الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
BSIs are severe diseases characterized by a high morbidity and mortality, which is
directly related with the delay in administration of the first appropriate antibiotic. Rapid
microbiological examination, identification of the causative agent and antimicrobial
susceptibility testing (AST) are therefore very important.
Blood cultures currently represent the main method to determine the etiology of a BSI
because they are easy to perform.
Microorganism identification can be accomplished using various methodologies
starting from biochemical reactions, detection of bacterial DNA directly from the positive
blood culture using nucleic acid-based methods and purification step to obtain a ‘bacterial
pellet’, suitable for a variety of approaches including MALDI-TOF MS.
The aim of this study was to compare biochemical methods to biotyping and molecular
methods for the detection and identification of bacteria in the blood of patients suspected
clinically to have bacteremia. BacT/ALERT® 3D system followed by VITEK® 2 was
compared with MALDI-TOF MS and detection of 16S rRNA genes by PCR.
The present study was conducted on 125 patients with clinical signs of possible
bacteremia. Blood samples were collected from each patient and inoculated in to
BacT/ALERT® blood culture bottles. All positive bottles were subcultured and grown
bacterial colonies were identified using VITEK® 2 system. Bacterial identification was
confirmed using MALDI-TOF MS analysis.
Conventional PCR was carried out for the detection of bacterial growth in
BacT/ALERT® bottles using universal primers targeting the 16S rRNA gene.
Sixty six (52.8%) blood cultures were positive among 125 blood samples and were
further identified by VITEK® 2 and 50 out of them were further tested by MALDI-TOF
MS.
Summary, Conclusion & Recommendation
102
A high percent of concordance (92%) was found between strains identified by
VITEK® 2 and MALDI-TOF MS. The discordance between the two methods was restricted
to subtypes.
The most frequent isolates were gram negative bacilli 52 (78.78 %) including (K.
pneumoniae 18 (27.27%), E. coli 12 (18.18%), A. baumannii 8 (12.12 %) Salmonella sp. 6
(9.09 %), Enterobacter species 5 (7.57%), P. aeruginosa 2(3.03%) and P. mirabilis 1 (1.5%)
while gram positive cocci were found only in 14 (21.2%) including S. aureus 10 (15.15%),
and E. fecalis 4 (6.06%).
Six Salmonella species isolates were isolated to the genus level by VITEK® 2 and
MALDI-TOF. Streptococci were not isolated among the 66 positive blood cultures included
in this study.
Eleven (91.67%) out of the 12 E coli isolates were sensitive to the carbapenem group
while only 7 (38.88%) out of the 18 k. pneumoniae isolates were sensitive to the same group.
Carbapenem resistance was detected in 6 (75%) out of the 8 Acinetobacter isolates and in
the 2 (100%) P. aeruginosa isolates. All K. pneumoniae isolates were ESBL producers
compared to 91.66% of E. coli isolates.
16S rRNA was amplified successfully from all the 26 BacT /Alert® positive bottles
that showed bacterial growth after subculturing. On the other hand, the gene was amplified
in only 10 (22.72%) out of the 44 BacT/Alert® positive bottles that did not show growth
after subculturing indicating that molecular detection of 16S rRNA has higher sensitivity in
comparison to conventional subculturing technique.
Surprisingly, 16S rRNA gene was amplified in 6 (60%) out of the 10 cases that were
positive on BacT /Alert® but showed no growth upon subculture. Blasting of their
sequencing results recognized 4 of them to the species level (K. pneumoniae, S. enterica, E.
cloacae and S. hominis). On the other hand, one was identified to the genus level
(Acinetobacter sp.) and one was diagnosed by as uncultured bacterium clone.
The 15 BacT/ALERT® Negative samples were also negative for the 16S rRNA
indicating that this test has a good specificity.
Summary, Conclusion & Recommendation
103
Conclusion
from the current study, we can conclude that:
1. Bacterial identification by VITEK® 2 and MALDI-TOF MS systems are concordant at
the genus level. Minor differences are present at the species level.
2. Gram Negative bacilli are more common in BSIs compared to Gram positive cocci.
3. The most prevalent Gram negative bacilli were K. pneumoniae followed by E. coli
and A. baumannii.
4. Salmonella species are important cause of bacteremia.
5. Among Gram positive cocci, only S. aureus and E. faecalis were isolated from BSIs.
All streptococci spp were not detected.
6. Almost all strains of K. pneumoniae and E. coli were ESBL producers.
7. The prevalence of MRSA among S. aureus was extremely high.
8. Amplification of 16S rRNA is more sensitive that subculture technique to confirm
bacterial growth in blood culture bottles.
9. Absence of growth after subculture from positive BacT/Alert® bottles does not
always indicate sterility.
10. Sequencing and blasting of 16S rRNA gene is an accurate technique for molecular
bacterial identification.
11. Blood culture using BacT/Alert® system is a specific technique with a good negative
predictive value.