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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. |