biology

Article
Emergence of Colistin and Carbapenem Resistance in
Extended-Spectrum β-Lactamase Producing Klebsiella
pneumoniae Isolated from Chickens and Humans in Egypt
Walid Elmonir 1, *, Norhan K. Abd El-Aziz 2, * , Yasmine H. Tartor 2, * , Samar M. Moustafa 3 ,
Etab M. Abo Remela 4,5 , Radwa Eissa 6 , Hosam A. Saad 7 and Ahmed Abdel Tawab 8

1 Department of Hygiene and Preventive Medicine (Zoonoses), Faculty of Veterinary Medicine,

Kafrelsheikh University, Kafrelsheikh 33516, Egypt
2 Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt
3 Department of Zoonoses, Faculty of Veterinary Medicine, Benha University, Benha 13518, Egypt;
samar.mmoustafa@yahoo.com
4 Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine,
Kafrelsheikh University, Kafrelsheikh 33516, Egypt; etab_mh@yahoo.com
5 Department of Biology, College of Science, Taibah University, Madina 42353, Saudi Arabia
6 Department of Microbiology and Immunology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt;



radwa.eissa80@yahoo.com

 7 Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
h.saad@tu.edu.sa
Citation: Elmonir, W.; Abd El-Aziz, 8 Department of Microbiology, Faculty of Medicine, Al Azhar University, Cairo 11884, Egypt;
N.K.; Tartor, Y.H.; Moustafa, S.M.;
tawwab.206@azhar.edu.eg
Abo Remela, E.M.; Eissa, R.; Saad,
* Correspondence: walid.elmonir@gmail.com (W.E.); nourhan_vet@zu.edu.eg (N.K.A.E.-A.);
H.A.; Tawab, A.A. Emergence of yasminehtartor@zu.edu.eg (Y.H.T.)
Colistin and Carbapenem Resistance
in Extended-Spectrum β-Lactamase Simple Summary: Carbapenems and colistin are reserved as the last-resort treatments of multidrug-
Producing Klebsiella pneumoniae
resistant (MDR) infections in humans. Consequently, the emergence of carbapenem and colistin-
Isolated from Chickens and Humans
resistant Klebsiella pneumoniae (K. pneumoniae) in poultry, contact workers and hospitalized patients is
in Egypt. Biology 2021, 10, 373.
of grave concern for therapeutic options, and no data are available supporting this assumption on
https://doi.org/10.3390/
a regional or countrywide scale. We investigated the frequency and typing of extended-spectrum
biology10050373
β-lactamase (ESBL) and carbapenemase-producing K. pneumoniae (ESBLK and CPK) in hospitalized
Academic Editors: patients, chickens from 10 poultry farms and their environment (water, food and litter) and farm
Chrissoula Voidarou, Athina S. Tzora workers in Egypt. All isolates from patients (13/90, 14.4%), workers (5/22, 22.7%), chickens (9/100,
and Georgios Rozos 9%) and the environment (10/60, 16.7%) harbored a single or multiple β-lactamase genes, blaSHV ,
blaTEM , blaCTX-M1 and blaOXA-1 , often in combination with carbapenemase genes (blaVIM , blaNDM-1 or
Received: 1 April 2021 blaIMP ; 45.9%), the mcr-1 gene (18.9%) or both (13.5%). Enterobacterial repetitive intergenic consensus
Accepted: 22 April 2021 (ERIC)-PCR genotyping highlighted potential inter and intraspecies clonal dissemination in the study
Published: 26 April 2021 area. The increased frequency and genetic relatedness of ESBLK and CPK from chickens and humans
pose a public health threat that urges more prudent use of antimicrobials in chicken farms to avoid
Publisher’s Note: MDPI stays neutral
the propagation and expansion of both ESBLK and CPK from the chicken sources to humans.
with regard to jurisdictional claims in
published maps and institutional affil-
Abstract: This study investigated the frequency of carbapenem and colistin resistance in ESBL-
iations.
producing K. pneumoniae (ESBLK) isolates recovered from chickens and their environment, contact
farm workers and hospitalized patients in Egypt. Further, the phenotypic and genotypic relationships
between the community and hospital-acquired K. pneumoniae isolates in the same geographical area
were investigated. From 272 total samples, 37 (13.6%) K. pneumoniae isolates were identified, of
Copyright: © 2021 by the authors.
which 20 (54.1%) were hypervirulent. All isolates (100%) were multidrug-resistant (MDR) with
Licensee MDPI, Basel, Switzerland.
multiple antibiotic resistance (MAR) indices ranging from 0.19 to 0.94. Colistin-resistant isolates
This article is an open access article
(18.9%) displayed colistin MIC values >2 µg/mL, all harbored the mcr-1 gene. All isolates from
distributed under the terms and
conditions of the Creative Commons
patients (13/90, 14.4%), workers (5/22, 22.7%), chickens (9/100, 9%) and the environment (10/60,
Attribution (CC BY) license (https:// 16.7%) harbored a single or multiple β-lactamase genes, blaSHV , blaTEM , blaCTX-M1 and blaOXA-1 , often
creativecommons.org/licenses/by/ in combination with carbapenemase genes (blaVIM , blaNDM-1 or blaIMP ; 45.9%), the mcr-1 gene (18.9%)
4.0/). or both (13.5%). Enterobacterial repetitive intergenic consensus (ERIC)–PCR genotyping revealed 24

Biology 2021, 10, 373. https://doi.org/10.3390/biology10050373 https://www.mdpi.com/journal/biology

Biology 2021, 10, 373 2 of 16

distinct ERIC types (ETs) with a discrimination index of 0.961. Six ETs showed clusters of identical
isolates from chicken and human sources. The increased frequency and genetic relatedness of ESBLK
and carbapenemase-producing K. pneumoniae (CPK) from chickens and humans pose a public health
threat that urge more prudent use of antimicrobials in chicken farms to avoid the propagation and
expansion of both ESBLK and CPK from the chicken sources to humans.

