Genotypic Characterization of Carbapenem-Resistant Klebsiella pneumoniae Isolated from an Egyptian University Hospital

Abstract

Citation: Taha, M.S.; Hagras, M.M.; Shalaby, M.M.; Zamzam, Y.A.; Elkolaly, R.M.; Abdelwahab, M.A.; Maxwell, S.Y. Genotypic Characterization of Carbapenem-Resistant Klebsiella pneumoniae Isolated from an Egyptian University Hospital. Pathogens 2023, 12, 121.

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Citation: Taha, M.S.; Hagras, M.M.;
Shalaby, M.M.; Zamzam, Y.A.;
Elkolaly, R.M.; Abdelwahab, M.A.;
Maxwell, S.Y. Genotypic
Characterization of Carbapenem-
Resistant Klebsiella pneumoniae
Isolated from an Egyptian University
Hospital. Pathogens 2023,12, 121.
https://doi.org/10.3390/
pathogens12010121
Academic Editor: Longzhu Cui
Received: 12 December 2022
Revised: 4 January 2023
Accepted: 9 January 2023
Published: 11 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pathogens
Article
Genotypic Characterization of Carbapenem-Resistant
Klebsiella pneumoniae Isolated from an Egyptian
University Hospital
Marwa S. Taha 1,* , Maha M. Hagras 2, Marwa M. Shalaby 1, Yosra Abdelmonem Zamzam 2, Reham M. Elkolaly 3,
Marwa A. Abdelwahab 1and Sara Youssef Maxwell 1
1Department of Medical Microbiology and Immunology, Faculty of Medicine, Tanta University,
Tanta 31527, Egypt
2Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
3Department of Chest, Faculty of Medicine, Tanta University, Tanta 31527, Egypt
*Correspondence: marwa.taha@med.tanta.edu.eg; Tel.: +20-1222253421
Abstract:
Globally, Klebsiella pneumoniae (K. pneumoniae) has been identified as a serious source of
infections. The objectives of our study were to investigate the prevalence of multidrug-resistant
(MDR) K. pneumoniae in Tanta University Hospitals, Gharbia Governorate, Egypt; characterize their
carbapenem resistance profiles; and identify their different capsular serotypes. We identified and
isolated 160 (32%) K. pneumoniae from 500 different clinical samples, performed antimicrobial suscep-
tibility testing, and then used multiplex PCR to detect carbapenemase genes and capsular serotypes
K1, K2, K3, K5, K20, K54, and K57. We detected phenotypic carbapenem resistance in 31.3% (50/160)
of the isolates; however, molecular assays revealed that 38.75% (62/160) of isolates were carrying
carbapenemase-encoding genes. Generally, bla
OXA-48
was the prevalent gene (15.5%), followed by
bla
VIM
(15%), bla
IMP
(7.5%), bla
KPC
(4%), and bla
NDM
(3.8%). Bla
VIM
and bla
OXA-48
correlated with
phenotypic resistance in 91.67% and 88% of the isolates that harbored them, respectively. Capsular
typing showed that the most prevalent pathotype was K1 (30.6%), followed by K57 (24.2%), K54
(19.35%), K20 (9.67%), and K2 (6.45%). A critical risk to community health is posed by the high
incidence of multidrug-resistant (MDR) virulent K. pneumoniae isolates from our hospital, and our
study examines this pathogen’s public health and epidemiological risks.
Keywords:
Klebsiella pneumoniae; carbapenem resistance; capsular serotypes; bla
OXA-48
;bla
VIM
;bla
KPC
;
blaNDM;blaIMP
1. Introduction
One of the biggest pressures on healthcare systems around the world is the rising
prevalence of antibiotics-resistant clinical bacterial isolates [
1
,
2
]. Understanding the genetic
factors of antibiotic resistance is essential to stop the spread of MDR bacteria [3].
Among these MDR bacteria, K. pneumoniae is regarded as one of the top six factors
contributing to healthcare-associated infections and drug resistance [
4
]. As an opportunistic
pathogen, K. pneumoniae consists of Gram-negative bacilli and is a member of the enterobac-
terales family that primarily affects people who are immunocompromised or are admitted
to hospitals. Numerous ailments, such as sepsis, bacteremia, pneumonia, and urinary tract
infections, are attributed to K. pneumoniae [5].
