Dissertation Ayi Corrected 03 09 2018 Copy [607271]

UNIVERSITY OF BUCHAREST
FACULTY OF BIOLOGY
PROGRAM STUDY APPLIED MICROBIOLOGY AND
IMMUNOLOGY
DISSERTATION
Scientific coordinator ,
Lect. Dr. Irina Gheorghe
Scientific adviser,
Lect. Dr. Carmen Curutiu

Student: [anonimizat] 2018
UNIVERSITY OF BUCHAREST

FACULTY OF BIOLOGY
PROGRAM STUDY APPLIED MICROBIOLOGY AND
IMMUNOLOGY
DISSERTATION
Antibiotic resistence features of some Klebsiella pneumoniae
strains isolated from hospitalized patients in Bucharest,
Romania.
Scientific coordinator ,
Lect. Dr. Irina Gheorghe
Scientific adviser,
Lect. Dr. Carmen Curutiu
Student: [anonimizat] 2018
DECLARATION
2

I, the undersigned Ayi Suwarayi, a candidate for the Master's Degree Exam at the Faculty
of Biology in University of Bucharest, specialize in Applied Microbiology and
Immunology, declare under my sole responsibility that this paper is the result of my work,
based on my research and on the basis of information obtained from sources quoted and
indicated, according to ethical norms, in the notes and in the bibliography. I declare that I
have not tacitly or illegally used the work of others and that no part of the thesis violates
the intellectual property rights of someone else, a natural or a legal person. I declare that
the work has not been presented under this form in any form to any higher education
institution in order to obtain a scientific or teaching degree or title.
Date: Signature,
Contents
3

Cover…………………………………………………………………………………………………………………… 1
Declaration3
Contents 4
Tables and Figures 6
Introduction8
Chapter 1. General aspects of Klebsiella pneumonia strains…………………………………… 9
1.1. Morphology, Taxonomy and Identification of Klebsiella pneumoniae strains
..9
1.2.Pathogenicity/Virulence Factors in Klebsiella pneumoniae strains12
1.3. Nosocomial infection produced by Klebsiella pneumoniae strains….6
1.3.1. Definition of Nosocomial Infection 13
1.3.2. Types of Nosocomial Infections 14
1.3.3. Factors that may affect nosocomial infections 15
1.3.4. Agents of Nosocomial Infections 16
1.3.5. Sources and Transmission of Nosocomial Infections 16
1.3.6. Nosocomial infections caused by Klebsiella pneumoniae strains
16
1.4. Antibiotic resistance mechanism in Klebsiella pneumoniae strains…17
1.4.1. Resistance to Beta-Lactam antibiotics 17
1.4.2. Resistance to Cephalosporins of third and fourth generation 21
1.4.3. Resistance to Quinolones 22
1.4.4. Resistance to Fluoroquinolones 23
1.4.5. Resistance to Aminoglycosides 25
1.4.6. Resistance to Tetracycline 25
1.4.7. Resistance to Polymyxins 26
1.4.8. Resistance to Tigecycline 26
1.4.9. Resistance to Colistin 27
Chapter 2. Characterization of the a ntibiotic resistance features of some Klebsiella
pneumoniae strains isolated from hospitalized patients in Bucharest, Romania
29
2.1. Aim and Objective ………………………………………………………………………….. 29
4

2.2.Materials and Methods 29
2.3. Results and Discussion 32
2.4. Conclusion 43
Selective References 45
Tables and Figures
5

Tables
Table 110
Table 218
Table 330
Table 431
Table 531
Table 633
Table 734
Table 836
Figures
Figure 19
Figure 211
Figure 311
Figure 411
Figure 512
Figure 616
Figure 717
Figure 818
Figure 921
Figure 1024
Figure 1126
Figure 1228
Figure 1333
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Figure 1434
Figure 1537
Figure 1637
Figure 1739
Figure 1840
Figure 1941
Figure 2042
Figure 2142
Figure 2243
Figure 2343
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Introduction
Klebsiella (K.) pneumoniae is a common environmental human and animal-
associated Gram-negative bacillus (rod shaped) that become one of the worldwide major
cause of nosocomial infection [1, 2]. The increasing of antibiotic resistance among members
of the Enterobaceriaceae family lead to the appearance of multidrug resistant (MDR)
strains, with a high frequency in Klebsiella. [3]
The emergence and worldwide expansion of multidrug resistant phenotyps of K.
pneumoniae have been increasingly reported in nosocomial infections. [1] Resistance of
penicillin was first reported early in 1940’s and after that beta-lactam resistance genes in
Klebsiella pneumoniae continuously being identified and reported. The first identification
of β-lactamase genes, blaSHV-1 and blaTEM-1. Twenty years later, the first extended
spectrum β-lactamase (ESBL) gene, blaSHV-2, was identified in Klebsiella pneumoniae.
Beside resistant to antibiotic from beta -lactam groups, Klebsiella pneumoniae have also
been reported as resistant to antibiotic from other groups: aminoglycosides, quinolones,
polymyxins, tigecycline . [4]
The production of K. pneumoniae carbapenemase (KPC) has become a prominent
mechanism and also become a potential clinical issue because these strains proved to be
multidrug resistant, lacking of the susceptibility also to betalactam antibiotics,
fluoroquinolones, and aminoglycosides. KPC-producing organisms are most frequently
reported in the United States, but also occurred in Israel and Greece. Beside those
countries, numerous of other countries have now also been affected by KPC-producing
organisms. The most frequent reported carbapenemases resistance in K. pneumoniae are
coded by genes from the blaKPC, blaIMP, blaVIM, blaNDM, blaTEM, blaCTX-M and
blaOxa groups. [3, 5]
Changes in K. pneumoniae cell permeability have been reported to be involved in
quinolone resistance, similar to their role in β-lactam resistance. Plasmid mediated
quinolone resistance (PMQR) was reported increasingly worldwide in K. pneumoniae.
There were five qnr proteins identified qnr A, qnr B, qnr C, qnr D and QnrS.[4,6]
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In the context of the numerous resistence genes identified in Klebsiella strains, the
aim of this study was to characterize the a ntibiotic resistence features of some Klebsiella
pneumoniae strains isolated from hospitalized patients in Bucharest, Romania.
CHAPTER 1. GENERAL ASPECTS OF KLEBSIELLA PNEUMONIAE STRAINS
1.1. Morphology, taxonomy and identification of Klebsiella pneumoniae
strains
The bacteria grouped in the genus Klebsiella are Gram-negative enterobacteria of
immobile bacillary morphology, capped from 0.3 to 1.5 x 0.6 to 6, μm, grouped in pairs
or short chains, and can be considered opportunistic bacteria [8]. The type species is
Klebsiella pneumoniae, which is found in the respiratory tract and feces of approximately
5-10% of healthy individuals, and is responsible for a small percentage of bacterial
pneumonias (Fig. 10). [8,9].
The first mention of a species of the Klebsiella genus was made by von Frisch in
1881, which referred to a capped bacillus observed in samples of patients with
rhinoscleroma; the Klebsiella genus was named after Trevisan in 1885, in honor of the
German microbiologist Edwin Klebs (1834-1913); the type species of this genus, K.
pneumoniae was described by Schroeter in 1886, and by Trevisan in 1887[10].

Fig 1. Klebsiella pneumoniae – Gram negative bacilii (Gram’s staining, 1000X) [9]
The Klebsiella taxonomy is characterized by a varied nomenclature that reflects
its complex taxonomic history. Initially the medical importance of the genus led to its
subdivision into three species, according to the diseases they caused: K. pneumoniae, K.
ozaena and K. rhinoscleromatis , and afterthat the taxonomy was improved due to the
9

development of new methodologies, the classification of species within the genus being
continuously revised. In present, the adoption of a consistent nomenclature is currently
difficult, although most European countries and the United States consider the
classification of ∅rskov, more widespread worldwide (table 1) [10,11].
Klebsiella spp. is ubiquitous in nature. It probably has two common habitats: the
environment, in which it is found in surface and wastewater, in soil and on plants; the
mucosal surfaces of mammals, and in human carriers, K. pneumoniae is found in the
upper respiratory tract and in the intestinal tract [10, 11].
Table 1. Classification of species of the genus Klebsiella according to two different taxonomic systems [11]
Cowan ∅rskov
K. aerogenes
K. edwardsii
subsp. Edwardsii
subsp. Atlantae
K. pneumoniae
K. ozaenae
K. rhinoscleromatisK. pneumoniae
subsp. Pneumoniae
subsp. Ozaenae
subsp. Rhinoscleromatis
K. oxytoca
K. terrigena
K. planticola (sin. K. trevisanii)
K. ornithinolytica
The genus Klebsiella is formed by Gram-negative, facultative anaerobic and
oxidase-negative bacilli and it belongs therefore to the Enterobacteriaceae family, a
family formed by a large number of bacterial species, and in addition to the
aforementioned traits, the bacteria of the genus Klebsiella are characterized by being
generally capped, non-motile, producers of the enzyme lysine decarboxylase but not of
the enzyme ornithine decarboxylase, and normally positive to the Voges-Proskauer (VP)
test. Klebsiella pneumoniae produces large dome shape, mucoid colony on BHI agar and
Lactose fermentative pink colony on Mac conkey agar (Fig. 2, 3). Its biochemical test
ussualy are: indole negative, methil red (MR) negative, VP positive, citrate positive,
oxidase negative and catalase positive (Fig. 4, 5). The percentage of guanine-cytosine of
DNA of species of the genus ranges between 53 and 58% [9,10,11].
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Fig 2: Mucoid dome shape colony on BHI agar [9]
Fig 3: Lactose fermentative colony- pink, dome shape colony on MacConkey agar[9]
Fig 4: Voges-Proskauer positive test in K. pneumoniae [9].
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Fig 5: IMViC test – 1. Indole-Negative 2. MR-Negative 3. VP-Positive 4. Citrate-Positive[9]
1.2. Pathogenicity/Virulence Factors in Klebsiella pneumoniae strains
Bacterial adherence to mammalian cell surfaces, mediated by bacterial pili or
fimbriae, has long been described. Pili-mediated attachment is correlated to the virulence
of the bacteria. The attachment is followed by colonization of the tissue, eventually
leading to intracellular penetration by the bacteria [12]. The bacterial surface
hydrophobicity and capsule also have known as factor that can influence the
virulence/pathogenicity of the bacteria. The bacterial surface hydrophobicity was studied
by two different methods: the first was hydrophobic interaction chromatography (HIC) in
phenyl sepharose, described by Källenius, another method used to analyze the surface
hydrophobicity was the bacterial adhesion to hydrocarbons (BATH), according to
Rosenberg [13].
K. pneumoniae utilizes a variety of virulence factors, especially capsule
polysaccharide (CPS), lipopolysaccharide (LPS), fimbriae, outer membrane proteins and
determinants for iron acquisition and nitrogen source utilization, for survival and immune
evasion during infection by penetrating and colonizes human mucosal surface[12,14].
The capsule encases the entire cell surface, accounts for the large appearance of
the organism on Gram stain, and provides resistance against many host defense
mechanisms. CPS is considered the most important virulence factor of K. pneumoniae. It
covers the bacterial surface and is responsible for its resistance to host phagocytes and
serum and promotes inflammation reaction.
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Members of the Klebsiella genus typically express 2 types of antigens on their cell
surface. The first is a lipopolysaccharide (O antigen); the other is a capsular
polysaccharide (K antigen). Both of these antigens contribute to pathogenicity [15].
LPS is a major component of the outer membrane of Gram-negative bacteria, and
consists of lipid A, core oligosaccharide, and a long chain polysaccharide (O antigen).
LPS is an important pathogenic determinant in K. pneumoniae, which causes pneumonia
and bacteremia. O antigen is responsible for bacterial resistance to complement-mediated
killing.
The colonization of mucous membranes by bacteria is the result of adhesion
between the bacterial capsule and the host’s mucous layer. The adhesion of K.
pneumoniae to mammalian tissue is mediated by two types of bacterial pili, type 1 and
type 3. Type 1 fimbriae play an important role in bacterial adhesion to the D-mannose
moiety on mammalian cell surfaces due to the specific affinity of fimbrial protein at the
tips of fimbriae. Type 3 fimbriae are characterized by their affinities for a range of
mammalian cells, which include bladder epithelial cells, uroepithelial cells, and
endothelial cells[16]. Expression of type 3 fimbriae has been described for many Gram-
negative pathogens and is commonly detected in isolates of the genera Klebsiella,
Enterobacter, Serratia , and Proteus. These fimbriae are characterized by their ability to
mediate agglutination of tannic acid-treated human erythrocytes and this
hemagglutination (HA) occurs in the presence or absence of D-mannose. Since
hemagglutinin was originally characterized in Klebsiella strains, the fimbrial adhesin has
been referred to as the mannose-resistant, Klebsiella-like (Mr/K) hemagglutinin[12].
1.3. Nosocomial infection produced by Klebsiella pneumoniae strains
1.3.1. Definition of Nosocomial Infection
A nosocomial infection can be defined as follows: an infection contracted in the
hospital by a patient and which was not present or incubating at the time of admission. An
infection that occurs in a patient hospitalized in a hospital or other health care
establishment in which the infection had not manifested or was in the incubation period at
the time of boarding. Nosocomial infection was defined as an infection acquired more
than 48 hours after being admitted to a hospital, 3 days of discharge or 30 days of an
operation. Patient care is provided in facilities that are available from well-equipped
clinics and university hospitals with advanced technology to primary care units with basic
services; despite the progress made in hospital and public health care, infections continue
13