Keywords: K. pneumoniae; colistin; ESBL; carbapenems; chickens; humans; environment

1. Introduction
Klebsiella pneumoniae (K. pneumoniae) is a pervasive nosocomial and community
associated-pathogen causing a wide range of infections in humans and animals [1,2].
Bacterial invasion in livestock animals poses a potential hazard to public health, as infected
animals act as a reservoir of multidrug-resistant (MDR) isolates [1,3]. In Egypt, no regula-
tions are controlling the use of antimicrobials in animals [4], which may be used as growth
promoters or for the prevention and treatment of zoonotic diseases. This supports the
evidence linking consumption of antimicrobials in livestock animals to their resistance in
humans [5].
The emergence of extended-spectrum β-lactamase-producing K. pneumoniae (ESBLK)
in Egypt is of serious concern. Previous studies suggested that food-producing animals,
particularly chickens, have been considered a possible source for transmission of ESBLK
to humans [6,7]. However, integrated studies on chickens and their environment, contact
workers and hospitalized patients in the same area are still missing.
Carbapenems and polymyxin E (colistin) are considered the last-resort for treating
infections caused by MDR K. pneumoniae isolates, particularly those producing extended-
spectrum β-lactamase (ESBL) enzymes [8,9]. Carbapenem-resistant K. pneumoniae exhibited
coresistance to a range of critically relevant antimicrobial classes resulting in few therapeu-
tic possibilities [10]. The emergence of carbapenemases together with the mcr-1 colistin
resistance gene constitutes a global risk to public health [11,12]. There are three mecha-
nisms by which K. pneumoniae employs carbapenem resistance: (i) enzymatic hydrolysis
via carbapenemases enzymes, (ii) overexpression of the efflux pump system and (iii) loss
of porin expression [13]. Carbapenemases (i.e., Ambler molecular classes A, B and D
β-lactamases) represent the most prevalent mechanism of carbapenem resistance. They
hydrolyze a wide variety of β-lactams including penicillins, cephalosporins, monobac-
tams, carbapenems and β-lactamases inhibitors through carbapenemase encoding genes,
mainly of class B metallo-β-lactamases (MBL), including imipenem metallo-β-lactamases
(blaIMP ), New Delhi metallo-β-lactamases (blaNDM ) and Verona integron-encoded metallo-
β-lactamases (blaVIM ) [14]. Carbapenemases are mostly detected in K. pneumoniae, with
a lower extent in other enterobacterial species [15]. Although carbapenem and colistin-
resistant K. pneumoniae isolates have been documented in numerous human studies in
Egypt [8,16–18], no data are available on this issue from a veterinary overview. More-
over, the role of poultry and their environment in the maintenance and transmission of
carbapenem-and colistin coresistant isolates is poorly explored.
Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)
has been documented to have a virtuous differentiation power for molecular characteriza-
tion and genotyping of bacterial isolates from human and animal origins [19,20]. Therefore,
this study aimed to determine the frequency of carbapenem and colistin resistance in
ESBLK isolates recovered from chickens and their environment, contact farm workers and
hospitalized patients in Kafrelsheikh Governorate, Egypt, and to investigate the phenotypic
and genotypic relationships between the community and hospital-acquired K. pneumoniae
isolates in the same geographical area.
Biology 2021, 10, 373 3 of 16

2. Materials and Methods

2.1. Study Design and Sampling
For determination of the potential risk for chicken-human cross-transmission, we
detected the occurrence of carbapenemase and colistin resistance in ESBLK in hospitalized
patients, workers who were in close contact with broiler chickens, broilers and their envi-
ronment. The protocol of this study was approved by the ethics committee of Kafrelsheikh
University, Kafrelsheikh, Egypt (KFS-2019/3). Written informed consent was attained from
the patients, poultry workers and owners of farms for the participation in this study and
the publication of any potentially identifiable data.
The sampling was carried out during the period from April 2019 to November 2019.
Sputum and urine samples were collected from 90 hospitalized patients showing man-
ifestations of respiratory and urinary tract infections. Moreover, 22 stool samples were
collected from contact workers from 10 different broiler farms in Kafrelsheikh Governorate,
Egypt. Additionally, 100 lung and trachea samples were collected from broilers showing
respiratory manifestations. Furthermore, 60 environmental samples were included: two
pooled samples for water, food and litter (n = 2 each) per farm were separately screened for
carbapenem and colistin resistance in ESBLK.

2.2. Isolation and Identification of K. pneumoniae

Samples were directly streaked onto HiCrome™ Klebsiella selective agar (Himedia,
India) at 37 ◦ C for 24 h. The presumptive purple colonies were confirmed by growth on
MacConkey’s agar and eosin methylene blue (Oxoid, Cambridge, UK) agar media and iden-
tified by biochemical tests [21]. The hypermucoviscous phenotype of colonies on an agar
plate has been defined by a positive string test [22]. The 16S-23S rDNA internal transcribed
spacer (ITS) of K. pneumoniae isolates were amplified with the species-specific primers
reported previously [23]. K. Pneumoniae isolates grown on Columbia sheep blood agar
(Oxoid, Cambridge, UK) plates for 24 h were subjected to matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS) (BioMeri’eux, Marcy I’Etoile,
France) identification using αcyano-4-hydroxycinnamic acid matrix solution as previously
described [24]. MALDI-TOF MS running MYLA 3.1.0-15 software (BioM’erieux, Inc., Marcy
I’Etoile, France) analyzed, compared the generated spectrum for each tested isolate with a
library of standard reference spectra and calculated the confidence values.

2.3. Antimicrobial Susceptibility Testing

The disc diffusion test [25] was used for testing the susceptibility of K. pneumoniae
isolates to a range of antimicrobials approved for both human and livestock use [26],
including ampicillin (10 µg), amoxicillin-clavulanic acid (30 µg), ceftazidime (30 µg),
cefepime (30 µg), amikacin (30 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), imipenem
(10 µg), azithromycin (15 µg), aztreonam (30 µg), gentamicin (10 µg), tetracycline (30 µg),
chloramphenicol (30 µg), nitrofurantoin (300 µg), trimethoprim-sulfamethoxazole (25 µg)
and colistin (25 µg).
The broth microdilution method [27] was used for the determination of the minimum
inhibitory concentrations (MICs) of colistin (Sigma-Aldrich, Seelze, Germany). A K. pneu-
moniae ATCC 700603 reference strain was included as quality control in the disc diffusion
test and the fully colistin-susceptible E. coli ATCC 25922 and the mcr-1-positive E. coli
NCTC 13846 with a colistin MIC of 4 mg/L were used in broth microdilution method.The
results of antimicrobial susceptibility testing were interpreted according to the Clinical and
Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility
Testing (EUCAST) joint subcommittee [28]. The multiple antibiotic resistance (MAR) index
for each isolate was calculated according to Tambekaret al. [29].