A sizeable portion of illnesses brought on by Klebsiella spp. is a result of two significant
pathotypes, notably the MDR and hypervirulent (hv), which eventually produce convergent
genetic copies, termed multidrug-resistant and hypervirulent (MDRhv) Klebsiella spp. [6].
New antimicrobial-resistance genes were initially found in K. pneumoniae, and they
later spread to further pathogens: carbapenem-resistant K. pneumoniae (CRKP) genes (bla
KPC
,
bla
OXA-48
and bla
NDM-1
) are examples [
7
]. The essential pathogenic component, known
Pathogens 2023,12, 121. https://doi.org/10.3390/pathogens12010121 https://www.mdpi.com/journal/pathogens

Pathogens 2023,12, 121 2 of 14
as the capsule, an extracellular polysaccharide structure that hinders the host immune
response and shields the invading pathogens from phagocytosis, is responsible for the
increasing death and morbidity rates linked to K. pneumoniae infections [8].
Klebsiella has at least 79 different capsular varieties, with each depicting the capsular
polysaccharide’s (CPS; the K antigen) molecular structure differently. These types have been
connected to the severity of the sickness and the type of infection [
9
]. Several capsular (K)
types, mainly K1, K2, K5, K20, K54, and K57, are correlated to invasive septicemia obtained
in the community, pneumonia, and liver abscesses [
10
]. Furthermore, K3 is attributed to
rhinoscleroma [11].
Information about capsule serotypes can be quickly retrieved from whole-genome
sequence (WGS) data by typing the relevant capsule (K) biosynthesis loci [
12
]. A chromoso-
mal region of 10–30 kbp and 10–30 genes make up the K locus. The preserved genes for the
export and synthesis of capsules are found in the 5
0
-(galF, cpsACP, wzi, wza, wzb, wzc)
and 3
0
-(ugd) most areas, and they surround the genes that code for the synthesis of capsule
sugar, namely Wzy repeat-unit polymerase and Wzx capsule-specific flippase [13].
Molecular capsular typing is the main technique used to categorize K. pneumoniae
isolates, and it has outstanding consistency and can distinguish between clinical isolates [
14
].
Multiplex PCRs have been successfully used to identify the capsule repeat-unit polymerase
Wzy genes [15].
Few studies on MDR K. pneumonia capsular typing have been conducted in Egypt
[16,17]
.
Consequently, we assessed the prevalence of nosocomial MDR K. pneumoniae infections in
our tertiary care hospitals and characterized their carbapenem resistance profiles.
2. Materials and Methods
2.1. Study Design
We carried out our cross-sectional study in the Tanta University Hospitals’ Clinical
Pathology and Medical Microbiology and Immunology Department over the course of a
year, from June 2021 to June 2022. The hospitals have a combined capacity of 2040 beds,
including 130 ICU beds, and serve over 190,000 patients annually. Our study received
permission from Tanta University’s Institutional Review Board for the Faculty of Medicine
in Egypt (Approval code 35789/9/22).
2.2. Study Subjects
A total of 500 patients from Tanta University hospital’s Pediatrics, Chest, Medicine,
and Intensive Care Unit (ICU) departments were enrolled in this study. The included
patients had hospital-acquired infections (HAIS). We studied 160 clinical isolates of Klebsiella
from 500 samples from different body sites (blood, CSF, urine, wound, and sputum) of
500 patients.
2.3. Identification of Bacterial Isolates
We gathered blood, CSF, urine, wounds, and sputum samples from different infection
sites and quickly sent them to the Microbiology Department laboratory for additional
processing. First, we codified the samples, and then we cultivated aerobically at 37
◦
C
on blood agar, nutrient agar, chocolate agar, and MacConkey agar plates (Oxoid, UK) for
24–48 h. We predominantly used routine microbiological methods for the phenotypic
detection of isolated pathogens [
18
]. Thereafter, we further processed only K. pneumonia.
We verified K. pneumonia using the Vitek-2 automated system (Biomérieux, Marcy-LÉtoile,
Paris, France) in accordance with the manufacturer’s recommendations. We kept all
K. pneumoniae isolates at
−
80
◦
C in brain–heart infusion broth (20% glycerol; Oxoid, UK)
until they were needed.