to manifest in hospitalized patients, which can also affect hospital staff. Many factors
favor infection in hospitalized patients: reduction of patients' immunity; the greatest
variety of medical procedures and invasive techniques, which create possible routes of
infection; and the transmission of drug-resistant bacteria in overcrowded populations in
hospitals, where poor control practices can help transmission [17, 18, 19 and 21].
1.3.2. Types of Nosocomial Infections
Based on clinical and biological criteria, CDC and National Healthcare Safety
Network (NHSN) categorize health care associated infection sites into 13 major types
which contain approximately 50 potentially specific infection sites for surveillance
purpose. The most common types of nosocomial infections that could occur in a hospital
set up are: Surgical wound and other soft tissue infections, urinary tract infections (UTI),
Respiratory infections, Gastroenteritis and Meningitis . However, with increased use of
invasive procedures for therapeutic and diagnostic purposes, cancer chemotherapy,
immunotherapy and advances in organ transplants, it is possible to observe change in the
distribution of nosocomial infection sites over time. WHO and other studies have also
shown that the highest prevalence of nosocomial infections occurs in intensive care units
and in surgical and orthopedic wards for acute care. The prevalence rates of infection are
higher in patients with greater vulnerability due to old age, underlying disease or
chemotherapy. [17, 19].
Nosocomial infections aggravate the functional disability and emotional stress of
the patient and, in some cases, can cause disabling disorders that reduce the quality of
life. They are one of the main causes of death. The economic costs are enormous; a
prolonged hospital stay of infected patients is the biggest contributor to that costs: one
study showed that the general increase in the period of hospitalization of patients with
surgical wound infections was 8.2 days and ranged from 3 days in cases of gynecological
surgery and 19, 8, in orthopedic infections; a prolonged stay increases not only the direct
costs for patients or payers, but also the indirect costs due to lost work [17, 18].
The increased use of medications, the need for isolation and the use of more
laboratory and other studies for diagnostic purposes also raise costs. Nosocomial
infections aggravate the imbalance between the allocation of resources for primary and
secondary care by diverting scarce funds towards the treatment of potentially preventable
conditions. The advanced age of patients hospitalized in these health care establishments,
the higher prevalence of chronic diseases in hospitalized patients and the greater use of
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therapeutic and diagnostic procedures that affect the host's defenses will constitute a
constant pressure in nosocomial infections in the future. The microorganisms that cause
nosocomial infections can be transmitted to the community by patients after hospital
discharge, health care personnel and visitors; if these microorganisms are multi-resistant,
they can cause serious illness in the community [17, 18].
1.3.3. Factors that may affect nosocomial infections.
Hospitalized patients suffer from minimal compromise immunity, undergo
invasive examinations and treatments, and patient care practices can contribute to the
transmission of microorganisms among them. The selective pressure exerted by the
intensive use of antibiotics promotes resistance to these products; although it has made
progress in the prevention of nosocomial infections and changes in the practice of
medicine, new forms of manifestation of infections occurs. The patient is exposed to a
wide variety of microorganisms during hospitalization. The contact between the patient
and a microorganism, in itself, does not produce necessarily a clinical disease, since there
are other factors that influence the nature and frequency of nosocomial infections; the
possibility of exposure leading to infection depends, in part, on the characteristics of the
microorganisms, including resistance to antimicrobials, intrinsic virulence and the amount
of infectious material (inoculum) (Fig. 6) [17].
Important factors for patients that influence the possibility of contracting an
infection include age, immunity status, any underlying disease, and diagnostic and
therapeutic interventions. In the extreme periods of life – childhood and old age –
resistance to infection usually decreases; patients with chronic disease, such as malignant
tumors, leukemia, diabetes mellitus, renal failure or acquired immunodeficiency
syndrome (AIDS) are more vulnerable to infections by opportunistic pathogens. These
bacteria are not infectious for a normal, healthy person, for example, could be part of the
normal bacterial microbiota of the human, but can become pathogenic when the immune
defenses of the organism are compromised. Immunosuppressive agents or irradiation can
reduce resistance to infection. Injuries to the skin or mucous membranes occur without
going through the natural defense mechanisms. Malnutrition also presents a risk for
nosocomial infection [17].
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Fig 6: Correlation between multiple factors that can caused nosocomial infection [17]
1.3.4. Agents of Nosocomial Infections
There is a large number of microorganisms responsible for hospital infections,
theoretically any microbiorganism have the capacity/ability to cause an infection in the
hospitalized patients. The major agent/organism of nosocomial infections are bacteria,
nevertheless mycobacteria, viral, fungal or protozoal agents being less commonly
involved. The bacteria that commonly cause nosocomial infections include
Staphylococcus aureus, Streptococcus spp., Bacillus cereus, Acinetobacter spp. ,
coagulase negative staphylococci, enterococci, Pseudomonas aeruginosa , Legionella and
members of the Enterobacteriaceae family such as Escherichia coli, Proteus mirabilis,
Salmonella spp., Serratia marcescens and Klebsiella pneumoniae [19].
1.3.5. Sources and Transmission of Nosocomial Infections
The nosocomial pathogens cause infections either from endogenous or exogenous
sources. Animate and inanimate sources of exogenous infections include hospital staff,
other patients, visitors, food, water, fomites, urinary catheter, intravenous devices,
respiratory equipment and other prostheses. The most important and major transmission
of nosocomial infections is by contact, usually directly but sometimes indirectly by
contact of secretions from the body. Air can also be a route and transmission source of air
borne-nosocomial pathogens[19].

1.3.6. Nosocomial infections caused by Klebsiella pneumoniae strains
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Nosocomial infections caused by extended-spectrum β-lactamase (ESBL) –
producing Klebsiella pneumoniae have been reported several times in USA, Canada,
Latin America. The emergence of ESBL-producing K. pneumoniae has been reported as
an important cause of nosocomial infection in the United States and Europe. The
prevalence of these strains in hospitals ranges from 5 to 25% in several parts of the
world [20,21]. Important manifestations of Klebsiella infection in the hospital setting include
UTI, pneumonia, bacteremia, wound infection, cholecystitis, and catheter-associated
bacteriuria. Other nosocomial infections in which Klebsiella may also be implicated
include cholangitis, meningitis, endocarditis, and bacterial endophthalmitis. Nosocomial
infections may affect adults or children, and they occur more frequently in premature
infants. The principal pathogenic reservoir for transmission of Klebsiella is the
gastrointestinal tract and the hands of hospital personnel. Because of their ability to
spread rapidly in the hospital environment, these bacteria tend to cause nosocomial
outbreaks[21,22].
The incidences of nosocomial infections in Intensive Care Units (ICU). ICU
were reported continuously, and it has been mentioned that in-patient population in ICU
has 2 to 5 times higher than in general or other department in-patient. Klebsiella spp is
one of the most causal bacteria for nosocomial infection in ICU. The most common
nosocomial infection occurred in ICU are ventilator-associated pneumonia (VAP), central
line-associated bloodstream infection (CLABSI), urinary catheter-related infection and
surgical site infection. [23, 24, 25].
1.4. Antibiotic Resistance Mechanisms in K. pneumoniae
17

Fig 7. Antibiotic Resistance Mechanisms in K. pneumoniae
1.4.1. Resistance to Beta-Lactam antibiotics
Worldwide, there is a widespread and recent spread of extended-spectrum beta-
lactamases (ESBLs) of various CTX-M subgroups which, in addition to generating
resistance to oximino-cefalosporines, are accompanied by transferable resistance
mechanisms to quinolones and aminoglycosides[6, 25]. Bacterial resistance to beta-lactam
antibiotics can be achieved by any of three strategies: the production of beta-lactam-
hydrolyzing beta-lactamase enzymes, the utilization of beta-lactam-insensitive cell wall
transpeptidases, and the active expulsion of beta-lactam molecules from Gram-negative
cells by efflux pumps. On the other hand, another worrying aspect is the production of
carbapenems, in plasmids, which has led to the transmission of these to other
enterobacteria and other non-fermenting bacilli, in addition to the association of
resistance to other antimicrobials. There are several mechanisms of resistance to
carbapenems: modifications in the permeability of the outer membrane, expression of
efflux pumps, production of beta-lactamases with activity of carbapenemases, alterations
of the PBPs, combination of these [4,25,26].
Two classifications of beta-lactamases are known, the Bush-Jacoby-Medeiros and
the Ambler (Table 2). The Ambler classes depend on the amino acid homology, are
classified in 4 molecular classes namely, A, B, C, and D. Molecular classes A, C, and D
include the β -lactamases with serine at their active site, whereas molecular class B stands
for metallo-beta-lactamases (MBLs), enzymes with zinc molecule in the active site. The
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Bush-Jacoby-Medeiros classification grouped the β -lactamases in 3 major groups and
sixteen subgroups. The classification is based on the inhibitors and substrates of the
enzymes[27,28]
Table 2.β-lactamases classification[29]
Extended spectrum beta-lactamases (ESBLs): K. pneumoniae produced the
most common type of ESBL; the transmissibility of plasmids encoding ESBLs allowed
the rapid expansion of this type of resistance to other Enterobacteriaceae as well as to
Pseudomonas (Ps.) aeruginosa[30]. One of the most frequent combinations in
Enterobacteriaceae is that of permeability disorders plus hyperproduction of AmpC beta-
lactamases or production of ESBLs. Today there are over 130 TEM derivatives, over 50
SHV and 12 OXA types[30]. The synergy between ESBL and cefotaxime, ceftazidime or
aztreonam allows in vitro detection of ESBL-producing strains. By blm genes mutations
type TEM, ESBLs have emerged confer resistance to beta-lactamase inhibitors (TRI).
These blm genes are spread to strains as E. coli, K. pneumoniae, Citrobacter (C.) fundi
and P. mirabilis.[4,25,26]
AmpC β-lactamases: are mainly chromosomally codified in Enterobacteriaceae
and they confer resistance to cephalothin, cefazoline, cefoxitin, most penicillin and to β
-lactamase inhibitor (clavulanic acid). Chromosomal AmpC enzymes are inducible and
can be showed at high levels via a mutation in ampD leading to AmpC hyper inducibility
or constitutive hyperproduction[31]. Overexpression confers resistance to extended-
spectrum cephalosporins including cefotaxime, ceftazidime, and ceftriaxone. AmpC
19

