminoglycosides 16S rRNA methyltransferase genes armA Amikacina, Gentamicinb, Tobramycinb
 Aminoglycoside modifying enzymes aphA6, aadB, and aacC1
Carbapenems Oxacillinases blaOXA-23 Imipenemc, Meropenemd, Ceftazidimee,
   Cefotaximee, Ceftriaxonee, Cefepimef,
   Piperacillin-tazobactamg,
   Ampicillin-sulbactamh
Fluoroquinolones Quinolones Resistance Determining gyrA gene mutation (Ser83Leu) Ciprofloxacini
 Region (QRDR)
Sulfonamides Dihydropteroate synthase Sul2 Sulfamethoxazole-Trimethoprimj
Tetracyclines Tetracycline efflux MFS transporter tetB Doxycyclinek
Table I
Resistance genes detected in the A. baumannii strain isolated in a urine sample from the urinary tract infection suffering cat.
a MIC 1024 µg/ml, b MIC 512 µg/ml, c MIC 16 µg/ml, d MIC 32 µg/ml, e MIC 64 µg/ml, f MIC 32 µg/ml, g MIC 128/4 µg/ml, h MIC 64/32 µg/ml, 
I MIC 16 μg/ml, j MIC 16/304 µg/ml, k MIC 128 µg/ml
Antibiotic category Mechanism Resistance associated gene Resistance phenotypes
XDR A. baumannii from pet cat2 233
national clonal lineage 2. The study conducted by 
Tada and his colleagues concluded that there is world-
wide dissemination of this clone also harboring the 
blaOXA-23 and armA genes but does not suggest the 
human-to animal transmission (Tada et al. 2015). Nota-
bly, the A. baumannii ST2 has been extensively isolated 
from humans, while some of the recent reports have 
also indicated the presence of ST2 in pets (Puntener-
Simmen et al. 2019). The carbapenem-resistant isolates 
in these studies were found to possess the intrinsic 
blaOXA-51 gene solely or accompanied by the acquired 
the blaOXA-23-like genes. Interestingly, the A. bau-
mannii isolates were reported among the pets living in 
the community (Lupo et al. 2017). Although the data 
is quite limited regarding the carriage of Acinetobacter 
species beyond the veterinary clinical settings, more 
than a few studies during the recent few years have 
detected the A. baumannii isolates in the community 
among domestic birds, dogs, livestock, and other large 
animals. These studies specify that the incidence of 
A. baumannii infections among animals is increasing 
and these animals may serve as a reservoir for A. bau-
mannii, particularly carbapenem-resistant strains, due 
to their selective advantage compared to the susceptible 
strains (Pomba et al. 2014; van der Kolk et al. 2019).
This study has reported an extensively drug-resistant 
A. baumannii, harboring the blaOXA-23 gene and other 
resistant associated genes isolated from a  companion 
animal previously treated with multiple empirical anti-
microbial courses. The infected pets may contribute to 
the pool of multidrug-resistant clinically relevant bacte-
ria and their interaction with the human may transmit 
these pathogens to humans. The extensive epidemio-
logical studies are essential for a better understanding of 
the extent of distribution, risk factors, and the directions 
of transmission of these multidrug-resistant strains.
   ORCID
Mohsin Khurshid https://orcid.org/0000-0002-3196-2857
Funding
This work was supported by the Higher Education Commis-
sion (HEC), Pakistan grant number 5679/Punjab/NRPU/R&D/
HEC/2016.
Conflict of interest
The authors do not report any financial or personal connections 
with other persons or organizations, which might negatively affect 
the contents of this publication and/or claim authorship rights to 
this publication.
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English

Extensively Drug-resistant Acinetobacter baumannii Belonging to International Clone II from A Pet Cat with Urinary Tract Infection; The First Report from Pakistan

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