2.4. Antimicrobial Susceptibility Testing and Phenotypic Detection of Carbapenemases
We performed the modified Kirby–Bauer disc diffusion method to assess the antibi-
otic susceptibility of all identified K. pneumoniae isolates on Muller–Hinton agar (Oxoid,

Pathogens 2023,12, 121 3 of 14
UK) plates. The antibiotics used were amoxicillin/ clavulanic acid (AMO) 20/10
µ
g,
ciprofloxacin (CIP) 5
µ
g, cefuroxime (CXM) 30
µ
g, piperacillin–tazobactam (TPZ) 110
µ
g,
cefoxitin (FOX) 30
µ
g, cefipime (FEP) 30
µ
g, ceftriaxone (CRO) 30
µ
g, ceftazidime (CAZ)
30
µ
g, cefotaxime (CTX) 30
µ
g, trimethoprim–sulfamethoxazole (SXT) 25
µ
g, imipenem
(IMI) 10
µ
g, ertapenem (ERT) 10
µ
g, and meropenem (MEM) 10
µ
g (Oxoid, UK). We used
the modified Hodge test (MHT) to check for carbapenemase production in isolates, which
showed intermediate or resistant zones for ertapenem according to CLSI guidelines [
19
].
We used E. coli ATCC 25922 as a susceptible strain and K. pneumoniae ATCC BAA-1705 as a
positive control. We interpreted data generated by the susceptibility assay using the CLSI
2021 guidelines [
19
]. The multiple antibiotic resistance (MAR) index of each isolate was
estimated according to Tambekar et al.’s method [20].
2.5. Multiplex PCR for Capsular Typing of K. pneumoniae and Detection of
Carbapenemases-Encoding Genes
We used two distinct multiplex PCR assays to carry out the molecular characterization
of the carbapenem resistance genes and capsular typing of K. pneumoniae. The K1, K2,
K5, K20, K54, K57, and K3 capsular antigens were the targets of the first multiplex PCR
typing [
21
] (Table 1). We utilized primer sets for the carbapenemases-encoding genes
blaVIM,blaIMP ,blaKPC,blaOXA-48 , and blaNDM in the second multiplex PCR [22]. (Table 1)
Table 1.
Primer sequences used in molecular detection of capsular genes and carbapenem resistance
genes of K. pneumoniae [23].
Primers Targeting Capsular-Encoding Genes
Target Genes Primer Sequence (50-30) Amplicon Size (bp)
khe F: TGA TTG CAT TCG CCA CTG G
R: GGT CAA CCC AAC GAT CCT G 428
WzyK1 F: GGT GCT CTT TAC ATC ATT GC
R: GCA ATG GCC ATT TGC GTT AG 1283
WzyK2 F: GAC CCG ATA TTC ATA CTT GAC AGA G
R: CCT GAA GTA AAA TCG TAA ATA GAT GGC 641
WzxK5 F: TGG TAG TGA TGC TCG CGA
R: CCT GAA CCC ACC CCA ATC 280
WzyK20 F: CGG TGC TAC AGT GCA TCA TT
R: GTT ATA CGA TGC TCA GTC GC 741
WzxK54 F: CAT TAG CTC AGT GGT TGG CT
R: GCT TGA CAA ACA CCA TAG CAG 881
Wzy57 F: CTC AGG GCT AGA AGT GTC AT
R: CAC TAA CCC AGA AAG TCG AG 1037
WzyK3 F: TAG GCA ATT GAC TTT AGG TG
R: AGT GAA TCA GCC TTC ACC T 549
Primers targeting carbapenemases-encoding genes
BlaKPC F-ATG TCA CTG TAT CGC CGT CT
R-TTT TCA GAG CCT TAC TGC CC 538
BlaIMP-1 F-TGA GCA AGT TAT CTG TAT TC
R-TTA GTT GCT TGG TTT TGA TG 139
BlaIMP-2 F-GGC AGT CGC CCT AAA ACA AA
R-TAG TTA CTT GGC TGT GAT GG 139
BlaVIM F-GAT GGT GTT TGG TCG CAT A
R-CGA ATG CGC AGC ACC AG 390
BlaNDM F-GGT TTG GCG ATC TGG TTT TC
R-CGG AAT GGC TCA TCA CGA TC 521
BlaOXA-48 F-TTG GTG GCA TCG ATT ATC GG
R-GAG CAC TTC TTT TGT GAT GGC 281

Pathogens 2023,12, 121 4 of 14
We obtained total genomic DNA using Qiagen DNA extraction kits (Qiagen, Hilden,
Germany) in accordance with the manufacturer’s instructions. Then, we kept the extraction
at −20 ◦C until the following stage.