2.4. Phenotypic Screening for ESBLs and Carbapenemases Production

ESBL production was screened using the CLSI ESBL confirmatory test (double disc
synergy test) with cefotaxime (CTX, 30 µg) and ceftazidime (CAZ, 30 µg) antimicrobial
Biology 2021, 10, 373 4 of 16

discs alone, and in combination with clavulanic acid (CA, 10 µg) (Becton, Dickinson, Sparks,
MD, USA). A positive ESBL production was considered when an increase in the inhibition
zone diameter for CTX or the CAZ disc + CA was ≥5 mm of the diameter around the CTX
or CAZ [30]. The modified Hodge test (MHT) was performed to screen for carbapenemase
production according to CLSI guidelines [31]. The EDTA-imipenem synergy test was used
for MBL carbapenemase identification [32]. K. pneumoniae ATCCBAA-1705 (carbapenemase-
positive) and ATCC700603 (SHV-18 producer) reference strains were used as positive
controls. A β-lactamase-negative E. coli ATCC25922 strain was used as a negative control.
The ESBL- and carbapenemases-producing isolates were subjected to further analysis
including detection of the resistance determinants, mcr-1gene in colistin-resistant isolates
and ERIC-genotyping.

2.5. Detection of ESBL, Carbapenemase-Encoding Genes, and Colistin Resistance

Determinant mcr-1
Genomic DNA was extracted from overnight cultures of all presumptive ESBLK and
carbapenemase-producing K. pneumoniae (CPK) isolates using QIAamp DNA Mini kit
(Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The pu-
rity and concentrations of extracted DNA were assessed by using a nanodrop™ 2000 and
2000c (Thermo Fisher Scientific, Waltham, MA, USA). The ESBL genes (blaSHV , blaCTX-M ,
blaTEM and blaOXA-1 ) were detected using the previously described multiplex PCR [33].
PCR amplification of carbapenemase genes (blaIMP , blaVIM and blaNDM1 ) was performed
using specific primers [34–36] listed in Table S1. The uniplex PCR was carried out in a
25-µL reaction mixture containing 12.5 µL of EmeraldAmp Max PCR Master Mix (Takara,
Shigino-higashi, Joto-ku, Osaka, Japan), 1 µL of each primer (20 pmol; Metabion GmbH,
Germany), 5.5 µL of nuclease-free water and 5 µL of DNA template using the Applied
Biosystems® 2720 thermal cycler (Thermo Scientific, Waltham, MA, USA). The PCR prod-
ucts were electrophoresed on 1.5% agarose gel (Applichem GmbH, Darmstadt, Germany)
containing 0.5 µg/mL ethidium bromide. The gel was photographed by Alpha Innotech gel
documentation system (Biometra GmbH, Göttingen, Germany) and the data were analyzed
through computer software.
Furthermore, colistin-resistant K. pneumoniae isolates were tested for the presence of
the mcr-1 gene using the specific primers (Table S1) as described previously [37]. Genomic
DNA from mcr-1-positive E. coli NCTC 13846 and colistin-susceptible E. coli ATCC 25922
were included in each run.

2.6. ERIC Genotyping

For analysis of fingerprinting profiles of different ESBL and carbapenemase-positive
isolates, ERIC-PCR was applied using the ERIC-1R and ERIC-2 primers (Table S1), as
previously described [38]. The ERIC-PCR band patterns were analyzed by GelJ software
v.2.0 [39]. The comparison between fingerprinting profiles was conducted using the Dice
coefficient, and a dendrogram was constructed using the unweighted pair group method
with arithmetic mean. Simpson’s discrimination index for ERIC genotyping was estimated
as previously described [40].

2.7. Data Analysis

Differences between frequencies of K. pneumonia in chicken and human samples
were assessed by the chi-squared test. A univariate logistic regression model was used
to estimate the odds ratios and significant associations between phenotypic and genetic
antimicrobial resistance profiles and source (humans versus chickens) of the K. pneumoniae
isolates. The analysis was done using SPSS v19 (IBM, Armonk, NY, USA), and associations
at a p-value ≤ 0.05 were considered significant. To determine the distribution of the isolates
from various hosts based on their antimicrobial resistance gene profiles, the occurrence of
a particular gene in the isolates was entered as binary data (0 = absent, 1 = present), and
this was used as inputs into non-metric multidimensional scaling (nMDS) analyses using
Biology 2021, 10, 373 5 of 16

the Sorensen distance. The nMDS biplot was produced to determine the association of the
genes and isolates.

3. Results
3.1. Prevalence of K. pneumoniae in Chickens, Their Environment, Contact Workers and
Hospitalized Patients in the Study Area
In total, 37 K. pneumoniae isolates were identified using the standard bacteriological
tests, PCR amplification of 16S-23S rDNA ITS region (fragment size 130 bp) and MALDI-
TOF MS. The latter provided correct species-level identification for K. pneumoniae isolates
with a confidence value of 99.9%. As presented in Table 1, K. pneumoniae isolates were
detected in 19 samples from diseased chickens (9/100; 9%) and their compartments (10/60;
16.7%), and 18 human samples including contact workers (5/22; 22.7%) and hospitalized
patients (13/90; 14.4%). Six of ten poultry farms (60%) were positive for K. pneumoniae (Ta-
ble S2). Of note, 20 isolates (54.1%) were hypervirulent, demonstrating a hypermucoviscous
phenotype as confirmed by the string test (Table 2).

Table 1. Frequency distribution of 37 multidrug-resistant K. pneumoniae isolates recovered from broiler farms and human
cases in the study area.

No. of Isolates (%) Chicken vs.

Chickens Humans Human
Isolates (%) *
C Ce TC Cw Hc TH
X2 p-Value
(n = 100) (n = 60) (n = 160) (n = 22) (n = 90) (n = 112)
MDR-K. pneumoniae (100) 9 (9) 10 (16.7) 19 (11.9) 5 (22.7) 13 (14.4) 18 (16.1) 0.9 0.3
ESBL-K. pneumoniae (100) 9 (9) 10 (16.7) 19 (11.9) 5 (22.7) 13 (14.4) 18 (16.1) 0.9 0.3
CR-K. pneumoniae (45.9) 1 (1) 2 (3.3) 3 (1.9) 4 (18.2) 10 (11.1) 14 (12.5) 12.7 >0.001
CTR-K. pneumoniae (18.9) 1 (1) 1 (1.7) 2 (1.25) 1 (4.5) 4 (4.4) 5 (4.5) 2.7 0.1
ESBL and CR-K. pneumoniae (45.9) 1 (1) 2 (3.3) 3 (1.9) 4 (18.2) 10 (11.1) 14 (12.5) 12.7 >0.001
ESBL and CTR-K. pneumoniae (18.9) 1(1) 1 (1.7) 2 (1.25) 1 (4.5) 4 (4.4) 5 (4.5) 2.7 0.1
CR and CTR-K. pneumoniae (13.5) 0 0 0 1 (4.5) 4 (4.4) 5 (4.5) - -
ESBL, CR and CTR-K. pneumoniae (13.5) 0 0 0 1 (4.5) 4 (4.4) 5 (4.5) - -
* Percentage was calculated from total isolates; C: diseased chicken; Ce: chicken environment samples; TC: total chicken samples; Cw:
chicken worker; Hc: human case; TH: total human samples; MDR: multidrug-resistant; ESBL: extended-spectrum β-lactamases; CR:
carbapenemases-producing; CTR: colistin resistant; X2 : chi-square.

