الفهرس 
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Abstract
The present study was concerned with the isolation of bacteria from cerebrospinal fluid CSF of Egyptian patients suffering from septic meningitis, and screening them against different antimicrobials agents commonly used to treat septic meningitis. Identifying the most common bacterial pathogens together with their resistance profile against major antibiotics used in the empirical treatment of septic meningitis was also included in the study. In addition, this study aimed at investigating the most common genes involved in bacterial resistance and to evaluate use of various antibiotic combinations for possible synergistic activities against the most clinically relevant pathogens causing septic meningitis particularly MDR.
In this study, a total of 1337 cerebrospinal fluid (CSF) specimens from suspected cases of meningitis were collected over a period of two years from September 2013 to September 2015. The majority of meningitis cases were possibly viral meningitis (573; 42.86%), followed by cases that exhibited diseases with symptomatic similarities to meningitis in which specimen’s cell count was reported “Zero” (467; 34.93%), followed by cases with CSF findings suggesting bacterial meningitis yet showed no positive culture growth (turbid no growth; 229; 17.13%) and finally cases that showed positive culture growth (68; 5.08%). from the positive culture CSF specimens, a total of 71 bacterial isolates were recovered from three different hospitals; (Abbassia Fever hospital, 54 isolates; Ain Shams University hospital, 15 isolates and Ain Shams Specialized hospital, 2 isolates). A total of 65 isolates (91.5%) were from specimens with single bacterial species, where 6 isolates (8.4%) were from mixed cultures. Specimens collected from males were 44 (64.7%), while only 24 specimens were collected from females (35.3%). Regarding age, 5 specimens (7.35%) were from infants (age from 1-12 months), 10 specimens (14.7%) were from children (>1-16 years) and the reset specimens (77.9%) were from adults (>16 Years). Using Gram stain, 48 isolates (67.6%) were found to be Gram-positive and 23 isolates (32.4%) were found to be Gram-negative. from 48 Gram-positive isolates the majority were S. pneumoniae constituting 31 isolates (64.6%, n=48) indicating that Gram-positive bacteria pneumococcal meningitis represents the highest contribution to bacterial meningitis in Egypt.
The susceptibilities of the recovered isolates to different antimicrobial agents commonly used to treat septic meningitis were tested. Results revealed that the highest resistance was observed to penicillin and ampicillin where 41 isolates (61.2%) were resistant to penicillin and 32 isolates (51.6%) were resistant to ampicillin. However, both bacterial categories recorded low resistance pattern to meropenem, levofloxacin and piperacillin / tazobactam.
The antimicrobial susceptibility testing of the Gram-positive isolates collected in this study (n=48) showed that the lowest resistance was observed to meropenem, ampicillin / sulbactam and piperacillin / tazobactam where only 3 isolates (8.57%, 8.57%, 9.1%) were resistant to each. Contrary to that, the highest resistance was observed with penicillin where 21 isolates (44.7%) showed resistance. The antimicrobial susceptibility testing of the 23 Gram-negative isolates collected in this study showed that the lowest resistance was observed to levofloxacin and gentamicin.
Among the 71 collected isolates, 26 isolates (36.6%) were found to be resistant to three or more antimicrobial classes, 18 of these isolates were Gram-negative (69.2%), while 8 isolates were Gram-positive (30.8%). Out of these 26 MDR isolates; 4 isolates were S. aureus (50%), 3 isolates were coagulase negative Staphylococcus (37.5%), one isolate was Enterococcus (12.5%), 7 (38.9%), 4 (22.2%), 2 (11.1%), 1 (5.5%), 1 (5.5%), 1 (5.5%), and 1 (5.5%) isolates were identified as Acinetobacter spp, K. pneumoniae, E. coli, Citrobacter spp, P. aeruginosa, N. meningitidis and Proteus spp, respectively.
All selected MDR isolates (n=26) showed resistance to penicillin and ampicillin. For MDR Gram-negative isolates the highest resistance was observed to penicillin and ampicillin, the lowest resistance was observed to levofloxacin, gentamicin and piperacillin / tazobactam. For MDR Gram-positive isolates the highest resistance was observed to penicillin and ampicillin and lowest resistance was observed to amikacin, piperacillin / tazobactam, and meropenem.
Ten MDR Gram-negative isolates and two MDR Gram-positive isolates were selected to be tested against different antibiotic combinations for possible synergistic activities against the MDR pathogens causing septic meningitis; fractional inhibitory concentration (FIC) values were calculated for each isolate.
MDR Gram-negative isolates were tested against four antibiotic combination (ampicillin / sulbactam plus cefepime; ampicillin / sulbactam plus amikacin; ampicillin / sulbactam plus levofloxacin and amikacin plus levofloxacin). It was found that ampicillin/sulbactam plus cefepime combination showed synergism against 8 tested MDR Gram-negative isolates (80%) and additive effect against two isolates (20%). While two MDR Gram-positive isolates were tested against five different combinations (ampicillin / sulbactam plus cefepime, vancomycin plus levofloxacin, doxycycline plus levofloxacin, ampicillin / sulbactam plus vancomycin and doxycycline plus amikacin) showed Synergism with doxycycline plus levofloxacin combination.
from the MDR isolates, in an attempt to detect weather antimicrobial resistance is plasmid mediated (acquired), or chromosomal mediated (intrinsic); plasmid bands were detected in only 8 out of 19 tested MDR isolates (42.1%) and all showed negative PCR results. Out of 19 MDR isolates subjected to PCR gene detection from extracted chromosome, only 9 isolates gave positive results (47.36%). Other isolates showed negative results despite their resistance profiles that suggest their harboring to tested genes. The prevalence of resistance genes investigated were: tem (21.1%), shv (15.8%), ctx-m (15.8%) coding TEM-, SHV, CTX-M extended spectrum β-lactmases (ESBLs), respectively; aac(6’)-Ib (26.32%) coding for aminoglycoside 6’-N-acetyltransferase type Ib responsible for ciprofloxacin resistant variant; and qnr B (5.3%) gene coding for quinolone resistance.
PCR products of the four genes suggestive for ctx-m, shv, tem, aac(6’)-Ib, obtained upon using chromosomal DNA extracted from certain Gram-negative isolates, were purified and sequenced from both directions. The obtained sequence files were assembled using Staden Package program, and the final consensus sequences were obtained, and their corresponding ORF were analyzed. This proved the existence of ctx-m, shv, tem, aac (6’)-Ib on the extracted chromosome. These genes were annotated and submitted to GenBank database where they were accepted and reported under the accession numbers, ”KX214665, KX214664, KX214663, KX214662”, respectively.
In conclusion, multiple drug resistance represents a serious threat to public health as it compromises the usage of many widespread and inexpensive antimicrobial agents. Most antibiotics including newer generations have reported increased resistant rates. Therefore it is obligatory to monitor antimicrobial usage, using the suitable drug at the appropriate dosage and for the necessary duration is one important method for decreasing the selective pressure that encourages the emergence of resistant microorganisms. Standard guidelines (Antimicrobial stewardship) for antimicrobial prescription have to be provided. Moreover, providing sufficient personnel and resources for infection control in all hospitals as well as developing newer antibacterial agents with different mechanism action that targets new target sites may limit the rabid spread of multidrug resistant microorganisms. Detecting and monitoring resistance pattern are of great importance to provide the healthcare practitioners with a clear picture and to guide them for prescription of more effective antimicrobial remedies. Guided prescription policies must be implemented globally to limit further spread of resistance. Public awareness should also be raised to limit the high level of antimicrobials misuse and to highlight the importance of hygienic practices.