We used Dream Taq TM Green PCR Master Mix (Fermentas, Waltham, MA, USA)
to amplify the tested gene as per the manufacturer’s directions using a Bio-Rad PTC-200
Thermal Cycler (Bio-Rad, Hercules, CA, USA). We created the PCR conditions for capsular
and carbapenemase genes molecular typing according to Ssekatawa et al.’s method [
23
].
We electrophoresed PCR products on a 1.5% agarose gel stained with ethidium bromide
and photographed with UV illumination. We used a 100-2000 base-pairs standard DNA
ladder (Biomatik, Wilmington, DE, USA) for sizing the PCR products.
2.6. Statistical Analysis
We analyzed the data with IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp,
New York, NY, USA, 2017). We utilized numbers and percentages to present qualitative
data. We used a p-value of ≤0.05 to determine statistical significance.
3. Results
3.1. Distribution of Isolated K. pneumoniae in Clinical Samples
We separated K. pneumoniae from distinct types of specimens collected from patients
admitted at Tanta university tertiary hospital. We collected 500 samples; however, only
160 specimens yielded K. pneumoniae, while the remaining specimens either yielded differ-
ent organisms or provided no growth. Regarding the 160 samples, 80 were isolated from
urine, 40 from pus swabs, 20 from sputum, 10 from tracheal aspirates, and 10 from blood
(Table 2).
Table 2. Prevalence of Klebsiella pneumoniae isolated from various clinical specimens.
Sample Type (Number) Klebsiella pneumoniae Isolates
Urine (216) 80 (50%)
Pus swab (103) 40 (25%)
Sputum (78) 20 (12.5%)
Tracheal aspirate (55) 10 (6.25%)
Blood (48) 10 (6.25%)
Total (500) 160 (100%)
3.2. Antibiotic Susceptibility Patterns and Phenotypic Detection of Carbapenemases
Based on the disc diffusion assay, the majority of the isolated K. pneumoniae showed
significant levels of resistance to used antibiotics. Overall, 99.4% of the isolates exhibited re-
sistance to cefotaxime, while 99% showed resistance to amoxicillin–clavulanic acid and cef-
tazidime. Furthermore, 98.1% of the isolates exhibited resistance to each of cefuroxime and
ceftriaxone, whereas 95% and 94.4% were resistant to trimethoprim–sulfamethoxazole and
cefepime, respectively. We observed resistance to piperacillin–tazobactam and ciprofloxacin
as the next highest among 81.8% of the isolates, followed by cefoxitin (60%). We found
the lowest resistance rate corresponding to imipenem and ertapenem (31.3%), followed by
meropenem (30%). All carbapenem-resistant isolates (100%) were MHT positive. The MAR
index ranged from 0.69 to 01.
3.3. Carbapenemase-Encoding Genes Distribution
Based on the results obtained by Multiplex PCR assay, out of 160 K. pneumoniae isolates,
38.75% (62/160) contained single or mixed carbapenemase genes (Tables 3and 4). Of those,
bla
OXA-48
was the most predominant, with a prevalence of (15.5%) (25/160), followed by
bla
VIM
(24/160 = 15%), bla
IMP
(12/160 = 7.5%), bla
KPC
(7/160 = 4%), and bla
NDM
(6/160 = 3.8%)
(Figure 1).

Pathogens 2023,12, 121 5 of 14
Table 3. Prevalence of carbapenemase-encoding genes in total Klebsiella pneumoniae isolates.
Carbapenemase Gene Tested Gene Prevalence in Total Klebsiella Pneumoniae Isolates
BlaKPC 7 (4%)
BlaIMP-1&2 12 (7.5%)
BlaVIM 24 (15%)
BlaNDM 6 (3.8%)
BlaOXA-48 25 (15.5%)
Total 74 (46.25%)
Table 4.
Distribution of single and mixed carbapenemase genes among the genotypically
resistant isolate.
Carbapenemase Gene Tested Number of Isolates Harboring Carbapenemases
BlaKPC 4
BlaIMP-1&2 8
BlaVIM 21
BlaNDM 2
BlaOXA-48 17
BlaNDM and BlaOXA-48 1
BlaKPC and BlaIMP-1&2 1
BlaKPC and BlaOXA-48 1
BlaIMP-1&2 and BlaOXA-48 2
BlaVIM and BlaOXA-48 2
BlaVIM and BlaNDM 1
BlaNDM,BlaKPC, and BlaOXA-48 1
BlaIMP-1&2,BlaNDM , and BlaOXA-48 1
Total 62
Figure 1. Gene prevalence in Klebsiella pneumoniae isolates.