Table 2. Antimicrobial resistance phenotypes, their associated genes and ERIC fingerprinting patterns of hypervirulent
ESBL and carbapenemase-producing K. pneumoniae isolates from chickens and humans in this study.

Pattern Code Antimicrobial Resistance MAR Colistin ERIC Fingerprint

Source Genotype
No. No. Pattern Index MIC (mg/L) (Band Size)
blaSHV , blaTEM ,
Amp, AMC, CAZ, FEP, IPM, NA, CIP, E12 (109, 312, 423,
1 Hc 34 0.94 16 blaVIM , blaNDM-1 ,
AK, TE, C, ATM, AZM, SXT, F, CT 878, 1000, 1225)
blaIMP , mcr-1
Amp, AMC, CAZ, FEP, IPM, NA, CIP,
2 Cw 22 0.88 8 blaSHV , blaVIM , mcr-1 E7 (872)
AK, TE, C, AZM, SXT, F, CT
Amp, AMC, CAZ, FEP, NA, IPM, CIP,
3 Cw 23 0.75 1 blaSHV , blaNDM-1 E7 (872)
TE, C, AZM, SXT, F
Amp, AMC, CAZ, FEP, NA, TE, SXT,
4 C 6 0.5 0.25 blaSHV E7 (878)
F
Amp, AMC, CAZ, FEP, CN, IPM, NA, blaSHV , blaCTX-M1 , E6 (298, 428, 860,
5 Hc 28 0.94 32
CIP, AK, TE, ATM, AZM, SXT, F, CT blaVIM , mcr-1 1148)
Amp, AMC, CAZ, FEP, CN, NA, CIP,
6 Hc 29 0.88 0.5 blaTEM E22 (157, 305, 401)
AK, TE, C, ATM, AZM, SXT, F
Biology 2021, 10, 373 6 of 16

Table 2. Cont.

Pattern Code Antimicrobial Resistance MAR Colistin ERIC Fingerprint

Source Genotype
No. No. Pattern Index MIC (mg/L) (Band Size)
Amp, AMC, CAZ, FEP, CN, IPM, NA,
7 C 4 0.81 0.25 blaTEM , blaNDM-1 E24 (159, 412)
AK, TE, C, ATM, SXT, F
Amp, AMC, CAZ, FEP, CN, IPM, NA, blaTEM , blaNDM-1 ,
8 Hc 25 0.81 1 E24 (150, 380)
CIP, AK, ATM, AZM, SXT, F blaIMP
Amp, AMC, CAZ, FEP, IPM, NA, CIP, blaSHV , blaCTX-M1 , E9 (127, 199, 294,
9 Ce 12 0.81 0.5
AK, TE, C, AZM, SXT, F blaNDM-1 , blaIMP 352, 436, 489, 748)
Amp, AMC, CAZ, FEP, IPM, NA, CIP,
10 Ce 15 0.81 0.25 blaSHV , blaVIM E20 (116, 168, 312)
AK, TE, C, ATM, SXT, F
Hc 30 2 E3 (144, 169, 320,
Amp, AMC, CAZ, FEP, CN, IPM, NA, blaSHV , blaVIM , 340, 432, 1205,
11 0.81
CIP, AK, TE, C, AZM, SXT blaNDM-1 1346)
Cw 20 1 E14 (107)
Amp, AMC, CAZ, FEP, CN, IPM, NA, blaSHV , blaCTX-M1 ,
12 Hc 32 0.81 2 E14 (117)
CIP, AK, ATM, AZM, SXT, F blaVIM , blaNDM-1
Amp, AMC, CAZ, FEP, CN, IPM, NA, blaSHV , blaOXA-1 , E19 (159, 244, 307,
13 Hc 26 0.88 16
CIP, AK, ATM, AZM, SXT, F, CT blaVIM , mcr-1 429, 680)
Amp, AMC, CAZ, FEP, CN, NA, CIP,
14 Hc 31 0.81 1 blaSHV E5 (1187, 1307)
AK, ATM, TE, AZM, SXT, F
Amp, AMC, CAZ, FEP, NA, TE, CIP,
15 Ce 19 0.69 2 blaSHV E5 (1205, 1353)
C, AZM, SXT, F
C 7 2 E5 (1196, 1320)
C 3 Amp, AMC, CAZ, FEP, CN, NA, CIP, 2 E5 (1214, 1353)
16 0.75 blaSHV
Ce 14 TE, C, AZM, SXT, F 1 E5 (1200,1333)
Cw 24 2 E5 (1214, 1353)
Amp, AMC, CAZ, FEP, NA, CIP, AK,
17 C 5 0.75 0.5 blaSHV , blaTEM E23 (157)
TE, C, AZM, SXT, F
Ce 10 0.5
18 Amp, AMC, NA, AK, TE, SXT, F 0.44 blaTEM E23 (159)
Ce 11 1
blaSHV , blaTEM ,
Amp, AMC, CAZ, FEP, CN, IPM, NA,
19 Hc 37 0.81 1 blaCTX-M1 , blaVIM , E11 (188, 306)
CIP, ATM, TE, C, AZM, SXT
blaNDM-1 , blaIMP
Amp, AMC, CAZ, FEP, NA, CIP, AK,
20 Ce 17 0.75 4 blaSHV , mcr-1 E4 (1360)
TE, C, SXT, F, CT
E18 (160, 254, 301,
Amp, AMC, CAZ, FEP, NA, AK, CN,
21 Hc 27 0.75 2 blaTEM 369, 424, 703, 884,
CIP, ATM, AZM, SXT, F
1197)
Amp, AMC, CAZ, FEP, TE, AK, C,
22 C 2 0.69 1 blaSHV E1 (679)
ATM, AZM, SXT, F
Amp, AMC, CAZ, FEP, NA, TE, C, blaTEM , blaCTX-M1 ,
23 C 9 0.69 8 E8 (891)
CN, SXT, F, CT blaOXA-1 , mcr-1
Amp, AMC, CAZ, IPM, FEP, NA, TE, blaCTX-M1 , blaVIM , E10 (107, 188, 377,
24 Hc 35 0.69 2
CIP, C, AZM, SXT blaIMP 492)
E17 (189, 266, 331,
Amp, AMC, CAZ, FEP, NA, TE, AK,
25 Ce 18 0.63 1 blaCTX-M1 373, 488, 565, 623,
C, SXT, F
964)
blaTEM , blaOXA-1 ,
Amp, AMC, CAZ, FEP, NA, IPM, CIP,
26 Hc 33 0.88 16 blaVIM , blaNDM-1 , E15 (167, 211, 316)
TE, C, ATM, AZM, SXT, F, CT
mcr-1
Amp, AMC, FEP, NA, IPM, CIP, TE, blaOXA-1 , blaVIM ,
27 Cw 21 0.63 0.25 E16 (396, 767, 1357)
AZM, C, SXT blaNDM-1
Amp, AMC, CAZ, FEP, NA, IPM, CIP, blaTEM , blaCTX-M1 ,
28 Hc 36 0.63 2 E13 (107, 878)
TE, C, SXT blaVIM , blaNDM-1
Biology 2021, 10, 373 7 of 16