enzymes existing on transmissible plasmids are commonly constitutively expressed in
bacteria that do not have or weakly expressing a chromosomal AmpC gene, such as
Escherichia (E.) coli, K. pneumoniae, and Proteus (P.) mirabilis. AmpC enzymes
encoded by both chromosomal and plasmid genes are capable of hydrolyzing broad-
spectrum cephalosporins more efficiently[31,32].
Carbapenemases: are β-lactamases with a broad hydrolytic spectrum. Almost all
hydrolyzable β-lactams including the carbapenems are inactivated by the action of these
enzymes[29,33].
Carbapenemases are in β-lactamases from Ambler classes A, B and D (Fig. 7, 8)
[34] K. pneumoniae produced carbapenems type metallo-beta-lactamase. As a single
enzyme they confer resistance to all beta-lactams less to monobactams (aztreonam). They
are also inhibited by EDTA. IMP-1 metallo-β-lactamase is a class of beta-lactamases,
detected in North Carolina in 1996 and designated as KPC-1 because it was identified for
the first time in K. pneumoniae, encoded in the blaKPC gene. Since then variants of this
enzyme have been detected worldwide (KPC-1/2 to KPC-11)[4]. The mobile nature of the
genetic element encoding KPC, Tn4401, which is found in a plasmid, had contributed to
the propagation of this enzyme that has been identified in numerous Enterobacteria. The
bla genes have been identified in long plasmids that vary in size and structure. These
plasmids generally carry resistance to aminoglycosides and have been associated with
other beta-lactamase genes such as the blaCTXM-15 gene[25,34]. Up to 7 types of beta-
lactamases were found associated with bla KPC in a single isolation of K. pneumoniae. KPC
enzymes can be confused with ESBLs, since they also confer resistance to cephalosporins
and are partially inhibited by clavulanic acid and tazobactam[4,25].
In class A, the prevalent carbapenemase is K. pneumoniae carbapenemase which
was mainly detected on plasmids of Klebsiella pneumonia[35,36]. Till today, K. pneumoniae
carbapenemase enzymes have fifteen different amino-acid variants [37] and possess
hydrolytic activity on the extended-spectrum cephalosporins, carbapenems and
aztreonam[27]. The NMC (non-metallo carbapenemase), IMI (imipenem hydrolyzing β-
lactamase) and SME ( Serratia marcescens enzyme) carbapenemases are parts of Ambler
class A and 2f in Bush-Jacoby-Medeiros classification. These enzymes are
chromosomally located in Enterobacter sp and in Serratia marcescens while they are
closely related to each other as imipenem hydrolyzing β-lactamase and non-metallo
carbapenemase have 97% amino acid similarity and they are homolog 70% to Serratia
20

marcescens enzyme[38,40]. All the 3 enzymes have a wide hydrolysis spectrum that
includes the penicillins, early cephalosporins, carbapenems, and aztreonam[33,40].
Fig 8: Carbapenems antibiotic.
Source: https://www.colourbox.com/vector/structural-chemical-formulas-of-beta-lactam-antibiotics-
carbapenems-vector-10787699
Fig 9: Phylogenetic tree of the metallo-carbapenemase and serine carbapenemase genes with their
mean percentage GC content, according to groups and phenotypic properties. The percentage GC values
correspond to the averages of each subgroup. ATM, aztreonam; CLA, clavulanic acid; R, resistant; S,
susceptible [34]
21

1.4.2. Resistance to Cephalosporins of third and fourth generation.
The β-lactams are broad-spectrum bacterial antibiotics that have a very low
toxicity for the human organism because their mode of action involves their union with
enzymes that participate in the cellular synthesis of bacteria, a structure that does not have
an equivalent in the eukaryotic cells; these antibiotics include bicyclic and monocyclic
compounds. Penicillin and cephalosporins are the first group, and mono-bactamics are the
second [41]. The enterobacterial cell wall has a peptidoglycan layer, a fundamental
component that allows a great mechanical resistance that prevents the osmotic effects that
could seriously modify the bacterial volume and therefore its viability [7]. The strength of
peptidoglycan depends on transpeptidation, which is the formation of cross-links between
the chains of the polymer, which is catalyzed enzymatically. The cephalosporins block
this process and leave the bacteria vulnerable to the osmotic alterations induced by the
medium, which easily kills them [4]. The main mechanism of resistance to β-lactams in
gram-negative bacteria involves β-lactamases, periplasmic enzymes capable of
hydrolyzing the β-lactam ring of the same antibiotics, which is related to their activity [7,
42].
During the last decades, the growth rates of the β-lactam levels, the growth rates
of the β-lactam levels, the substrate spectrum of the β-lactamases, remained constant in
the bacterial population acting inactivating the antibiotic pioneers but not those that were
going to develop over time. Thus, in the early eighties, β-lactamases, which easily
hydrolyzed ampicillin, virtually did not act on third generation cephalosporins
(cefotaxime, ceftriaxone and ceftazidime) or on aztreonam (the first monobactam
produced) [7, 43].
In 1983 the isolation of a strain of Klebsiella ozaenae was reported in Germany
with a plasmid encoding a new β-lactamase, which was capable of hydrolyzing
cefotaxime. Over time, other β-lactamases were found that conferred resistance to varying
degrees to cefotaxime, ceftazidime and aztreonam but which were still sensitive to
imipenem and cephamycin. These enzymes were called “extended spectrum β-
lactamases”. However, later expanded forms appeared in its spectrum of substrates, which
acted on imipenem or on cefoxitin and other cephamycin [42]. The molecular analysis of
some extended spectrum β-lactamases revealed that, compared to the original enzymes,
they had mutations that determined the alteration of the primary structure in one or
several positions of the protein which indicated that the bacteria had remodeled the active
site of the β-lactamases they produced, so that they were now able to recognize several
22

new substrates which, of course, maintained a certain chemical-structural analogy with
the pioneering antibiotics [41]. Third-generation cephalosporin-resistant (3GC-
R) Enterobacteriaceae have spread throughout the world, initially in the hospital setting
and more recently in the community. This resistance is mainly mediated by acquired
extended-spectrum β-lactamase (ESBL) genes located on mobile genetic elements such
as plasmids or transposons[42,43].
1.4.3. Resistance to Quinolones
Quinolones are a group of broad-spectrum synthetic antimicrobials whose “target”
is the synthesis of DNA; they have been widely used for the treatment of intra- and extra-
hospital infections, being a very important resource for developing countries due to the
high availability of generics that drastically reduce the cost of treatment. Its effectiveness
is due to its high bioavailability, level of safety and form of administration that can be
both enteral and parenteral [44, 45]. However, they have been used indiscriminately in the
field of agriculture and in food processing, which makes the increase in resistance to
quinolones huge problem associated with the constant exposure of various
microorganisms [46].
The term “quinolone” is used in a generic sense to refer to the class of inhibitors
of DNA synthesis that include: Naphthyridines, quinolones, isothiazole quinolones,
quinazolines and related agents. The screening of synthetic quinine analogues in search of
new antimalarial drugs led to the discovery of 7-chloroquinoline. The investigation of
similar compounds, such as 1,8 naphthyridines resulted in the discovery of nalidixic acid
(1-ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) in 1962, becoming the first
synthetic quinolone with antimicrobial activity, especially for the treatment of
uncomplicated urinary tract infections[46,47]. This led to the development of other
antimicrobials based on the 4-quinolone ring, such as oxalinic acid, cinoxacin and
flumequine, used clinically to treat infections caused by Gram-negative bacteria[48].
K. pneumoniae resistome combines all the resistance mechanisms known for
quinolone resistance in Gram-negative bacteria involving target-site gene mutations,
increased production of MDR efflux pumps, modifying enzymes and/or target protection
proteins[46,47]. The first and major resistance mechanism is chromosomal mutations in the
quinolone binding targets, DNA gyrase ( gyrA-gyrB subunits) and topoisomerase IV
(parC-parE subunits). Mutations in gyrA and parC in K. pneumoniae were recognized
earlier than mutations in gyrB and parE. Cell permeability changes in K. pneumoniae
23

have been reported to be involved in quinolone resistance, similar to their role in β-lactam
resistance[4]. These included OmpK36 deficiency, overexpression of acrAB, the multidrug
efflux pump gene and consistent production of kdeA. Another important pump in K.
pneumoniae, OqxAB, presumed to originate from the K. pneumoniae chromosome, has
now been identified to get involved in plasmid-mediated quinolone resistance (PMQR),
and to spread between other bacteria. Efflux pump regulators were also identified to get
involved in quinolone resistance in K. pneumonia. Another group of quinolone resistance
genes include PMQR factors, which occur in K. pneumoniae, as in other
Enterobacteriaceae are members of the qnr genes, which encode a family of proteins that
physically protect DNA gyrase and topoisomerase IV from the inhibitory activity of
quinolones[4,46,47].
1.4.4. Resistance to Fluoroquinolones
Fluoroquinolones is quinolones with a fluorine atom (Fig. 9); they are pefloxacin,
ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, moxifloxacin and gemifloxacin. The
microorganisms sensitive to fluoroquinolones are very numerous. Fluoroquinolones
inhibit the activity of topoisomerases, enzymes responsible for DNA super-aspiration
(DNA-gyrase) and relaxation of super spiral DNA (topoisomerase IV), both enzymes
have a similar way of action [47, 50]. The target of fluoroquinolone action is DNA-gyrase in
Gram-negative bacteria and topoisomerase IV in Gram-positive bacteria. Fluoroquinolone
resistance arises through specific mutations within the target proteins DNA gyrase and
topoisomerase IV, within a region termed the quinolone-resistance determining region [50].
In members of the Enterobacteriaceae family, the most common changes occur at
position 83 or 87 within DNA gyrase A and position 80 or 84 within the ParC subunit of
topoisomerase IV. Mutations at all the positions mentioned above have been reported in
fluoroquinolone-resistant Klebsiella pneumonia isolates and a fifth mutation at position
78 in parC has recently been identified. Besides topoisomerase mutations, energy-
dependent efflux and porin loss have also been shown to confer a fluoroquinolone
resistance phenotype in K. pneumoniae. These two changes often occur together in the
majority of multidrug-resistant Klebsiella isolates. [3, 4, 47, 50]
24