Pathogens 2023,12, 121 6 of 14
3.4. Correlation between Genotypic and Phenotypic Assays
We detected variations between the genotypic and phenotypic resistance of the iso-
lates. A total of 24 isolates harbored the VIM gene, and 22 (91.67%) showed phenotypic
carbapenem resistance. This was followed by OXA-48, which showed phenotypic resis-
tance in 22 (88%) of the isolates, then Kpc in 5 (71.43%), IMP-1&2 in 9 (75%), and NDM in
4 (66.67%) (Table 5).
Table 5. Correlation between genotypic and phenotypic resistance.
Carbapenemase-
Encoding
Genes
Number of Isolates
Harboring the Gene
Number of Isolates
Harboring the Gene
and Phenotypically
Resistant
Number of Isolates
Harboring the Gene
and Phenotypically
Sensitive
Percentage of
Resistance Conferred
by Gene Presence
BlaKPC 7 5 2 71.43%
BlaIMP-1&2 12 9 3 75%
BlaVIM 24 22 2 91.67%
BlaNDM 6 4 2 66.67%
BlaOXA-48 25 22 3 88%
3.5. Prevalence of Capsular Types in Isolates Harboring Carbapenemases-Encoding Genes
Our multiplex PCR assay results showed that out of 62 carbapenem-resistant isolates,
19 (30.6%) harbored capsular gene K1, followed by the K57 (15; 24.2%), K54 (12; 19.35%),
K20 (6; 9.67%), and K2 genes (4; 6.45%). However, we did not detect the K3 and K5 genes
in any of the collected isolates (Figure 2).
Figure 2. Prevalence of capsular types in carbapenem genotypically resistant isolates.
3.6. Correlation between Source, Antimicrobial Resistance Pattern, Multiple Antibiotic Resistance
(MAR) Index, Distribution of Carbapenemase-Encoding Genes, and Capsular Types
The comprehensive correlation between an isolate’s source, antimicrobial resistance
pattern, MAR index, carbapenemases genes, and capsular serotypes are displayed in
Table 6. We found no significant relations when correlating the different carbapenemase
genes detected during our study with capsular serotypes (Table 7).

Pathogens 2023,12, 121 7 of 14
Table 6.
Correlation between source of samples, antimicrobial resistance pattern, MAR index, car-
bapenemase genes, and capsular genes.
Pattern
Number
Code
Number Antimicrobial Resistance Pattern MAR
Index
Carbapenemase
Genes
Capsular
Genes
1 1 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaKPC K1
2 3 U AMO, SXT, CXM, TPZ, CRO, FEB,
CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaVIM K54
3 7 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CIP, CTX, IMI, MEM, ERT 01 blaIMP-1&2 K1
4 9 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaOXA-48 K20
5 17 U AMO, CXM, TPZ, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaVIM K1
6 19 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaOXA-48 K54
7 23 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM K1
8 27 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaVIM K54
9 31 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaOXA-48 K1
10 33 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM K20
11 43 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaVIM K57
12 45 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaIMP-1&2 K54
13 48 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaKPC,blaIMP-1&2 K1
14 54 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaVIM K57
15 58 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaNDM K2
16 64 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaOXA-48 K57
17 67 U AMO, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.77 blaOXA-48 K54
18 75 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaVIM K54
19 77 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaVIM K1
20 79 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaIMP-1&2,
blaOXA-48 -
21 91 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaVIM K20
22 107 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaVIM,blaOXA-48 K1
23 110 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaVIM K57

Pathogens 2023,12, 121 8 of 14
Table 6. Cont.