Table 2. Cont.

Pattern Code Antimicrobial Resistance MAR Colistin ERIC Fingerprint

Source Genotype
No. No. Pattern Index MIC (mg/L) (Band Size)
29 Ce 13 Amp, AMC, CAZ, NA, AK, C, SXT, F 0.5 0.5 blaTEM E21(170, 320, 459)
Amp, AMC, CIP, NA, AK, ATM, SXT,
30 Ce 16 0.5 1 blaTEM , blaOXA E21(169, 320, 462)
F
31 C 8 AMP, NA, TE 0.19 0.25 blaOXA-1 E21(170, 320, 466)
E2 (297, 409, 700,
32 C 1 Amp, AMC, CAZ, FEB, TE, C, F 0.44 1 blaSHV , blaCTX-M1
1158)
PT: Phenotypic resistance pattern; AMP: ampicillin; AMC: amoxicillin-clavulanic acid; CAZ: ceftazidime; FEB: cefepime; IPM: imipenem;
NA: nalidixic acid; CIP: ciprofloxacin; AK: amikacin; CN: gentamicin; TE: tetracycline; C: chloramphenicol; ATM: aztreonam; AZM:
azithromycin; SXT: trimethoprim-sulfamethoxazole; F: nitrofurantoin; CT: colistin; MAR: multiple antibiotic resistance index; Hc: human
case; Cw: chicken worker; C: chicken; Ce: chicken environment. Patterns with bold numbers were found in more than one isolate. Isolates
with bold code numbers are hypervirulent.

3.2. Antimicrobial Resistance of K. pneumoniae Isolates

All K. pneumoniae isolates were MDR, being resistant to 3–15 of the 16 tested antimi-
crobials with MAR indices ranged from 0.19 to 0. 94. The MDR isolates were assigned
to a total of 32 distinct resistance patterns (Table 2). All hospitalized patients´ isolates
showed high levels of MDR being resistant to 10–15 antimicrobials (MAR indices ranged
from 0.63–0.94) and the farm workers’ isolates were resistant to 10–14 antimicrobials (MAR
indices ranged from 0.63–0.88). The majority of chicken isolates (66.7%) were resistant
to 11–13 antimicrobials with MAR indices 0.68–0.81. Moreover, 60% of the isolates from
environment samples were resistant to 10–13 antimicrobials (MAR indices = 0.63–0.81).
Overall, the isolates exhibited full resistance to ampicillin (100%) and 97.3% resistance
to amoxicillin-clavulanic acid. Moreover, 94.6% of the isolates exhibited resistance to
each of nalidixic acid and trimethoprim-sulfamethoxazole, whilst 86.5% were resistant to
ceftazidime and cefepime. The resistance to tetracycline was found among 83.8% of the
isolates, followed by nitrofurantoin (81.1%), ciprofloxacin (72.9%), chloramphenicol (70.3%),
azithromycin (64.9%), amikacin (59.5%) and imipenem (45.9%). The lowest resistance rate
was found to colistin (18.9%) followed by aztreonam (37.8%) and gentamicin (43.2%).
A univariate logistic regression model analysis (Table S3) showed that the acquisition
of carbapenemase encoding genes (blaVIM and blaNDM ) were more likely to be associated
with human isolates than chicken ones (OR = 10.6–36, p = 0.002–0.01). A similar association
was recorded for imipenem phenotypic resistance (77.8% vs. 15.8%, OR = 18.7, p = 0.001).
Additionally, human isolates were more likely resistant to azithromycin, aztreonam and
gentamicin antimicrobials than chicken isolates (OR = 4.4–29.1, p = 0.003–0.04) (Table S3).
There was no significant association with regard to other antimicrobial resistance genes or
tested antimicrobials among detected isolates.

3.3. Distributions of ESBLs, Carbapenemase Genes and Colistin Resistance Determinant mcr-1
among K. pneumoniae Isolates
Molecular testing revealed that among the 37 phenotypically positive isolates for
ESBLs, 17 blaSHV , 14 blaTEM , 9 blaCTX-M1 and 6 blaOXA-1 producers were identified (Table 2).
Seventeen isolates with decreased susceptibility to imipenem, and showed positive MHT,
produced MBL carbapenemases and possessed blaVIM (n = 13), blaNDM-1 (n = 12) and blaIMP
(n = 5). As depicted in Tables 1–3 and Figure 1, all CPK isolates harbored carbapenemase
genes in combination with other ESBL genes. As presented in Table 3, 25 profiles were
found for the distributions of ESBL and carbapenemase genes among ESBLK and CPK
isolates. Importantly, five human isolates (13.5%) harbored ESBLs, carbapenemases genes
and colistin resistance determinant mcr-1. Moreover, two isolates with blaTEM , blaCTX ,
blaOXA-1 and blaSHV genotype profiles from chicken and their environmental samples also
harbored the mcr-1 gene (Tables 1–3). Figure 1 presents the ESBL and carbapenemases
genes found in humans, broilers and environment samples. In the broiler farms, the
Biology 2021, 10, 373 8 of 16

predominant ESBL and carbapenemases of ESBLK and CPK isolates from chickens and
their environment were blaSHV (11/19; 57.9%) and blaNDM (2/19, 10.5%); blaSHV (4/5, 80%),
blaVIM and blaNDM (3/5; 60% each) were predominant in poultry workers. blaSHV , blaTEM
(7/13; 53.8% each) and blaVIM (9/13; 69.2%) were the most frequent genes in hospitalized
patients in the study area. As presented in Figure 1, the nMDS analysis of K. pneumoniae
isolates (n = 37) displayed a non-specific clustering pattern, as the chicken and human
isolates largely overlapped. However, 12 isolates among all analyzed origins were omitted
from the analysis as they were identical and demonstrated unique patterns.