Fig 10: General structure of fluoroquinolones, using the accepted numbering scheme for positions on the
molecule. Source: https://www.researchgate.net/figure/General-structure-of-fluoroquinolones-using-the-
accepted-numbering-scheme-for-positions_fig1_221817604
1.4.5. Resistance to Aminoglycoside
Aminoglycosides selectively disrupt the synthesis of bacterial proteins by binding to the
ribosomal 30S subunit, affecting the elongation of the peptide being formed. This
mechanism of action is complex, involving inhibition of peptidyl-tRNA transfer from site
A to the P site and alteration of the reading process that controls the accuracy of the
translational process: the latter process leads to premature termination of protein synthesis
[51]. The final effects vary to some extent from one compound to another, which may
explain differences in bactericidal rates; aberrant proteins can be inserted into the cell
membrane, resulting in alteration of permeability and subsequent stimulation of
aminoglycoside transport. Resistance to aminoglycosides occurs through three
mechanisms: defects accumulation, target modification, synthesis of inactivating
enzymes. 16S rRNA methylase, belonging to the armA gene family, that encodes
enzymes prevents aminoglycosides to bind to their 16S rRNA target [4]. These genes are
plasmid-encoded in K. pneumoniae, and drug-modifying enzymes have a narrow
spectrum activity. 16S rRNA methylases confer resistance to practically all
aminoglycosides, including plazomicin, the most recent aminoglycoside compound
developed. Plasmid mediated 16S rRNA methylases including Rmt family and NpmA
were also found in K. pneumoniae with no evidence of chromosomal location.
25

Chromosomal resistance mechanisms against aminoglycosides in K. pneumoniae include
modifications in cell permeability due to alterations in AcrAB-TolC and KpnEF efflux
pump systems, and due to loss of putative porin, KpnO [3,4,7,51].
1.4.6. Resistance to Tetracyclines
Tetracyclines inhibit aminoacyl-tRNA binding to the ribosome A site. RNAs 7S
and 16S proteins have the best affinity for tetracyclines and therefore are the main
pharmacological targets [52]. This binding inhibits the fixation of a new amino acid-
ribosome tRNA. At high concentrations, tetracyclines also bind to 23S RNA, which is
part of the ribosomal peptidyl transferase; resistance to tetracycline is widespread.
Numerous determinants of resistance to tetracycline (Tet) and to oxytetracycline (Otr)
have been identified, found more frequently in plasmids; thus, resistance to tetracycline
occurs rather by gaining Tet determinants than mutations in existing chromosomal genes
(Fig.10)[52]. The main mechanisms involved in bacterial resistance are: reduction of the
antibiotic concentration in the cytoplasm, which can be achieved by two mechanisms;
reduction of the permeability of the bacterial wall. It is found only in Gram-negative
bacteria and is associated with other mechanisms to determine high levels of resistance;
the energy-dependent efflux determines high levels of resistance. [52,53,54].
Fig 11: Mechanism of T et(O)‐mediated tetracycline resistance. Source: http://emboj.embopress.org/content/22/4/945
1.4.7. Resistance to Polymyxin
Polymyxins act as detergents and alter the permeability of the cytoplasmic
membrane, in the outer membrane, by binding to the negatively charged
lipopolysaccharides and leading to cell lysis. T he major resistance gained to these
antibiotics is chromosomal mechanism and results from a decrease in external membrane
permeability, referred to as the LPS modification system. Strains equipped with this
multifaceted system alter the LPS structure resulting secondary changes in its
biochemical composition which is decreasing of anionic charge interfering with
26

polymyxins binding . Low sensitivity bacteria are characterized by a decrease in the
phospholipid / lipid ratio and an increased concentration of divalent cations (Ca2 +, Mg2
+) [55]. These changes in LPS are provided by mutations in several core genes, responsible
for the maturation of lipid A ( lpxM and its regulator ramA) and by neutralization of lipid
A, by additional binding of amino arabinose ( pbgP, pmrE), phosphoethanolamine ( pmrC)
or palmitate (pagP) [4]. Resistance also involves increased activity of numerous LPS-
modifying gene regulators, such as phoPQ, pmrA and pmrD. Mutation in one of two other
regulation genes, leading to pmrB overexpression or mrgB deactivation, these being
already sufficient to cause resistance to polymyxins . Although it remains exceptional, the
resistance to these antibiotics has recently been described in multiresistant strains to beta-
lactams and aminoglycosides [55, 56].
1.4.8. Resistance to Tigecycline
Tigecycline is bacteriostatic against most susceptible strains, it generally exhibits
bacteriostatic activity against a wide spectrum of aerobic and anaerobic bacteria,
including MDR organisms. It exerts its action by joining the 30s subunit of the bacterial
ribosome; thus, it blocks the entry of the aminoacyl RNAt into the A site of the ribosome,
thereby inhibits the synthesis of bacterial proteins[57]. Tigecycline, the first member of
glycylcyclines, can overcome the two main resistance mechanisms of tetracycline
(ribosomal protection and activity of efflux pumps) due to its long side chain and high
affinity to ribosome [58]. The AcrAB efflux pump is regulated by its local transcriptional
repressor, AcrR, and a global transcriptional activator, RamA, in tigecycline-
nonsusceptible Klebsiella pneumoniae (TNSKP) isolates. High-level expression
of acrAB can result from mutation in acrR and upregulation of ramA [59]. The latter can be
caused by a mutation in ramR, which encodes a local transcriptional repressor of ramA.
Moreover, overexpression of RarA, functioning as a transcriptional activator of the efflux
pump OqxAB, can confer low-level resistance to tigecycline in Klebsiella pneumoniae as
well. In summary, the RamA and AcrAB pathway and RarA together with the AcrAB and
OqxAB pathways have been implicated mainly in tigecycline resistance in Klebsiella
pneumoniae [4,57,58,59].
1.4.9. Resistance to Colistin
Infections caused by Gram-negative bacilli have become a growing problem in
clinical practice due to the emergence of mechanisms of resistance to almost all
27

antibiotics used for their management, which has led to more serious infections, longer
and complexes treatment schedules. The most common mechanism of resistance to this
bacterial group is the production of extended-spectrum beta-lactamases (ESBL), a group
of enzymes capable of hydrolyzing most beta-lactam antibiotics, with the exception of
carbapenems, making from the last ones the best option used for tretment. However, in
the last decade there has been a global increase in the report of Enterobacteriaceae
infections, and non-fermenting Gram-negative bacilli such as Pseudomonas aeruginosa
and Acinobacter baumannii resistant to carbapenems, generating a great clinical impact
by substantially reducing therapeutic options, much more when the development and
research of new molecules has declined throughout the world[60,61,62]. This situation led to
the use of polymyxins, specifically polymyxin E or colistin, as the treatment of choice to
treat infections by these multiresistant microorganisms[63].
Colistin has been used clinically since 1950 in the treatment of Gram-negative
bacterial infections. The mechanism of action is explained by its polycationic nature with
hydrophilic and lipophilic fractions, which act through electrostatic interactions with the
outer membrane of Gram-negative bacteria competitively displacing divalent cations
from the lipids of the membrane, which generates changes in permeability and favors the
release of intracellular content [64]. In principle, the use of the antibiotic was restricted due
to its association with cases of nephrotoxicity and neurotoxicity; however, two reasons
have led to its use: recent studies showing that the toxicity is much lower than initially
believed and[65] the emergence of multiresistant bacteria, specifically K. pneumoniae,
Pseudomonas aeruginosa and Acinobacter baumannii that present susceptibility only to
this agent (Fig. 11)[65]. Taking into account the above, colistin was been for long time a
reserve drug used for the treatment of carbapenem resistant Klebsiella pneumonia strains
in some endemic countries; In addition, in the literature it is common to find texts
recommending its use for specific cases such as treatment of liver abscess due to
multiresistant Pseudomonas aeruginosa , infection with carbapenem-resistant Acinobacter
baumannii and carbapenem-resistant Klebsiella pneumonia endocarditis[66]; with the use
of colistin, reports with resistance to this antibiotic has begun to appear in different
countries of the world, such as Italy, Spain, France, Algeria, United Kingdom and
Switzerland, as well as in different bacterial species such as K. pneumoniae,
Pseudomonas aeruginosa and Acinobacter baumannii [62,64,66].
The experience has shown that hospital outbreaks generate important impact in
terms of morbidity and mortality, in the closure of hospital wards and of the costs for the
28

patients and also for the hospial, so the knowledge of the characteristics of infections
outbreaks caused by these resistant bacteria is very important [66].
Fig 12: Colistin- resistant A.
baumannii strain s do not produce LPS. The
positions of standard molecular mass
markers are shown on the left. Source:
https://aac.asm.org/content/54/12/4971.full
CHAPTER 2. CHARACTERIZATION OF THE A NTIBIOTIC RESISTENCE
FEATURES OF SOME KLEBSIELLA PNEUMONIAE STRAINS ISOLATED
FROM HOSPITALIZED PATIENTS IN BUCHAREST, ROMANIA
2.1.Aim and objectives
2.1.1.Aim
The aim of this study was to analyse the antibiotic resistance features of some
Klebsiella pneumoniae strains isolated from hospitalized patients in Bucharest, Romania
2.1.2.Objectives
1. Analysis of phenotipic antibiotic resistence profile of some Klebsiella
pneumoniae strains
2. Molecular investigation of the antibiotic resistance features in 21 K.
pneumoniae MDR+ strains. Molecular detection of antibiotic resistance genes (ARGs)
using simplex and multiple PCR assays for:
– Carbapenems encoded by blaNDM, blaOXA48, blaCTX-M, blaTEM, blaKPC
– Quinolons encoded by qnrA, qnrB, and qnrS.
2.2.Materials and methods
2.2.1.Materials
This study was conducted on a total number of 21 strains identified as MDR K.
pneumoniae strains isolated from patients hospitalized in 2017 in ICU and in other
clinical departments of Emergency Institute of Cardiovascular Disease “Prof. CC Iliescu”.
Others materials
29

