الفهرس 
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Abstract
Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety
of infections, especially in hospitalized and immunocompromised patients. The severe
morbidity and mortality caused by MDR K. pneumoniae, particularly in intensive care
units and pediatric/neonatal wards, causes healthcare costs burden worldwide. The
increased drug resistance of K. pneumoniae over the last few decades has rendered the
infection by these strains very challenging to treat.
The pathogen utilizes different mechanisms to establish resistance against
antimicrobials. This includes production of β-lactamase enzymes, formation of biofilm and
the employment of efflux pumps. Biofilm formation is considered an important virulence
factor for the pathogenesis of K. pneumoniae. Biofilm producing bacteria exhibit resistance
to antibiotics by various methods such as restricted penetration of antibiotics into the
complex biofilm-structure, decreased growth rate of the bacteria and by the expression of
resistance genes.
Rapid and discriminative typing methods are useful for determining the clonality and
genetic relatedness of the isolates in hospital outbreaks. Several methods were described
for K. pneumoniae typing; these include biotyping, serotyping, ribotyping, PFGE, as well
as PCR-based typing methods, such as RAPD-PCR and ERIC-PCR. Other methods, such
as microarrays and sequencing-based methods, are also available, but are more expensive.
The aim of the present study was to determine prevalence of K. pneumoniae
infections among pediatric and neonatal patients, and to determine the association of their
antimicrobial resistance patterns, biofilm formation ability and their molecular genotype.
A total of 46 isolates were collected from different types of clinical samples from
pediatric and neonatal inpatients that attended El-Anfoushy pediatric hospital (Ministry of
Health) over a period of one year. Clinical samples included blood, sputum, urine,
Endotracheal tube (ETT) aspirates, Bronchoalveolar lavage (BAL), and Cerebrospinal
fluid (CSF).
Identification of K. pneumoniae was done by conventional methods, including
morphology, culture characteristics, capsule stain and biochemical tests. Susceptibility of
K. pneumoniae isolates to different antibiotics was carried out by Kirby-Bauer disk
diffusion method. The minimum inhibitory concentration (MIC) values of colistin
antibiotic were determined by the broth microdilution method. Biofilm-formation ability
was then determined by using the crystal violet microtiter plate test. Finally, genotyping of
the clinical isolates was performed by the Enterobacterial Repetitive Intergenic Consensus-
Polymerase Chain Reaction (ERIC-PCR) method. The ERIC-PCR fingerprints of the
isolates were compared and a dendrogram was constructed using a software.
The present study showed the following results:
K. pneumoniae was the most commonly isolated organism among pediatric/neonatal
patients, with a prevalence of 36%. Other bacteria isolated were E. coli (27%),
Enterobacter spp. (4 %), Citrobacter spp. (1 %), Pseudomonas spp. 7 (5 %),
Acinetobacter spp. (3 %), Staphylococcus aureus (8 %), Coagulase-negative
Staphylococci (14 %) and Enterococcus spp. (2 %).
Male predominance was noted, with a percentage of 69.6%.
The majority of the K. pneumoniae isolates were obtained from blood (45.6%). This
was followed by sputum (23.9%), urine (15.2%), ETT aspirate (10.9%) and finally
CSF and BAL (2.2%).
56.5% of the K. pneumoniae isolates were collected from the neonatal intensive care
unit, followed by the pediatric intensive care unit 28.3%, internal unit 10.9% and
lastly the operation unit 4.3%.
80.4% of the K. pneumoniae isolates were able to produce biofilm, out of which
10.9% were strong biofilm-producers, 36.9% were moderate biofilm-producers, and
32.6% were weak biofilm-producers.
Antimicrobial resistance patterns revealed a high level of resistance towards Cefadroxil,
Ceftriaxone and Cefepime (89.1%). Ceftazidime (87%), Ampicillin/Sulbactam (80.4%),
Amoxicillin/Clavulanic acid (78.3%), Piperacillin / Tazobactam (76.1%) and
Cefoxitin (73.9%), followed this. Gentamicin and Aztreonam had also shown a very
high degree of resistance (76.1% and 73.9% respectively). Resistance towards the
carbapenems Imipenem and Meropenem was 52.2% and 56.5% respectively. The
highest level of sensitivity was encountered for Colistin (71.7%) and Tigecycline
(67.4%).
In light of the previous results, only 10.9 % of the isolates were found to be of the
―sensitive‖ type, while 41.3% were multi-drug resistant (MDR), and 47.8% were
extensively drug-resistant (XDR).
Regarding the relationship between biofilm-producing ability and the drug-resistance
type, it was found that 37%, 34.8% and 8.7% of the isolates were biofilm producers
showing MDR, XDR and S antibiotic resistance patterns respectively, while 4.3%,
13% and 2.2% of the isolates were non-biofilm producers showing MDR, XDR and
S resistance patterns respectively. No statistically significant association was found
between biofilm-producing ability and the drug-resistance type (p = 0.375).
The only sample obtained from BAL was a strong-biofilm former. Among the blood
samples, 23.8% were non-biofilm formers, 19% were weak biofilm-formers, 47.6%
were moderate biofilm-formers, and 9.5% were strong biofilm-formers. The single CSF
sample was non-adherent. Among the ETT samples, 60% were weak biofilm-formers,
and 40% were moderate biofilm formers. More than 90% of sputum samples were
biofilm-formers. Urine samples had a widely distributed biofilm-forming ability, where
28.6% were non-adherent, 42.9% were weak biofilm-formers, 14.3% were moderate
biofilm-formers, and 14.3% were strong biofilm-formers. No significance was found
between degree of biofilm formation and specimen type (p = 0.344).
Regarding the antibiotic resistance profiles in biofilm negative and –positive isolates,
there was no significant association found between biofilm-forming ability and
resistance to specific antibiotics.
Genotypic analysis using ERIC-PCR produced a range of one to six bands on gel
electrophoresis per each K. pneumoniae isolate. The size of the PCR products ranged
from 90 to 950 bp.
ERIC-PCR fingerprints classified our 46 isolates into 3 clusters and 30 genotypes,
where 11 (23.9%) isolates were grouped cluster A, 17 (37%) in cluster B, and 18
(39.1%) in cluster C. High genetic diversity was observed among the isolates.
No significant correlation was found between ERIC-PCR cluster distribution and the
type of specimen, hospital ward, biofilm-producing ability and drug-resistance type.
from the data obtained throughout this study, the following was concluded:
K. pneumoniae is a very common pathogen affecting pediatric and neonatal wards.
Most of the clinical samples from which K. pneumoniae was isolated were blood.
A high level of antibiotic resistance including MDR and XDR was detected among
the 46 K. pneumoniae isolates.
Resistance towards Carbapenems is notably increasing in Egyptian hospitals.
Although resistance towards Colistin and Tigecycline is increasing, they are still
promising for treating MDR and XDR K. pneumoniae isolates, however, extreme
care should be taken when dealing with pediatric/neonatal patients due to the grave
drug toxicities they may cause.
K. pneumoniae has a profound ability to form biofilms, which enhances its
virulence and hence pathogenesis.
ERIC-PCR is a rapid, affordable, accessible and interpretable method that can
easily be used for the genetic characterization of bacterial isolates, with excellent
discriminatory power. However, the interlaboratory reproducibility is low, as it
lacks the availability of guidelines for the elucidation of the banding patterns.
K. pneumoniae isolates included in our study were extremely diverse according to
cluster analysis, which negates any cross-contamination or outbreak.