Table 3. Distribution of extended-spectrum β-lactamase, carbapenemase genes and colistin resistance determinant mcr-1
among 37 K. pneumoniae isolates from chickens and humans.

Chickens Humans
No. Isolates Group and Genotypes Total
C Ce TC Cw Hc TH
ESBL-producing isolates
1 blaSHV 8 (21.6) 4 2 6 (75) 1 1 2 (25)
2 blaSHV , blaCTX-M1 1 (2.7) 1 1 (100)
3 blaSHV , blaTEM 1 (2.7) 1 1 (100)
4 blaTEM 5 (13.5) 3 3 (60) 2 2 (40)
5 blaTEM , blaOXA-1 1 (2.7) 1 1 (100)
6 blaCTX-M1 1 (2.7) 1 1 (100)
7 blaOXA-1 1 (2.7) 1 1 (100)
ESBL- and CR-producing isolates
8 blaSHV , blaVIM 1 (2.7) 1 1 (100)
9 blaSHV , blaNDM-1 1 (2.7) 1 1 (100)
10 blaSHV , blaVIM , blaNDM-1 2 (5.4) 1 1 2 (100)
11 blaSHV , blaCTX-M1 , blaVIM , blaNDM-1 1 (2.7) 1 1 (100)
12 blaSHV , blaCTX-M1 , blaNDM-1 , blaIMP 1 (2.7) 1 1 (100)
13 blaSHV , blaTEM , blaCTX-M1 , blaVIM , blaNDM-1 , blaIMP 1 (2.7) 1
14 blaTEM , blaNDM-1 1 (2.7) 1 1 (100)
15 blaTEM , blaNDM-1 , blaIMP 1 (2.7) 1 1 (100)
16 blaTEM , blaCTX-M1 , blaVIM , blaNDM-1 1 (2.7) 1 1 (100)
17 blaCTX-M1 , blaVIM , blaIMP 1 (2.7) 1 1 (100)
18 blaOXA , blaVIM , blaNDM-1 1 (2.7) 1 1 (100)
ESBL-producing and CTR isolates
19 blaSHV , mcr-1 1 (2.7) 1 1 (100)
20 blaTEM , blaCTX-M1 , blaOXA-1 , mcr-1 1 (2.7) 1 1 (100)
ESBL-, CR- producing and CTR isolates
21 blaSHV , blaVIM , mcr-1 1 (2.7) 1 1 (100)
22 blaSHV , blaCTX-M1 , blaVIM , mcr-1 1 (2.7) 1 1 (100)
23 blaSHV , blaOXA-1 , blaVIM , mcr-1 1 (2.7) 1
24 blaSHV , blaTEM , blaVIM , blaNDM-1 , blaIMP , mcr-1 1 (2.7) 1
25 blaTEM , blaOXA-1 , blaVIM , blaNDM-1 , mcr-1 1 (2.7) 1 1 (100)
ESBL: extended-spectrum β-lactamase; CR: carbapenemases-producing; CTR: colistin resistant; C: chicken; Ce: chicken environment; TC:
total chicken samples; Cw: chicken worker; Hc: human case; TH: total human samples.

In the univariate analysis, blaVIM and blaNDM were significantly associated with human
isolates with increased odds (p = 0.002, 0.01; OR = 36, 10.6; and 95% CI = 3.8–337.9, 1.9–60.2,
respectively).
Biology
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Figure 1. Non-metric
Figure 1. Non-metric multidimensional multidimensional
scaling biplot scaling distribution
showing the overall biplot showing the overall
of isolates fromdistribution
various hostsofbased
isolates
fromofvarious
on the frequency distribution hosts based
antimicrobial on thegenes.
resistance frequency
Eachdistribution
dot refers toofone
antimicrobial
isolate and resistance
the arrowsgenes. Each
refer to thedot
refers to one isolate and the
association of each gene with either dimension 1 or 2. arrows refer to the association of each gene with either dimension 1 or
2.