Pattern
Number
Code
Number Antimicrobial Resistance Pattern MAR
Index
Carbapenemase
Genes
Capsular
Genes
24 114 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM K1
25 116 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaVIM,blaOXA-48 K54
26 121 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaVIM K1
27 124 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 K1
28 128 U AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaKPC -
29 129 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaNDM,blaKPC&,
blaOXA-48 K54
30 134 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 K20
31 137 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaIMP-1&2 -
32 139 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, ERT 0.92 blaVIM K1
33 144 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM K1
34 156 U AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaVIM,blaNDM K54
35 4 P AMO, CXM, TPZ, FOX, CRO, FEB, CAZ, CTX,
IMI, MEM, ERT 0.85 blaOXA-48 K57
36 15 P AMO, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.77 blaNDM, blaOXA-48 K57
37 35 P AMO, SXT, CXM, TPZ, CRO,
FEB, CAZ, CTX, CTP, IMI, MEM, ERT 0.92 blaKPC K57
38 42 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 K2
39 50 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaOXA-48 K20
40 66 P AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP 0.69 blaVIM K2
41 69 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaIMP-1&2 K1
42 71 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 -
43 82 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaKPC K57
44 87 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM -
45 89 P AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaOXA-48 K57
46 96 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaIMP-1&2,
blaOXA-48 K1

Pathogens 2023,12, 121 9 of 14
Table 6. Cont.
Pattern
Number
Code
Number Antimicrobial Resistance Pattern MAR
Index
Carbapenemase
Genes
Capsular
Genes
47 98 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM -
48 106 P AMO, SXT, CXM, TPZ, FOX, CRO FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaIMP-1&2 K54
49 113 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, ERT 0.92 blaOXA-48 K20
50 120 P AMO, SXT, CXM, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.85 blaIMP-1&2 K57
51 122 P AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, CIP, IMI, MEM, ERT 0.92 blaIMP-1&2 K54
52 130 P AMO, SXT, CXM, TPZ, FOX, CRO FEB, CAZ,
CTX, CIP 0.77 blaVIM K1
53 135 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CTP, IMI, MEM, ERT 01 blaIMP-1&2, blaNDM,
blaOXA-48 K57
54 140 P AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 K54
55 40 S AMO, SXT, CXM, TPZ, CRO,
FEB, CAZ, CTX, CTP, IMI, MEM, ERT 0.92 blaNDM K1
56 62 S AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP, IMI, MEM, ERT 01 blaKPC,blaOXA-48 K1
57 101 S AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, CIP 0.77 blaOXA-48 K57
58 151 S AMO, SXT, CXM, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaIMP-1&2 K57
59 60 B AMO, SXT, CXM, TPZ, FOX, CRO, FEB, CAZ,
CTX, IMI, MEM, ERT 0.92 blaVIM K57
60 84 B AMO, CXM, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.69 blaOXA-48 K1
61 10 T AMO, CXM, TPZ, FOX, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaVIM K2
62 24 T AMO, SXT, CXM, TPZ, CRO,
FEB, CAZ, CTX, IMI, MEM, ERT 0.85 blaOXA-48 K57
U: urine; P: pus; S: sputum; B: blood; T: tracheal aspirate; MAR: multiple antibiotic resistance; —: samples
negative for tested capsular types; AMO: amoxicillin/clavulanic acid; CIP: ciprofloxacin; CXM: cefuroxime, TPZ:
piperacillin tazobactam; FOX: cefoxitin; FEP: cefipime; CRO: ceftriaxone; CAZ: ceftazidime; CTX: cefotaxime; SXT:
trimethoprim–sulfamethoxazole (SXT); IMI: imipenem (IMI); ERT: ertapenem; MEM: meropenem.
Table 7. Correlation between carbapenemases and capsular genes.
BlaOXA-48
(n = 25)
BlaVIM
(n = 24)
BlaIMP1&2
(n = 12)
blaKPC
(n = 7)
blaNDM
(n = 6) χ2p-Value
K1 6 (24%) 9 (37.5%) 4 (33.3%) 3 (42.9%) 1 (16.7%) 2.170 0.733
K2 1 (4%) 2 (8.3%) 0 (0%) 0 (0%) 1 (16.7%) 2.876 0.481
K20 4 (16%) 2 (8.3%) 0 (0%) 0 (0%) 0 (0%) 2.663 0.554
K54 5 (20%) 5 (20.8%) 3 (25%) 1 (14.3%) 2 (33.3%) 1.152 0.932
K57 7 (28%) 4 (16.7%) 3 (25%) 2 (28.6%) 2 (33.3%) 1.752 0.817
χ2: Chi-square test.

Pathogens 2023,12, 121 10 of 14
4. Discussion
K. pneumoniae has been identified as one of the most popular causes of infections
developed in hospitals and the community [
24
]. The appearance of, MDR and hvKP strains,
as well as their rapid clinical propagation, is particularly concerning [
25
] because their resis-
tance propagation is associated with mobile genetic components, which may additionally
hold virulence factors, such as the capsule, siderophores, fimbriae, and lipopolysaccharides
(LPS) [
26
]. Therefore, when highly pathogenic bacteria develop antibiotic resistance, the
situation deteriorates [23].