Beside K. pneumoniae strains, many other materials were needed and used while
conducting this study, such as:
-Sterile physiological water, sterile laboratory tampons, sterile inoculation loop,
sterile pins;
-Agar Muller-Hinton media;
-Antibiotics;
-0,5 Mcfarland standard;
-Sterile test tubes, eppendorf tubes, and sterile pipettes;
-Agarose Gel Electrophoresis, TBE buffer (TRIS, EDTA, boric acid);
-PCR Master Mix 2x, Thermo Scientific; the Primers (Biozyme, Romania);
-Solution of NaOH (sodium hydroxide) and SDS (sodium dodecyl sulphate);
-Autoclave, Incubator, Centrifuge, Refrigerator, Thermo block, Thermo Cycler,
Electrophoresis Tanc Machine;
-Bunsen Burner;
-Laminar Flow Hood;
-Safety equipment (gloves, masker, and laboratory coat).
2.2.2. Methods
Molecular detection of antibiotic resistance genes
The genetic support of the ARGs was investigated by s implex and multiplex PCR,
using a total volume 20 µl of reaction mix containing 10 µl PCR Master Mix 2x Thermo
Scientific (Table 3), 7 or 8 µl and 1 µl of bacterial DNA extracted by the alkaline
extraction method.
Table 3. The composition of the total reaction mix.
The
genesConcentration Total
Final
Volume
PrimerMgCl2DNTPDNA
Taq-polReaction
bufferDNA
NDM –
OXA480.5µM1.2mM2µM0.2Uµl1x1 µl20 µl
CTX-M
TEM0.5µM1.2mM2µM0.2Uµl1x1 µl20 µl
KPC
0.5µM1.2mM2µM0.2Uµl1x1 µl20 µl
qnrA
qnrB0.5µM1.2mM2µM0.2Uµl1x1 µl20 µl
qnrS0.5µM1.2mM2µM0.2Uµl1x1 µl20 µl
30

DNA extraction
In this study, for DNA extraction from the strains the alkaline lysis method was
used by performing following steps:[67] 1-5 colonies of bacterial cultures were suspended
in 1.5 ml eppendorf tube containing 20 µl solution of 0.05 M NaOH (sodium hydroxide)
and 0.25% SDS (sodium dodecyl sulphate), heated on a thermo block at 95°C for 15
minutes, for the permeabilization of bacterial cell wall . The next step was the addition
of 180 µl of TE buffer (TRIS+EDTA) 1X. TE buffer was prepared by dissolving Tris-
HCl (10mM) and Na2-EDTA (1mM) in distilled water and adjusting the pH to 8.0 with
HCl. The buffer was stored at room temperature and diluted as required. After that, the
samples were centrifuge at 13000 rpm for 3 minutes, then the supernatant that contains
only the DNA was collected, transferred in a new tube freeze and maintained at -4 °C.
PCR assay
All PCR reactions were performed using the Thermal Cycler machine Applied
Biosistem. Genomic DNA was used as a template for the PCR detection of ARGs –
carbapenems encoded by blaOXA -48, blaNDM, blaTEM, blaCTX-M, blaKPC, and quinolones
encoded by qnrA, qnrB, and qnrS. The PCR reactions were initiated with different
protocols (table 4) and primers (table 5).
Table 4.The amplification program.
Gene Amplification program
Total
VolumeInitial
denaturationNumbers
of cycle Denaturation
in each cyclePrimer
attachment Extension
primer
blaOXA-
4820 µl95°C for 5
min 3095°C,30 sec55°C,40 sec72°C,50 sec72°C,5
min
blaNDM20 µl95°C for 5
min 3095°C,30 sec55°C,40 sec72°C,50 sec72°C,5
min
blaCTX-
M20 µl95°C for 5
min 3095°C,30 sec59°C,30 sec72°C,1 min72°C,
10 min
blaTEM20 µl95°C for 5
min 3095°C, 30 sec59°C, 30 sec72°C,1 min72°C,
10 min
blaKPC20 µl94°C for 10
min 3694°C, 30 sec52°C, 40 sec72°C, 50 sec72°C,5
min
qnrA20 µl95°C for 10
min 3095°C, 1 min54°C, 1 min72°C,1 min72°C,
10 min
qnrB20 µl95°C for 10
min 3095°C, 1 min54°C, 1 min72°C,1 min72°C,
10 min
qnrS20 µl95°C for 10
min 3095°C, 1 min54°C, 1 min72°C,1 min72°C,
10 min
31

Table 5. The nucleotide sequence of the primers
The genePrimerNucleotide sequence Amplicon sizeReferences
blaOXA-48OXA48- F
OXA48-R5´-GCGTGGTTAAGGATGAACAC-3´
5´-CATCAAGTTCAACCCAACCG-3´43868
blaNDMNDM-F
NDM-R5´-GGTTTGGCGATCTGGTTTTC-3´
5´-CGGAATGGCTCATCACGATC-362168
blaCTX-MCTX-M-F
CTX-M-R5'-CGCTGTTGTTAGGAAGTGTG-3'
5'–GGCTGGGTGAAGTAAGTGAC-3'73069
blaTEMTEM-F
TEM-R5'-ATGAGTTTTCAACATTTTCG-3'
5'-TTACCAATGCTTAATCAG TG-3'86170
blaKPCKPC-F
KPC-R5'-GTATCGCCGTCTAGTTCTGC-3 '
5'-GGTCGTGTTTCCCTTTAGCC-3'63871
qnrAqnrA-F
qnrA-R5´-AGAGGATTTCTCACGCCAGG -3´
5´-TGCCAGGCACAGATCTTGAC58069
qnrBqnrB-F
qnrB-R5´-GGMATHGAAATTCGCCACTG -3´
5´-TTTGCYGYYCGCCAGTCGAA -3´26469
qnrSqnrS-F
qnrS-R5´-GCAAGTTCATTGAACAGGGT -3´
5´-TCTAAACCGTCGAGTTCGGCG -3´42869
Gel Electrophoresis
Agarose gel electrophoresis is the most effective way of separating DNA
fragments of varying sizes ranging from 100 bp to 25 kb.[72] In this study the amplification
products were visualized by electrophoresis on a 1% agarose gel using one of the most
common gel running buffers which is TBE buffer pH=8. Specific weight marker (100pb,
Thermo Scientific) was also used.
2.3. Results and discussion
Identification and antibiotic susceptibility testing (automated VITEK 2
system).
In table no 6 there have been revealed the isolation source, hospital department
of isolation and the antibiotic phenotype (automated VITEK 2 system) of 21 strains of K.
pneumoniae MDR strains isolated from patients hospitalized in 2017 in ICU (Intensive
Care Unit) and in other clinical departments of Emergency Institute of Cardiovascular
Disease “Prof. CC Iliescu from Bucharest, Romania.
32

Tabel 6. The bacterial strains and their isolation sources
Department Isolation SourceCoding and Species/Strains Phenotype
Cardiology Urine1. K. pneumoniae 1440MDR +
ICU 2 Urine2. K. pneumoniae 2306MDR +
ICU Vasculary Urine3. K. pneumoniae 2335MDR +
ICU 2 Tracheal
Secretion4. K. pneumoniae 5243MDR +
ESLB +
ICU 2 Tegumentary
Portraiture5. K. pneumoniae 2544MDR +
Vascular Surgery Feces6. K. pneumoniae 2569MDR +
ICU 2 Feces7. K. pneumoniae 2576MDR +
Vascular Surgery Feces8. K. pneumoniae 2583MDR +
ICU 1 Tracheal
Secretion9. K. pneumoniae 1562MDR +
ICU 2 Urine10. K. pneumoniae 2268MDR +
ICU 1 Tracheal
Secretion11. K. pneumoniae
2201/EO8915MDR +
ICU 1 Tracheal
Secretion12. K. pneumoniae 2277MDR +
Cardiology Urine13. K. pneumoniae I 2284MDR +
ICU Vasculary Tracheal
Secretion14. K. pneumoniae 2489MDR +
ICU 2 Feces15. K. pneumoniae 2460MDR +
ICU 2 Blood (Venous
Catheter) 16. K. pneumoniae 1526MDR +
ICU 1 Urine17. K. pneumoniae 2181MDR +
ICU 2 Feces18. K. pneumoniae 2372MDR +
ICU 2 Feces19. K. pneumoniae 1511MDR +
ICU 1 Tracheal
Secretion20. K. pneumoniae 1315MDR +
ICU 1 Tracheal
Secretion21. K. pneumoniae 3038MDR +
Regarding the distribution of the analyzed strains by department hospital (Fig.
13), study reveakled that most of the strains were isolated from ICU 2 (43%) followed
by ICU 1 (30%), ICU Vasculary (9%), Cardiology (9%), and vascular Vurgery (9%). If
all the ICU’s results are put toghether (ICU 1, ICU 2, ICU Vasculary), then the most of
the strains were isolated from ICU (82%).
Fig 13: Graphic representation of the strains distribution according isolation department of the hospital
Regarding the isolation sources, the strains were recovered from urine (n=6),
tracheal secretion (n=7), feces (n=6), blood (n=1) and tegumentary portraiture (n=1).
Most of K. pneumoniae strains were isolated from tracheal secretion (34%) followed by
urine and feces with same percentage (28%), blood (5%) and tegumentary portraiture
(5%) (Fig. 14).
Fig 14: Graphic representation of analyzed K. pneumonia strains distribution by the isolation sources.
33

The incidences of nosocomial infections in ICU were reported continuously, and it
has been mentioned that in-patient population in ICU has 2 to 5 times higher than in
general or other department in-patient. Klebsiella spp. is one of the most causal bacteria
for nosocomial infection in ICU. The most common nosocomial infection occurred in
ICU are ventilator-associated pneumonia (VAP), central line-associated bloodstream
infection (CLABSI), urinary catheter-related infection and surgical site infection, this can
be correlated with the result of this study in which majority of specimen/samples were
isolated from tracheal secretion (33%) and urine (28%), (fig 13) [23, 24, 25].
The strains identification and antibiotic susceptibility testing (automated VITEK 2
system) were performed in the Microbiology Laboratory of the above mentioned hospital,
with the automated VITEK 2 system and the results could be seen in table 7.
Table 7. Number of resistance starins to antibiotics.
Antibiotics Number of resistance strain
BETA LACTAMSPIP (PIPERACILIN) 21
AMP-SUL ( AMPICILIN-SULBACTAM) 21
PIP- TAZ (PIPERACILIN-TAZOBACTAM) 21
CFT-TAZ (CEFTOLOZANE-TAZOBACTAM) 21
AMX-AC. CLA (AMOCICILIN-
AC.CLAVULANIC)21
CFZ (CEFAZOLIN) 21
CFT (CEFOTETAN) 21
CFR (CEFUROXIM) 21
CTZ (CEFTAZIDIME) 21
CFP (CEFEPIME) 21
CTR (CEFTAROLIN) 21
ERT (ERTAPENEM) 19
IMP (IMIPENEM) 12
MRP (MERONPEM) 5
DRP (DORIPENEM) 5
QUINOLONES/
FLOUROQUINOLONE
SCIPRO (CIPROFLOXACIN) 20
LEVO (LEVOFLOXACIN) 20
NOR (NORFLOXACIN) 5
AC.NAL (AC. NALIDIXIC) *
AMINOGLYCOSIDETOB (TOBRAMYCIN) 20
AMC (AMIKACIN) 8
OTHER TYPE
ANTIBIOTICSTET (TETRACICLIN) 20
COL (COLISTIN) 20
TGC (TIGECICLIN) 0
CPE (CLORAMFENIKOL) 21
GMC (GENTAMICIN) 20
NTR (NITROFURANTOIN) 21
34

FOS (FOSFOMICIN) 3
TMP (TRIMETHOPRIM) *
TMP/SMX (TRIMETHOPRIM
/SULFAMETHOXAZOLE) 20
35

Table 8. Antibiotic susceptibility testing results (automated VITEK 2 system).