3.4.Genotyping
3.4. Genotypingand andEpidemiological
EpidemiologicalAssociation
AssociationofofChicken
ChickenandandHuman
HumanIsolates
Isolates
Thegenetic
The genetic relatedness
relatedness and and homology
homologyofofESBLKESBLK and
and CPKCPKisolates from
isolates chickens,
from their
chickens,
environment,
their environment,contact workers
contact and hospitalized
workers patients
and hospitalized in the study
patients in thearea
studywere investigated
area were in-
using ERIC-PCR.
vestigated As revealed
using ERIC-PCR. Asin Figure 2,
revealed inERIC
Figuregenotyping classified 37
2, ERIC genotyping K. Pneumoniae
classified 37 K.
isolates intoisolates
Pneumoniae three ERIC
into branches
three ERIC (EB): EBI, EBII
branches andEBI,
(EB): EBIII containing
EBII and EBIII 13,containing
14 and 10 isolates
13, 14
of chickens
and andofhumans,
10 isolates chickensrespectively.
and humans,These EBs contained
respectively. These 24 EBsdistinct ERIC24types
contained (ET)
distinct
with band sizes ranged from 100 to 1400 bp with a discrimination index
ERIC types (ET) with band sizes ranged from 100 to 1400 bp with a discrimination index of 0.961. Six ETs
(E5, E7, E14, E21, E23 and E24) showed clusters of two to six identical
of 0.961. Six ETs (E5, E7, E14, E21, E23 and E24) showed clusters of two to six identicalisolates per each ET
(Figureper
isolates 2). each
Of these, E5 contained
ET (Figure 2). Of four
these,isolates (two from
E5 contained fourchickens, one from
isolates (two fromthe chicken
chickens,
environment
one and one
from the chicken from farm workers)
environment that shared
and one from identicalthat
farm workers) antimicrobial resistance
shared identical an-
pattern (P16) (Figure 2).
timicrobial resistance pattern (P16) (Figure 2).
 Biology
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Figure 2. ERIC typing dendrogram of K. pneumoniae isolates from humans and chickens in the study area and their
Figure
associated genetic and phenotypic 2. ERIC typing
antimicrobial dendrogram
resistance of EB:
patterns. K. pneumoniae isolates
ERIC branch; fromtype;
ET: ERIC humans
blackand
dot:chickens
positiveinforthe
study area and their associated genetic and phenotypic antimicrobial resistance patterns.
antibiotic resistance gene; black square: positive for antibiotic resistance phenotype; AMR: antimicrobial resistance; AMP: EB: ERIC
ampicillin; AMC: amoxicillin-clavulanic acid; CAZ: ceftazidime; FEB: cefepime; IPM: imipenem; NA: nalidixic acid; CIP: for
branch; ET: ERIC type; black dot: positive for antibiotic resistance gene; black square: positive
antibiotic
ciprofloxacin; AK: amikacin; CN: resistance
gentamicin; phenotype; AMR:
TE: tetracycline; antimicrobial resistance;
C: chloramphenicol; AMP: ampicillin;
ATM: aztreonam; AMC: amoxicil-
AZM: azithromycin;
lin-clavulanic acid; CAZ: ceftazidime; FEB: cefepime; IPM: imipenem; NA: nalidixic acid; CIP:
SXT: trimethoprim-sulfamethoxazole; F: nitrofurantoin; CT: colistin.
ciprofloxacin; AK: amikacin; CN: gentamicin; TE: tetracycline; C: chloramphenicol; ATM: aztre-
onam; AZM: azithromycin; SXT: trimethoprim-sulfamethoxazole; F: nitrofurantoin; CT: colistin.
4. Discussion
The emergence of colistin resistance in K. pneumoniae is a global public health concern,
4. Discussion
since this
Theantibiotic
emergence is the
of last defense
colistin line against
resistance in K. carbapenem-resistant
pneumoniae is a global isolates
public[41]. So far,
health con-
there are no published data on colistin and carbapenem resistance in ESBLK
cern, since this antibiotic is the last defense line against carbapenem-resistant isolatesisolated from
[41].
chickens and their environment, community settings and hospitals in the same geographical
So far, there are no published data on colistin and carbapenem resistance in ESBLK iso-
region. In this study, K. pneumoniae was isolated from 9% of diseased chickens and 16.7%
lated from chickens and their environment, community settings and hospitals in the same
of environment samples, which is inconsistent with the findings reported in a previous
geographical region. In this study, K. pneumoniae was isolated from 9% of diseased chick-
study in Egypt, where K. pneumoniae was recovered from 35% of broiler chickens and 25%
ens and 16.7% of environment samples, which is inconsistent with the findings reported
of water samples [42]. However, the prevalence of K. pneumoniae among contact poultry
in a previous study in Egypt, where K. pneumoniae was recovered from 35% of broiler
workers (22.7%) and hospitalized patients (14.4%) in the same region was lower than lately
chickens reports
published and 25%inofEgypt
water[7,42,43]
samplesor [42 ]. However,
abroad, theChina
both in prevalence
[44] andof Taiwan
K. pneumoniae among
[45]. Hence,
contact poultry workers (22.7%) and hospitalized patients (14.4%) in the
the direct transmission may be facilitated via close contact between chickens and humans same region was
lower than lately published reports in Egypt [7,42,43] or abroad, both in
as evidenced previously by Davis et al. [46]. The prevalence differences may be attributed China [44] and
toTaiwan [45]. Hence,
the geographical the direct
location, transmission
climatic may be
circumstances, facilitated via
environmental close contact sample
contamination, between
chickens and humans as evidenced previously by
types, chicken breed, management systems and growth conditions. Davis et al. [46]. The prevalence differ-
ences may be attributed to the geographical location, climatic circumstances,
Antimicrobial resistance is an emergent affair of concern in human and veterinary environmen-
tal contamination,
medicine. sample
In this study, types, chicken
K. pneumoniae breed,
resistance was management
detected most systems
commonlyand growth condi-
to ampicillin
tions.
(100%), followed by amoxicillin-clavulanic acid (94.6%), nalidixic acid and trimethoprim-
Biology 2021, 10, 373 11 of 16

sulfamethoxazole (94.6% each) and ceftazidime and cefepime (86.5% each). Almost half
of the isolates (45.9%) showed imipenem resistance, whereas the lowest resistance rates
were observed against colistin (18.9%), aztreonam (37.8%) and gentamicin (43.2%). In
China, a previous study reported higher resistance rates of K. pneumoniae human isolates to
β-lactams, quinolones and carbapenems, while all isolates exhibited 100% susceptibility to
colistin [47]. Even worse, an earlier study in Shandong province, China documented pan
drug-resistant K. pneumoniae isolates from both patients and chickens [48]. This disparity
in resistance may be related to the antimicrobial agent frequently used for treatment in
diverse geographical areas.
K. pneumoniae is one of the foremost imperative causes of MDR infections all over
the world, resulting in inflated healthcare expenses and high mortalities [49]. Here, all
K. pneumoniae isolates were MDR, being resistant to 3–15 commonly used antimicrobial
agents either in Egypt or worldwide of diverse antimicrobial classes. Similarly in Indonesia,
all K. pneumoniae isolates from chicken farms showed an MDR pattern [50], whereas the
proportion of MDR K. Pneumoniae in Indonesian hospitalized patients was 54.49% [51].
However, 96% of K. pneumoniae isolates from humans and livestock including poultry
demonstrated a nonwild-type phenotype to ≥ three antimicrobial classes in rural Cam-
bodia [52]. The MDR pattern may be accredited to the unregulated use of antimicrobials
in human and animal medicine [53], the facility in purchasing antimicrobials freely with-
out prescription and the vertical or horizontal transmission of antimicrobial resistance
genes via plasmids from humans to animals and vice versa [54]. Thus, the high level of
MDR K. pneumoniae isolates warrants a commitment to establish purposeful antimicrobial
stewardship.
ESBL-producing Enterobacteriaceae are virtually resistant to all β-lactams and may
compel the use of last-resort antimicrobials as carbapenems and colistin [55]. Initially,
hospital-associated K. pneumoniae was reported as an ESBL producer and, thereafter,
community-acquired infections have emerged worldwide [56,57]. Herein, irrespective
of the source, all K. pneumoniae isolates (n = 37) were phenotypically confirmed as ESBLs.
According to PCR results, blaSHV was the slightly predominant β-lactamase gene (45.95%),
followed by blaTEM (37.84%), blaCTX-M1 (24.32%) and blaOXA-1 (16.22%). Generally, it was
documented that most K. pneumoniae isolates harbored SH-based non-ESBL β-lactamases
as blaSHV [58]. In Germany, all K. pneumoniae isolates from broilers and their environ-
ment carried the same ESBL-gene, blaSHV−2 [59]. In contrast, in Indonesia, blaTEM (100%)
was detected in all K. pneumoniae isolates of chicken origin, followed by blaCTX-M (90.9%).
However, only 9.1% of the isolates harbored blaSHV [50].
In the current study, 17 out of 37 K. pneumoniae isolates were imipenem-resistant
as well as MHT-positive (45.95%). Our CPK isolates possessed MBL carbapenemases
comprising blaVIM (76.47%), blaNDM-1 (70.59%) and blaIMP (29.41%). Concerning the source,
the dominant enzymatic mechanism of carbapenemase resistance was blaNDM (10.5%) in
broiler farms, whereas blaNDM and blaVIM (60% each) were the most frequent in poultry
workers. Moreover, blaVIM (69.2%) was the predominant gene in the hospitalized patients
in the study area. Thus, blaVIM was frequently involved in causing carbapenem resistance
in humans. Likewise, CPK occurred at a relatively high rate amongst broilers (42.86%),
drinking water (60%) and contact workers (56%) at poultry farms in Egypt, all of them
possessed blaNDM [42]. However, an earlier report in Iran [60] was discordant with our
results, in which blaNDM-1 was mostly detected in K. pneumoniae isolates from humans
(75%), while blaVIM was not found in any examined CPK isolates.
Globally, in hospitalized patients, an elevated rate of carbapenem resistance was
reported in Greece (68%; NDM, OXA-48 and KPC), followed by India (54%; NDM, OXA-48
and KPC) and eastern Mediterranean regions (54%; NDM and OXA-48) [61], the USA and
China (11% each; NDM, OXA−48 and KPC) and Africa (4%; NDM and OXA-48) [62].
In this study, cocarriage of ESBLs and carbapenem resistance genes in K. pneumoniae
isolates was 45.9%, ESBLs and colistin resistance genes was 18.9% and carbapenem and
colistin resistance genes was 13.5%. As reported previously, 90% of CPK isolates had
Biology 2021, 10, 373 12 of 16