Therefore, we analyzed the frequency of carbapenem-resistant pathogenic K. pneumo-
niae in our tertiary care hospitals to better understand its dangers. Our survey findings
show that 50% of K. pneumoniae isolates were found in urine, 25% in pus swabs, 20% in
sputum, and 6.25% in both blood and tracheal aspirates. Our results are similar to those
of a study conducted at Al-Azhar University, Egypt [
27
]. Additionally, further research
carried out in Uganda concluded that most K. pneumoniae isolates were obtained from urine,
pus, and blood [23].
However, a study in New York conducted by Parrott et al. [
28
] confirmed that most
K. pneumoniae isolates were recovered from blood culture, followed by wound swabs.
Additionally, Palmeiro et al. [
29
] found that blood specimens yielded the highest number of
isolates. Furthermore, Sedighi P et al. [
30
] found that throat, urine, and tracheal swabs were
the most prevalent samples, while wound, blood, sputum, and abscess cultures showed
the least amounts of isolates.
This variation in results may be explained by variations in sample type and case count,
sampling conditions, sampling times, sampling locations, sampling countries, and patient
general health.
We determined that the isolates we detected in our study were MDR because of
their resistance to several types of antibiotics. Meropenem had a 30% resistance rate,
whereas imipenem and ertapenem both had a 31.3% resistance rate. This outcome was
consistent with the research conducted by Farhadi et al. [
31
], who observed that 33% of
the K. pneumoniae isolates were resistant to imipenem. Furthermore, Pereira et al. [
32
]
found that 73 Klebsiella isolates found in samples of a urinary tract infection were extremely
resistant to IMP.
Moreover, Moghadas et al. [
33
] found that only 7.5% of their isolates were resistant
to IMP, and their survey of North and West Africa highlighted a noticeably increased
phenotypic resistance to carbapenems (>50%) [
34
–
37
]. Additionally, a bigger study that
examined the South African provinces of Gauteng, KwaZulu-Natal, Western Cape, and
Free State found that imipenem, meropenem, and doripenem had overwhelmingly high
phenotypic resistance rates of between 47 and 50%, while ertapenem had rates between
84% and 89%.
The disparity in sensitivity patterns between the aforementioned studies may be
attributed to various antibiotic policies, the emergence of resistant strains because of
indiscriminate antimicrobial therapy, the patient’s immune status, various infection control
strategies, or frequent hospitalization.
We must determine whether the K. pneumoniae isolate produces carbapenemase in
order to conduct epidemiological research and choose the best course of treatment for
infections [
38
]. Regarding the PCR-based carbapenemase gene identification, bla
OXA-48
was
the most prevalent, with a genotypic frequency of (15.5%), followed by bla
VIM
type (15%),
bla
IMP
(7.5%), bla
KPC
(4%), and bla
NDM
(3.8%). Our findings were consistent with another
Egyptian study conducted by Raheel et al. [
39
], who demonstrated that the bla
OXA-48
gene
(96.2%) was the most frequently present gene, while the bla
KPC
gene (7.5%) was the least
common. Additionally, our result is consistent with recent research that identified the
OXA-48 gene and its variations as the most popular gene [35,40–42].
OXA- 48 was initially discovered in a K. pneumoniae strain from Turkey in 2003. OXA-
48 intermittently reached neighboring nations in the southern and eastern Mediterranean

Pathogens 2023,12, 121 11 of 14
Sea, as well as North Africa [
43
]. This explains why OXA-48 is more common in Tunisia
and Egypt than anywhere else [35,41].
Nevertheless, Lopes et al. and Hussein et al. [44,45] found that carbapenem-resistant
K. pneumoniae isolates had a higher level of bla
KPC
expression. Furthermore, El-Monir
et al. [
46
] reported that both bla
VIM
and bla
NDM-1
were the most prevalent genes detected in
Egypt. Additionally, further studies showed that the most abundant genes in East Africa
were VIM and IMP [40,47], whereas NDM was the most common in South Africa [47–50].