Fig 15. Graphic representation of number resistance strains from antibiotic susceptibility testing
results (part 1)
Fig 16. Graphic representation of number resistance strains from antibiotic susceptibility testing
results (part 2)
In this study, the antibiotic susceptibility testing (automated VITEK 2 system)
for carbapenems antibiotics showed that 19 (90.47%) of K. pneumoniae isolates were
resistant to ertapenem, 12 (57.14%) of the isolation were resistant to imipenem, and only
5 (23.8%) of the isolates were resistant to meropenem, and intermediate resistance to
meropenem was observed (23.8%); similar results from another study present the first
characterization of carbapenem-non-susceptible K. pneumoniae isolates by means of a
structured six-month survey performed in Romania as part of an Europe-wide
investigation. In that study, the results of the Kirby-Bauer disk-diffusion method showed
that 72 (96%) of K. pneumoniae isolates were resistant to ertapenem, 40 (50.3%)
exhibited meropenem resistance, whereas only 8 (10.7%) of the isolates were imipenem
resistant [73]. Different results were observed in a study conducted in Tehran, Iran. The
antibiotic susceptibility testing was performed by the disk diffusion method with the
antibiotic discs MAST Company, UK according to Clinical and Laboratory Standards
Institute (CLSI) guideline. This study showed that only 15% isolates were resistant to
ertapenem, 13% isolates were resistant to imipenem, 14% isolates were resistant to
meropenem, and intermediate resistance to meropenem was not observed (0%)[74].
For the third generation cephalosporin antibiotic, this study revealed that 21
(100%) of K. pneumoniae isolates were resistant to ceftazidime, similar with the result
showed in the study conducted in Iran by Hosseinzadeh et al, in which 66.8% isolates
were resistant to ceftazidime[70].
In this study, the antibiotic susceptibility testing for quinolone antibiotics
showed that 20 strains (95.23%) of K. pneumoniae isolates were resistant to ciprofloxacin
and levofloxacin, and 5 strains (23.8%) were resistant to norfloxacin. Similar results were
obtained in a study that was conducted on eleven K. pneumoniae clinical isolates in 2008
from a major tertiary teaching hospital, in Dublin. In that study, all the isolates were
found to be resistance to β-lactam-based compounds including cephalosporins, along with
quinolones and fluoroquinolones [75]. Different results were observed in another study

conducted in Romania, in which were only 6.9% of K. pneumoniae strains were resistant
to ciprofloxacin[69].
Molecular detection of antibiotic resistance genes
Based on the results from the phenotypic antibiotic susceptibility testing
(automated VITEK 2 system) on 21 strains of K. pneumoniae MDR+ strains, detection of
ARGs was performed – blaNDM, blaOXA48, blaCTX-M, blaTEM, blaKPC, and qnrA,
qnrB, and qnrS- codifying for quinolons, using simplex and multiple PCR assay and gel
electrophoresis in the Microbiology Laboratory of Biology Faculty, University of
Bucharest.
The results of PCR assays revealed different distribution of carbapenemases
encoded by blaNDM, blaOXA48, blaCTX-M, blaTEM, blaKPC in K. pneumoniae
isolates (Fig. 17,18,19,20).
Fig 17: Graphic representation of ESLBs and carbapenemases disreibution presented in analyzed
strains.
This study demonstrated that all K. pneumoniae strains were negative for blaKPC
gene. Similar result showed in study conducted in Iran by Bina et all,- all investigated K.
pneumoniae strains (100%) were negative for amplification of the bla KPC gene using
the PCR method[74]. This results were also consistent with the results from another study
conducted in Romania, in 2013-2014 by Lixandru et all., that found only four (5.6%)
isolates of K. pneumoniae having the blaKPC-2gene, this study presenting the first
description of K. pneumoniae strains harbouring blaKPC-2 and blaVIM-1 genes in Romania.
[73]. Whereas, other studies from the USA [76], China [77], Italy [78], and in the Middle East,
revealed that significant epidemic of KPC-producing CPE has evolved[79], and confirm
the presence of the bla-KPC gene by PCR in contrast with the present study. This
contrast can be due to reduced susceptibility to at least one extended-spectrum
cephalosporin and another mechanism such as carbapenem resistance as a result of a
combination of an ESBL or AmpC-type enzyme with porin loss[74].

Fig. 18: Electrophoresis gel for carbapenemases KPC. L- Marker (Thermo Scientific), strains no1; 2-2; 3-
3; 4-4; 5-5; 6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15, 16-16; 17-17; 18-18; 19-19; 20-20; 21-21. All
strains were negative blaKPC gene.
Regarding detection of blaOXA48 gene responsible for class D carbapenemases,
this study revealed the presence of the gene in 100% of the strains, similarly result were
conducted in Romania:79% of carbapenemase-producing K. pneumoniae strains. Since
2001, this carbapenemase has been identified in many other countries. OXA-48 had first
been identified from a clinical K. pneumoniae isolate recovered in Istanbul, Turkey.
Further, an increasing number of outbreaks due to OXA-48-producing K. pneumoniae
isolates were reported, not only in Turkey but also in Belgium, France, Greece, the
Netherlands and Spain. The successful spread of OXA-48 producers is now considered an
epidemic threat as it represents an important source of multidrug resistance
in K. pneumoniae in Europe[73]. Different results showed from study conducted in Iran.
The presence of blaOXA-48-like gene in that study was detected in only 2 (0.9%) isolates of
carbapenem-resistant K. pneumoniae and confirmed by sequencing of amplicons[80].

Fig 19: Electrophoresis gel for carbapenemases blaOXA-48 and NDM. L- Marker (Thermo Scientific),
Strains no.1; 2-2; 3-3; 4-4; 5-5; 6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15, 16-16; 17-17; 18-18; 19-
19; 20-20; 21-21, C- Control Negative. All isolates were positive for blaOXA-48 gene and negative for
blaNDM gene.
In case of analysis for detection of blaNDM gene which encode New Delhi
metallo-beta-lactamase, this study revealed the absence of blaNDM, in contrast with
results from another two different study conducted in Romania. One study, in 2015
showed eight (15%) isolates of K. pneumoniae positive for blaNDM-1 gene[73], and another
study in 2013 showed that the majority of the carbapenem-resistant strains exhibited the
blaNDM gene (66.7-100%)[69]. Also, the presence of blaNDM gene was reported by a study
conducted in Iran in 27 (10.9%) isolates, 23 of them being carbapenem-resistant K.
pneumoniae strains [80].
Regarding the detection of blaCTX-M gene responsible for resistance to 3rd
generation of cephalosprine, this study revealed the presence of 95.23% of blaCTX-M
gene in K. pneumoniae, similar to the result of the study conducted by Aljanaby et all. in
Al-Najaf Province-Iraq: 22 strains (51.16%) were positive for blaCTX-M gene in
Klebsiella pneumoniae isolates[81] . Another study conducted in Romania by Marinescu et
al, revealed that blaCTX-M was present predominantly in Klebsiella sp. strains (33.33%)
[69].
55.14% of K. pneumoniae isolates showed the presence of blaTEM for ESBL-
producing this our study, and this is similar and consistent with another study conducted
in northeast India by Ajrit Bora et al, the most prevalent genotype was found
blaTEM in K. pneumoniae strains (77.58%)[82], and twelve strains (48%) of Klebsiella
pneumoniae isolates were also detected in a study conducted in Poland[83].

Fig 20: Electrophoresis gel for ESLB CTX-M and TEM. L- Marker (Thermo Scientific) strain no1; 2-2; 3-
3; 4-4; 5-5; 6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15, 16-16; 17-17; 18-18; 19-19; 20-20; 21-21.
Positive isolates for blaCTX-M gene:2, 3, 4, 5, 11, 13, 14, 15, 16, 17, 18, 19, 20 ; and positive isolates for
blaCTX-M gene: . 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.
The quinolone resistance was also revealed at genotypical level in K. pneumoniae
isolates (Fig. 18, 19, 20).
Fig 21: Graphic representation of the distribution of quinolone resistance markers in analyzed strains.
Regarding the detection of qnr genes resposible for the plasmid-mediated
quinolone resistance, the study revelead the presence of the most prevalent qnr genes in
analyzed K. pneumoniae strains:19 of them (90.47%) were positives for qnrS gene
followed by 2 isolates (9.52%) with qnrB gene positive, similar with study conducted in
pediatric hospital in China: qnrS was the most prevalent (15.1%), followed
by qnrB (6.1%)[84]. All the strains were negative for qnrA gene in analized K.
pneumoniae strains, similar to the study conducted by Marinescu et al. in Romania[69], and
the study conducted by Abduljabar et al. in Iraq[81]. Different result have been
demonstrated in one study conducted by Magesh et al, in Chennai, according whom
69.5% of the isolates were positives for qnrA and followed by qnrB genes – 47.8% [85].
Fig 22: Electrophoresis gel for quinolons qnrA and qnrB. L- Marker (Thermo Scientific) strain no1; 2-2; 3-
3; 4-4; 5-5; 6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15, 16-16; 17-17; 18-18; 19-19; 20-20; 21-21. All
strains were negative for qnrA gene to and there have been founded positive isolates for qnrB gene: 1, 7
This study revealed that the most prevalent qnr genes in K. pneumoniae strains
were qnrS gene, 90.47% positive in analyzed strains.

Fig 23: Electrophoresis gel for quinolos qnrS. L- Marker (Thermo Scientific) strain no1; 2-2; 3-3; 4-4; 5-5;
6-6; 7-7; 9-9; 10-10; 12-12; 13-13; 14-14; 15-15, 16-16; 17-17; 18-18; 19-19; 20-20; 21-21. Positive
isolates for qnrS gene:1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21.
2.4. Conclusion
1. The most of the strains were isolated from ICU (82%), that confirm the fact
that incidences of nosocomial infections in-patient population in ICU were 2 to 5 times
higher than in general or other department in-patient. This high incidence is due to the
treatments or procedures that ICU’s in-patient received such as installation of ventilator,
central line, urinary catheter or the surgical site infection. This incidence also can be
correlated with the results of the isolation source, the most of the samples being isolated
from tracheal secretion (33%) and urine (28%).
2. Using Vitek for antibiotic phenotype caracterisation of K. pneumoniae isolates,
high rate of resistance to ertapenem (90.47%) were observed, followed by resistance to
imipenem (57.14%) and only 23.8% resistance to meropenem; in case of the third
generation cephalosporin antibiotic, 100% of K. pneumoniae isolates were resistant to
ceftazidime and regarding quinolones antibiotics, 95.23% of the strains were resistant to
ciprofloxacin and levofloxacin, and 23.8% were resistant to norfloxacin.
3. The molecular screening of ESBLs and carbapenem resistance genes revealed
the presence blaOXA48 gene in all the tested strains, 95.23% were positives for blaCTX-

M gene, and 55.14% for blaTEM. The most prevalent ESBLs and carbapenemases
encoding genes among analyzed K. pneumoniae strains were blaOXA48 gene, blaCTX-
M gene and blaTEM genes.
The molecular screening of quinolone resistance markers revealed the presence of
the plasmid-mediated quinolone resistance, and the most prevalent qnr genes in K.
pneumoniae was qnrS (90.47% of the strains) followed by qnrB (9.52%).
Selective References
1.Moradigaravand, D., Martin, V., Peacock, S.J., Parkhill, J. 2017. Evolution and
Epidemiology of Multidrug-Resistant Klebsiella pneumoniae in the United
Kingdom and Ireland. In: American Society for Microbiology . 8;1-13.
2.Yan, J., Pu, S., Jia, X., Xu, X., et al. 2017. Multidrug Resistance Mechanisms of
Carbapenem Resistant Klebsiella pneumoniae Strains Isolated in Chongqing,
China. In: Clinical Microbiology Annals of laboratory medicine . 37;398-407.
3.Seibert, G., Hörner, R., Meneghetti, B.H., et al. 2014. Nosocomial infections by
Klebsiella pneumoniae carbapenemase producing enterobacteria in a teaching
hospital. 12(3);282-6.
4.Navon-Venezia1, S., Kondratyeva, K., Carattoli, A. 2017. Klebsiella pneumoniae :
a major worldwide sourceand shuttle for antibiotic resistance. In: FEMS
Microbiology Reviews fux013. 41;252-257.
5.Sidjabat, H., Nimmo, G.R., Timothy, R., et al. 2011. Carbapenem Resistance in
Klebsiella pneumoniae Due to the New Delhi Metallo-b-lactamase. In: Brief
Report CID. 52;481-4.