the carbapenemase phenotype, of which 50% were ESBL-producers [63]. In France, the
coxistence of ESBL genes and the mcr-1 colistin resistance gene was reported in a hospital-
ized patient [64]. Also, a recent study in Iran encountered chromosomal-mediated colistin
resistance among CPK isolates in hospitalized patients with bloodstream infections [65].
Consistent with our results, a recent study in the United States documented the coexistence
of carbapenem and colistin resistance in 13% of K. pneumoniae isolates from hospitalized
patients [66]. The most striking finding in this study was the cocarriage of ESBLs, car-
bapenem and colistin resistance genes in 13.5% of K. pneumoniae isolates from poultry
workers (n = 1/22; 4.5%) and hospitalized patients (n = 4/90; 4.4%), as the first report
in Egypt. The extensive use of carbapenems results in selection pressure and simplifies
the spread of carbapenem-resistant isolates, for which colistin is one of the last treatment
choices. Further colistin resistance in CPK, such as the reported outbreak in hospitalized
patients in Germany [67], leaves limited treatment options, among them fosfomycin and
tigecycline. Thus, regimens of colistin combined with carbapenem display a high level of
synergism even in the existence of colistin resistance [68].
In this study, we present the first report in the genetic relatedness of community and
hospital-acquired K. pneumoniae isolates using ERIC-PCR. In this context, data fingerprints
revealed pronounced similarities between K. pneumoniae isolates recovered from chicken
farms and hospitalized patients. These may originate from a common ancestral strain,
demonstrating cross-transmission between chickens and humans. A recent study in Iran
showed immense genetic diversity among K. pneumoniae isolates from hospitalized pa-
tients [69]. Moreover, another Indian study demonstrated heterogeneous K. pneumoniae
human isolates while using ERIC-PCR, suggesting the involvement of multiple subtypes
in infection [70]. In light of our results, additional studies are needed to confirm the
transmission between poultry and humans and to explain the direction and machinery of
transmission.
The limitation of this study is due to restricted funding resources. Therefore, we
investigated the genetic relatedness among the community and hospital-acquired ESBL
producing K. pneumoniae isolates based on ERIC-PCR typing, which is a less expensive
and less laborious approach in routine diagnosis that allows rapid genotyping with a high
discriminatory potential, and thus is more suitable on a local scale in developing countries.
However, using advanced techniques such as multilocus sequence typing (MLST) and
pulsed-field gel electrophoresis (PFGE) would add more sensitivity and accuracy to the
results and would allow comparison among different typing methods simultaneously.
Future studies are warranted in this direction.

5. Conclusions
This is the first, at least in Egypt, of the coexistence of ESBLs, carbapenem and col-
istin resistance in K. pneumoniae isolates recovered from poultry workers and hospitalized
patients. Moreover, our findings demonstrate great similarities between community and
hospital-acquired K. pneumoniae isolates, which warrants the need to improve farm man-
agement strategies and implementation of proper hygiene practices in the study area.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/biology10050373/s1.Table S1: Oligonucleotide primer sequences used for PCR assays. Table S2:
Frequency distribution of K. pneumoniae isolates recovered from broiler farms and human workers in
the study area. Table S3: Source associated variations in phenotypic and genetic antibiotic resistance
traits of K. pneumoniae isolates from chickens and humans in this study.
Author Contributions: Conceptualization, W.E., N.K.A.E.-A. and Y.H.T.; methodology, W.E., N.K.A.E.-A.
and Y.H.T.; validation, S.M.M., E.M.A.R., R.E., H.A.S. and A.A.T.; formal analysis, W.E.; investigation,
W.E., N.K.A.E.-A., Y.H.T., S.M.M., E.M.A.R., R.E., H.A.S. and A.A.T.; data curation, W.E., N.K.A.E.-A.
and Y.H.T.; writing—original draft preparation, N.K.A.E.-A. and Y.H.T.; writing—review and editing,
N.K.A.E.-A., Y.H.T. and W.E.; project administration and funding acquisition, H.A.S. and A.A.T. All
authors have read and agreed to the published version of the manuscript.
Biology 2021, 10, 373 13 of 16

Funding: This research received no external funding.

Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Ethics Committee of Kafrelsheikh University,
Kafrelsheikh, Egypt (KFS-2019/3).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study. Written informed consent has been obtained from the patient(s) to publish this paper.
Acknowledgments: We thank Taif University researchers supporting project TURSP-2020/07, Taif
University, Taif, Saudi Arabia. We thank the staff members of Faculty of Veterinary Medicine,
Kafrelsheikh University and Zagazig University, Egypt for their technical support during this study.
Conflicts of Interest: The authors have no conflict of interest to declare.

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