We recovered more than one resistance gene in 12 K. pneumoniae isolates, which is in
accordance with many previously published studies that demonstrated that A. baumannii
and K. pneumoniae carry several genes, increasing their likelihood of being multi- or pan-
drug resistant [
49
,
51
–
54
]. However, this can be contested because of the possibility of
resistance spreading and the restricted accessibility of antibiotics useful for therapy, as well
as the diminishing effectiveness of older antibiotics, such as colistin [55,56].
Our study found that genotypic resistance was generally higher than overall phe-
notypic resistance. For example, 25 isolates harbored the OXA-48 gene, and 22 (88%) of
them showed phenotypic carbapenem resistance. This can be explained by many reports
that described OXA-48 and its variant genes’ oxacillinases as having limited hydrolyzing
activity for carbapenems [43,57,58].
The capsule is a key element affecting K. pneumoniae’s pathogenicity. Numerous
investigations revealed that the virulence of infections generated by K. pneumoniae is
influenced by the capsular forms [
59
,
60
]. In several strains of Klebsiella spp., the gene cluster
architecture responsible for producing capsular polysaccharide (CPS) has been previously
analyzed [
61
]. The Wzy and Wzx genes, which generate the proteins necessary for the
polymerization and assembly of the various CPS subunits, are situated in a variable region
in the center of the CPS locus. As a result, the foundation of PCR capsular typing assays is
the significant sequence diversity of the Wzy gene among the various capsular types [
62
].
Considering this, we identified and characterized the K. pneumoniae capsular serotypes that
were most clinically relevant using the Wzy gene.
Our results revealed that (30.6%) of K. pneumoniae isolates harbored capsular gene K1,
followed by the K57 (24.2%), K54 (19.35%), K20 (9.67%), and K2 genes (6.45%); however,
we did not detect the K3 and K5 genes in the collected isolates.
Ssekatawa et al. [
23
] found that K1, K2, K3, K5, and K20 made up 46.7% of the
K. pneumoniae
isolates; according to capsular typing by heptaplex PCR, while none of the
isolates had K54 or K57.
These findings correspond to research conducted by Fung et al. and Chuang et al. [
60
,
63
],
who concluded that the greatest virulent capsular forms of K. pneumoniae K1 and K2 were
responsible for septicemia and liver abscesses. Furthermore, according to two surveys
conducted in Taiwan by Fang et al. and Lin et al. [
59
,
62
], the K1, K2, K3, K5, and K20 genes
were the most common capsular types in pneumonic and liver abscess patients. Moreover,
Paczosa and Mecsas [
64
] reported that among the 519 invasive strains they investigated,
K2 isolates were found in the largest numbers. In addition, Choi et al. [
65
] found that K24
was the most prevalent capsule type.
We evaluated the correlation between capsular serotypes and the presence of carbapen-
emase genes. Our results revealed that carbapenemases genes could not be related to any
capsular serotypes (data were statistically not significant). Nonetheless, Soltani et al. [
66
]
found a correlation between blaOXA-48 and K20 in a study conducted in Iran.
5. Conclusions
Our research highlighted high incidence rates for carbapenem-resistant K. pneumoniae
in our tertiary care hospital. Although our study did not seek to identify other viru-
lence determinants, the considerable prevalence of carbapenem resistance among capsu-
lar serotypes that we found raises the possibility of carbapenem-resistant hypervirulent
K. pneumoniae, which must be assessed in further studies.

Pathogens 2023,12, 121 12 of 14
Author Contributions:
Conceptualization, M.S.T. and S.Y.M.; data curation, Y.A.Z.; formal analy-
sis, M.M.H., Y.A.Z. and R.M.E.; investigation, M.A.A.; methodology, M.S.T., M.A.A. and S.Y.M.;
resources, M.S.T. and M.M.S.; software, M.M.S., Y.A.Z. and R.M.E.; supervision, M.S.T.; validation,
M.M.H., M.M.S., Y.A.Z. and R.M.E.; visualization, M.M.H.; writing—original draft, M.S.T. and S.Y.M.;
writing—review
and editing, M.M.H., M.M.S. and M.A.A. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
The Institutional Review Board of Tanta University Faculty
of Medicine in Egypt gave the study its approval (approval code 35789/9/22). All techniques were
conducted in accordance with the ethical recommendations of the relevant committee on human
experimental research (institutional and national), as well as the principles outlined in the Helsinki
Declaration (1975), as updated in (2013).
Informed Consent Statement:
All participants or their parents (in the case of pediatric patients)
provided written informed permission.
Data Availability Statement: Data are accessible upon request from the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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