6.Moghadasi, M. , Mirzaee, M. , Mehrabi, M. R., 2016. Frequency of Quinolone
Resistance and qnrB and qnrC Genes in Clinical Isolates of Klebsiella
pneumoniae. In: J Med Bacteriol. 5;39-45.
7.Mihai, D.C., Chifiriuc, C., Dițu, L.M., Banu, O., Mihăescu, G. 2018. Phenotypic
Profile Virulance Factors in Multiresistant Bacterial Strains Isolated from
Hospitalized Patients. In: Section Advance in Biotechnology, 18 International
Multidisciplinary Scientific GeoConference SGEM 2018;415-20.
8.Vuoto,C., Longo, F., Balice, M.P., Donelli, G., Varaldo, P.E. 2014. Antibiotic
Resistance Related to Biofilm Formation in Klebsiella pneumoniae . In: Pathogens.
3;743–758.
9.Patel, S.S., H.C. Chauhan, A.C. Patel, M.D. Shrimali, K.B. Patel, B.I. Prajapati,
J.K. Kala, M.G. Patel, Manish rajgor and Patel, M.A. 2017. Isolation and
Identification of Klebsiella pneumoniae from Sheep-Case Report Int. J. Curr.
Microbiol. App. Sci. 6(5);331-334.
10.Carter, K.C. Koch’s postulates in relation to the work of Jacob Henle and Edwin
Klebs. 1985. In: Medical History. 1985. 29;353–74.
11.Mortimer, Starr, P. 1986, Edwards and Ewing's Identification of
Enterobacteriaceae. In: International Journal of Systematic and Evolutionary
Microbiology. First Published Online: 01 October 1986. 36;581-582.
12.Fatma, I., Sonbol, Tarek, E., et al. 2012. Conjugative plasmid mediating adhesive
pili in virulent Klebsiella pneumoniae isolates. In: Archives of Clinical
Microbiology. 3(6);4.
13.Camprubí, S., Merino, S., Benedí, J. et al. 1992. Physicochemical surface
properties of Klebsiella pneumoniae . In:Current Microbiology. 24;31-3.
14.Paczosa, M.K, Mecsas, J. 2016. Klebsiella pneumoniae : Going on the Offense
with a Strong Defense. In: American Society for Microbiology. Vol 80 No
3.P:629-61.
15.Sikarwar, A.S., Batra, H.V.2011. Identification of Klebsiella Pneumoniae by
Capsular Polysaccharide Polyclonal Antibodies. In: International Journal of
Chemical Engineering and Applications. 2;130-4.

16.Huynh, D. T. N., Kim, A.-Y., & Kim, Y.-R. 2017. Identification of Pathogenic
Factors in Klebsiella pneumoniae Using Impedimetric Sensor Equipped with
Biomimetic Surfaces. 17(6);1406.
17.Ducel, G. et al. Hospital-acquired infections A PRACTICAL GUIDE 2nd edition.
WHO/CDS/CSR/EPH/2002.12.
18.Nazir, A., Kadri, S.M. 2014. An overview of hospital acquired infections and the
role of the microbiology laboratory. In: International Journal of Research in
Medical Sciences. 2;21-7.
19.Bereket, W., Hemalatha, K., Getenet, B., et al. 2012. Update on bacterial
nosocomial infections. In: European Review for Medical and Pharmacological
Sciences. 16;1039-1044.
20.Marra, A.R., Wey, S.B., Castelo, A., et al. 2006. Nosocomial bloodstream
infections caused by Klebsiella pneumoniae : impact of extended-spectrum β-
lactamase (ESBL) production on clinical outcome in a hospital with high ESBL
Prevalence. In: BMC Infectious Disease. 6(3);1-8.
21.Hadžić, S., Čustović, A., Smajlović, J., Ahmetagić, S. 2012. Distribution of
nosocomial infections caused by Klebsiella pneumoniae ESBL strain. In: Journal
of Environmental and Occupational Science. 1(3);141-146.
22.Dasgupta, S., Das, S., Chawan, N.S., Hazra, A. 2015. Nosocomial infections in
the intensive care unit: Incidence, risk factors, outcome and associated pathogens
in a public tertiary teaching hospital of Eastern India. In: Indian J Crit Care Med.
19(1);14–20.
23.Esfahani, B. N., Basiri, R., Mirhosseini, S. M. M., Moghim, S., & Dolatkhah, S.
2017. Nosocomial Infections in Intensive Care Unit: Pattern of Antibiotic-
resistance in Iranian Community.In:Advanced Biomedical Research. 6;54.
24.Majumdar, Suman S. Padiglione. 2012. Nosocomial infections in the intensive
care unit. In: Anaesthesia & Intensive Care Medicine. 13(5);204-208.
25.Podschun, R., Ullmann, U.1998. Klebsiella spp. as Nosocomial Pathogens:
Epidemiology, Taxonomy, Typing Methods, and Pathogenicity. In: Factors Clin
Microbiol Rev. 11(4);589–603.

26.Bush, K., Jacoby, G.A., Medeiros, A.A., 1995. A functional classification scheme
for beta-lactamases and its correlation with molecular structure. Antimicrob
Agents Chemother. 39(6);1211-33.
27.Bush K, Fisher JF. 2011. Epidemiological expansion, structural studies, and
clinicalchallenges of new ß-lactamases from Gram negative bacteria. Annu Rev
Microbiol. 65;455-478.
28.Ambler RP. 1980. The structure of β-lactamases. Philos. Trans. R. Soc. Lond.
Biol. Sci. 289: 321–331
29.Kanj, S. S., & Kanafani, Z. A. 2011. Current Concepts in Antimicrobial Therapy
Against Resistant Gram-Negative Organisms: Extended-Spectrum β-Lactamase–
Producing Enterobacteriaceae, Carbapenem-Resistant Enterobacteriaceae, and
Multidrug-Resistant Pseudomonas aeruginosa . Mayo Clinic Proceedings , 86(3),
250–259
30.Rawat, D., Deepthi, N.J. 2010. Extended-spectrum β-lactamases in Gram
Negative Bacteria. In: Glob Infect Dis. 2(3);263–274.
31.Jacoby GA. 2002. Clin Microbiology Review AmpC-type ß-lactamases. 46;1-11.
32.Schmidtke AJ, Hanson ND. 2006. Model system to evaluate the effect of ampD
mutations on AmpC-mediated beta-lactam resistance. Antimicrob. Agents
Chemother. 50;2030–2037.
33.Queenan AM, Bush K. 2007. Carbapenemases: the versatile ß-lactamases. In: Clin
Microbiol Rev. 20;440–458.
34.Diene, S.M., Rolain, J.M. 2014. Carbapenemase genes and genetic platforms in
Gram-negative bacilli: Enterobacteriaceae,
Pseudomonas and Acinetobacter species. In: Clinical microbiology and infection.
20(9);831-838.
35.Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward
CD, Alberti S, Bush K, Tenover FC. 2001. Novel carbapene-hydrolyzing ß-
lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae .
Antimicrob Agents Chemother. 45;1151-1161.
36.Nordmann P, Naas T, Poirel L 2011. Global spread of carbapenemase-producing
Enterobacteriaceae. Emerg Infect Dis. 17;1791-1798.

37.Bush, K. 2017. ß-Lactamase Classification and Amino Acid Sequences for TEM,
SHV and OXA Extended-Spectrum and Inhibitor Resistant Enzymes. [Cited on
22th August 2018]. From https://www.lahey.org/studies/
38.Rasmussen, B. A., K. Bush, D. Keeney, Y. Yang, R. Hare, C. O’Gara, and A.
Medeiros 1996. Characterization of IMI-1 betalactamase, a class A carbapenem-
hydrolyzing enzyme from Enterobacter cloacae. Antimicrob. Agents Chemother.
40;2080–2086.
39.Naas, T., L. Vandel, W. Sougakoff, D. M. Livermore, and P. Nordmann 1994.
Cloning and sequence analysis of the gene for a carbapenem-hydrolyzing class A
beta-lactamase, Sme-1, from Serratia marcescens S6. Antimicrob. Agents
Chemother. 38; 1262–1270.
40.Mariotte-Boyer, S., Nicolas-Chanoine, M. H., Labia, R. 1996. A kinetic study of
NMC-A beta-lactamase, an Ambler class A carbapenemase also hydrolyzing
cephamycins. FEMS Microbiol. Lett.143; 29–33.
41.Kliebe, C., Niesm, B. A., Meyer, J. F., Tolxdorff-Neutzling, R. M., Wiedemann,
M. 1985. Evolution of Plasmid-coded Resistance to Broad-spectrum
Cephaloscherichia coli and Klebsiella spp. in Canadian Hospitals”. Antimicrobial
Agents and Chemotherapy. 48; 1204-1214.
42.Kothari, S., Mishra, V., Ranjan, N., Singh, A. 2013. Third generation
cephalosporin-resistance in Klebsiella pneumoniae isolates: an emerging threat.
In: International Journal of Basic and Clinical Pharmacology. 12(1); 56-60.
43.Van der Steen, M., Leenstra, T., Kluytmans, J.A.J.W., Van der Bij, A.K. 2015.
Trends in Expanded-Spectrum CephalosporinResistant Escherichia coli and
Klebsiella pneumoniae among Dutch Clinical Isolates, from 2008 to 2012. PLoS
ONE 10(9);1-12.
44.Heeb, S., Fletcher, M.P., Chhabra, S.R., Diggle,S.P., Williams, P., Cámara M.,
Section (editor) Dieter Haas. 2011. Quinolones: from antibiotics to autoinducers.
FEMS Microbiol Rev. Mar. 35(2); 247–274.
45.Casas, M.R. Camargo, C.H., Soares, F.B., da Silveira W.D., Fernandes S.A. 2016.
Presence of plasmid-mediated quinolone resistance determinants and mutations in
gyrase and topoisomerase in Salmonella enterica isolates with resistance and

reduced susceptibility to ciprofloxacin.In: Diagn Microbiol Infect Dis. 2016 May.
85(1); 85-89.
46.Fàbrega,A., Madurga,S., Giralt,E., Vila1,V. 2009. Mechanism of action of and
resistance to quinolones. Microb Biotechnol. 2(1); 40–61.
47.Drlica,K., Hiasa,H., Kerns,R., Malik,M., Mustaev,A., Zhao, X.1997. DNA gyrase,
topoisomerase IV, and the 4-quinolones.In: Journal ListMicrobiol Mol Biol Revv.
61(3); 377–392.
48.Pallecchi L, Bartoloni A, Riccobono E, Fernández C, Mantella A, Magnelli D, et
al. 2012. Quinolone resistance in absence of selective pressure: the experience of a
very remote community in the Amazon Forest. PLoS Negl Trop Dis 2012. 6 (8);
1790.
49.Drlica, K., Hiasa, H., Kerns, R. 2009. Quinolones: action and resistance updated.
In: Curr Top Med Chem. 9; 981-98.
50.Mehlhorn, A.J., Brown, D.A. 2007. Safety concerns with fluoroquinolones. Ann
Pharmacother. 41; 1859-6
51.Matt, T., Leong, Ng., C., Lang, K., Sha, S.H., Akbergenov, R., Shcherbakov, D.,
Meyer, M., Duscha, S., Xie,J., Dubbaka, S.R., Fernandez, D.P., Vasella, A.,
Ramakrishnan, V., Schacht, J., Böttger, E.C., 2012. Dissociation of antibacterial
activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-
deoxystreptamine apramycin In: PNAS July 3. 109 (27); 10984-10989.
52.Bergeron J, Ammirati M, Danley D et al. 1996. Glycylcyclines bind to the high-
affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-
mediated ribosomal protection. Antimicrob Agents Chemother. Sep. 40(9); 2226-
2228.
53.Chopra I, Roberts M. 2001. Tetracycline antibiotics: mode of action, applications,
molecular biology, and epidemiology of bacterial resistance. In: Microbiol Mol
Biol Rev. Jun. 65(2); 232-60
54.Fluit, A.C., Florijn, A., Verhoef, J., et al. 2005. Presence of tetracycline resistance
determinants and susceptibility to tigecycline and minocycline. Antimicrob
Agents Chemother. Apr. (4); 1636-1638.

55.Abiola, O., Morand, O.M., Rolain, J.M. 2014. Mechanisms of polymyxin
resistance: acquired and intrinsic resistance in bacteria. In: Front Microbiol. 2014;
643.
56.Falagas, M.E., Rafailidis, P.I., Matthaiou, D.K. 2010. Resistance to polymyxins:
Mechanisms, frequency and treatment options Drug resistance updates. 13;132-
138.
57.Spanu T, Angelis GD, Cipriani M, et al. 2012. In Vivo Emergence of Tigecycline
Resistance in Multidrug-Resistant Klebsiella pneumoniae and Escherichia coli.
In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology.
56;4516 – 4518.
58.Sheng, Z.-K., Hu, F., Wang, W., Guo, Q., Chen, Z., Xu, X., Wang, M. 2014.
Mechanisms of Tigecycline Resistance among Klebsiella pneumoniae Clinical
Isolates. Antimicrobial Agents and Chemotherapy. 58(11); 6982–6985.
59.Chiu, S.K., Chan, M.C., Huang, L.Y., Lin, Y.T., Lin, J.C., et al. 2017. Tigecycline
resistance among carbapenem-resistant I: Clinical characteristics and expression
levels of efflux pump genes. PLOS ONE 12(4): e0175140.
60.National Nosocomial Infections Surveillance System. National Nosocomial
Infections Surveillance (NNIS) System Report, data summary from January 1992
through June 2004, issued October 2004. Am J Infect Control. 2004 Dec. 32(8);
470-85
61.Paterson, D.L., Sagnimeni, A.J., Hansen, D.S., Gottberg, A.V., Mohapatra, S.
2002. Community -Acquired Klebsiella pneumoniae Bacteremia. Global
Differences in Clinical Patterns. In: Emerg Infect Dis.2;1-8.
62.Tacconelli, E., Cataldo, M.A., Dancer, S.J., Angelis, G.De., et al. 2014. Escmid
Publications. ESCMID guidelines for the management of the infection control
measures to reduce transmission of multidrug-resistant Gram-negative bacteria in
hospitalized patients. In: Clinical Microbiology and Infection . 20;1-55.
63.Higuita-Gutiérrez, L.F., et al. 2017. Brotes hospitalarios de bacterias resistentes a
colistina: revisión sistemática de la literatura. In: Infection. 21(4);214-222.

64.Li, J., Nation, R.L., Turnidge, J.D., Milne, R.W., Coulthard, K., Rayner, C.R.,
Paterson, D.L. 2006. Colistin: the re-emerging antibiotic for multidrug-resistant
Gram negative bacterial infections. In: Lancet Infect Dis. 6(9); 589-601.
65.Lim, L.M., Ly, N., Anderson, D., Yang, J.C., Macander, L., Jarkowski, A. 3rd, et
al. 2010. Resurgence of colistin: a review of resistance, toxicity,
pharmacodynamics, and dosing. In: Pharmacotherapy. 30(12); 1279-91.
66.Falagas, M.E., Kasiakou, S.K. 2005. Colistin: the revival of polymyxins for the
management of multidrug-resistant gram-negative bacterial infections. In: Clin
Infect Dis. 40(9); 1333-41.
67.Natarajan, V.P., Zhang, X., Morono, Y., Inagaki, F., Wang, F. 2016. A Modified
SDS-Based DNA Extraction Method for High Quality Environmental DNA from
Seafloor Environments. Front. Microbiol. 7; 986.
68.Poirel, L., Walsh, T.R., Cuvillier, V., Nordmann, P. 2011. Multiplex PCR for
detection of acquired carbapenemase genes. 70(1); 119-123.
69.Marinescu, F., Marutescu, L., Savin, I., Lazar, V. 2015. Antibiotic resistance
markers among Gram-negative isolates from wastewater and receiving rivers in
South Romania. In: Romanian Biotechnological Letters. 20(1); 10055-69.
70.Efrekar, R.F., Hosseini-Mazinani, S.M., Ghandili, S., Hamraz, M., Zamani, S.
2005. PCR detection of plasmid mediated TEM, SHV and AmpC β-lactamases in
community and nosocomial urinary isolates of Escherichia coli. In: Iranian J
Biotech. 3(1); 48-54.
71.Hong, S. S., Kim, K., Huh, J. Y., Jung, B., Kang, M. S., & Hong, S. G. 2012.
Multiplex PCR for Rapid Detection of Genes Encoding Class A
Carbapenemases. In: Annals of Laboratory Medicine. 32(5); 359–361.
http://doi.org/10.3343/alm.2012.32.5.359.
72.Lee, P.Y., Costumbrado, J., Hsu, C.Y., Kim, Y.H. 2012. Agarose Gel
Electrophoresis for the Separation of DNA Fragments. In: J. Vis. Exp. (62); 1-5.
doi:10.3791/3923.
73.Lixandru, B. E., Cotar, A. I., Straut, M., Usein, C. R., Cristea, D., Ciontea, S.,
Damian, M. 2015. Carbapenemase-producing Klebsiella pneumoniae in Romania:

A six-month survey. In: PLoS ONE. 10(11); 1-10.
https://doi.org/10.1371/journal.pone.0143214 .
74.Bina, M., Pournajaf, A., Mirkalantari, S., Talebi, M., & Irajian, G. 2015.
Detection of the Klebsiella pneumoniae carbapenemase (KPC) in K. pneumoniae
Isolated from the Clinical Samples by the Phenotypic and Genotypic Methods. In:
Iranian Journal of Pathology. 10(3); 199–205.
75.Anes, J., Hurley, D., Martins, M., & Fanning, S. 2017. Exploring the Genome and
Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical
Origin. Frontiers in Microbiology. 8; 1913.
http://doi.org/10.3389/fmicb.2017.01913
76.Munoz-Price LS, Hayden MK, Lolans K, Won S, Calvert K, Lin M, et al. 2010.
Successful Control of an Outbreak of Klebsiella pneumoniae Carbapenemase–
Producing Klebsiella pneumoniae at a Long-Term Acute Care Hospital. Infect
Control Hosp Epidemiol. 31; 341–347.
77.Chen, S., Hu, F., Xu, X., Liu, Y., Wu, W., Zhu, D., et al. 2011. High Prevalence
of KPC-2-Type Carbapenemase Coupled with CTX-M-Type Extended-Spectrum-
Lactamases in Carbapenem-Resistant Klebsiella pneumoniae in a Teaching
Hospital in China. Antimicrob Agents Chemother . 5; 2493–2494.
78.Mosca, A., Miragliotta, L., Prete, D.R., Tzakis, G., Dalfino, L., Bruno, F., et al.
2013. Rapid and sensitive detection of bla -KPC gene in clinical isolates of
Klebsiella pneumoniae by a molecular real-time assay.SpringerPlus. 2; 31.
79.Duin, V.D., Doi, Y. 2017. The global epidemiology of carbapenemase-producing
Enterobacteriaceae. Virulence. 8(4); 460–469.
80.Hosseinzadeh, Z., Ebrahim-Saraie, H., Sarvari, J., et al. 2018. Emerge
of blaNDM-1 and blaOXA-48- like harboring carbapenem-resistant Klebsiella
pneumoniae isolates from hospitalized patients in southwestern Iran. In Journal of
The Chinese Medical Association. 81; 536-540.
81.Aljanaby, A.A.J., Alhasnawi, H.M.R.J. 2017. Phenotypic and Molecular
Characterization of Multidrug Resistant Klebsiella pneumoniae Isolated from
Different Clinical Sources in Al-Najaf Province-Iraq. In: Pakistan Journal of
Biological Sciences. 20(5); 217-32.

82.Bora, A., Hazarika, N.K., Shukla, S.K., Prasad, K.N., Sarma, J.B., Ahmed, G.
2014. Prevalence of blaTEM , blaSHV and blaCTX-M genes in clinical isolates
of Escherichia coli and Klebsiella pneumoniae from Northeast India. Indian J
Pathol Microbiol. 57; 249-54.
83.Ojdana, D., Sacha, P., Wieczorek, P., et al. 2014. The Occurrence of blaCTX-
M, blaSHV, and blaTEMGenes in Extended-Spectrum β-Lactamase-Positive Strains
of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis in Poland. In:
International Journal of Antibiotics. 2014; 1-7
84.Wang, A., Yang, Y., Lu, Q., Wang, Y., Chen, Y., et al. 2008. Occurrence of qnr-
positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-
type β-lactamase from five pediatric hospitals in China, FEMS Microbiology
Letters. 283(1); 112–116. https://doi.org/10.1111/j.1574-6968.2008.01163.x
85.Magesh, H., Kamatchi, C., Vaidyanathan, R., Sumathi, G. 2011. Identification of
plasmid-mediated quinolone resistance genes qnrA1, qnrB1 and aac(6')-1b-cr in a
multiple drug-resistant isolate of Klebsiella pneumoniae from Chennai. In:Indian J
Med Microbiol. 29; 262-8.