الفهرس 
INTRODUCTION & AIM OF THE WORKOnly 14 pages are availabe for public view
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Abstract
Acinetobacter species are free-living microorganisms, present in clinical samples, and different environmental conditions. They are Gram-negative bacteria, strictly aerobic, diplo-coccobacilli, catalase, and citrate utilization positive, oxidase-negative, non-fermenting, and grown easily on common laboratory media. A. baumannii can survive for prolonged periods under a wide range of environmental conditions and on surfaces making it a frequent cause of nosocomial infections and outbreaks especially in ICU, CCU, and inpatients. This organism targets immunocompromised patients and causes a wide spectrum of infections, e.g., bacteremia, respiratory tract, urinary tract, wounds, and associated catheter infections. One of the most striking features of A. baumannii is its unusual ability to develop multiple resistance mechanisms against most of the antibiotic classes, resulting in the emergence and spread of MDR, XDR, and PDR.
This study aimed at evaluation of nosocomial infection rates, the antibiotic-resistant profile of A. baumannii recovered from hospitalized patients of two major University Hospitals in Upper Egypt (Al-Azhar and Assiut University Hospitals) followed by studying fluoroquinolone resistance mechanisms. Furthermore, the study aimed to evaluate the use of various CIP or LEV combinations with various antimicrobial and non-antimicrobial agents as an attempt to control this life-threatening pathogen. A total of 1600 Gram-negative isolates were recovered from different clinical specimens. Out of these, 977 isolates (61.06%) were identified as lactose fermenters and 623 isolates (38.94%) were identified as non-lactose fermenters. The clinical isolates were identified as E. coli (667; 41.69%), Klebsiella spp. (310; 19.38%), P. aeruginosa (486; 30.38%), Proteus spp. (22; 1.38%), A. baumannii (100; 6.25%), and Serratia marcescens (15; 0.94%). The recovered A. baumannii isolates represented 6.25% of the total collected isolates. The specimens that showed the highest percentage (61%, 61/100) of A. baumannii contamination were obtained from the respiratory tract (ETT, 29%; nasal, 17%; sputum, 13%; and throat, 2%) while 9% and 12% isolates were recovered from urinary tract (not catheter-associated) and blood infections, respectively. Wounds, skin, urinary tract, and central venous catheter specimens gave 6%, 2%, 8%, and 2% A. baumannii isolates, respectively.
The isolates of A. baumannii were isolated from the cardiac care units (CCU), intensive care units (ICU), and wards of inpatient (IP) in two different hospitals. The highest percentage (77%) of isolates was obtained from different types of ICU (general ICU, chest, gyno, neuro, and ortho ICU) followed by IP (14%) and the lowest percentage (9%) was from CCU. Males were more susceptible to A. baumannii infection than females. The mean age of the patients was 47.9 years. A. baumannii infection occurred among ages ranged from 6 months to 85 years. The highest percentage (38%) of isolates was obtained from patients of ages ranged from 18-40 years while the lowest percentage (2%) was recorded among patients at the initial years of their life. Most A. baumannii isolates (71%) were recovered from patients treated with fluoroquinolones, while the rest (29%) of these isolates were recovered from patients treated with other antimicrobial agents such as cephalosporins, imipenem, and amoxicillin or combination (amoxicillin-clavulanic acid). The illnesses of patients from whom A. baumannii isolates were recovered included surgical, catheterization, ETT, and chronic diseases such as diabetes, bronchial asthma, and heart failure. The average patient stays time in hospitals was 6 days. A. baumannii infection occurred within 3 to 13 days stay period in hospital.
The antimicrobial susceptibility patterns of 100 A. baumannii isolates were determined which revealed a high resistance prevalence of ≥ 78 % for A. baumannii isolates tested against 19 antimicrobial agents except for DO and CT which showed 57% and 5% resistance prevalence, respectively. Out of the 100 isolates, 86 isolates were resistant to ciprofloxacin, MICs range extended from 8 to 64 μg/ml while 14 isolates were susceptible to ciprofloxacin (MICs less than 0.125 µg/ml). Eighty-two isolates were resistant to levofloxacin, their MICs ranged from 4 to 32 μg/ml. On the other hand, the left 18 isolates were susceptible (MICs less than 0.125 μg/ml). Resistance profile analysis of the resulting A. baumannii (100 isolates) showed that two isolates (2%) were scored as pan drug-resistant (PDR), 68% isolates as extensive drug-resistant (XDR), and 30% isolates as multidrug-resistant (MDR). PDR and most of XDR- A. baumannii isolates were isolated from Assiut University hospitals. The susceptibility profile of the tested isolates against the 19 antimicrobial agents showed diversity in their resistance. They were divided into 12 major profiles according to the number of antimicrobial agents to which they were resistant. The resistance scope ranged from 6 to 19 antimicrobial agents. The first profile is PDR (two isolates), represented isolates resistant to 19 antimicrobial agents. The second profile is XDR (32 isolates), represented isolates resistant to 18 antimicrobial agents. On the other hand, profiles number 3 to 12 represented A.baumannii isolates resistant to 17 to 6 of the 19 tested antimicrobial agents.
Determination of the epidemiology of hospital-acquired infection occurred by A. baumannii isolates was carried out by the genotyping method using ERIC-PCR technique. Clonal analysis was revealed that the recovered isolates were not clonal. The phylogenetic dendogram of ERIC-PCR showed that the isolates can be divided into three major clusters. The high diversity into three major clusters may be due to the multiple contamination sources of A. baumannii. This finding is an indication of clonal expansion and microbial colonization from different sources.
Investigations of the mechanisms responsible for the resistance of A. baumannii isolates to FQs included target site mutation, ESBLs production, and plasmid-associated resistance. We found that, firstly, target site mutation analyses of the selected twelve A. baumannii isolates subjected to sequencing represent 12 profiles. Two isolates were susceptible to CIP and had the wild-type profile. Ten isolates were CIP-resistant, 9 of them (9/10; 90%) had 1 gyrA /1 parC mutations (Ser 81→Leu mutation for gyrA gene and Ser 84→Leu mutation for parC gene). The remaining CIP-resistant isolate (1/10; 10%) had 0 gyrA /1 parC mutation (Ser 84→Leu mutation for parC gene). All tested isolates had a silent mutation in one or more positions of their gyrA and parC or both gyrA and parC. Secondly, ESBLs resistant genes detection of the 86 CIP-resistant isolates for three genes showed that these isolates carry CTX-M (33.72%; 29/86), SHV (48.84%; 42/86), and Tem (66.28%; 57/86). Additionally, the CIP-susceptible isolates harbor also ESBLs resistance genes; CTX-M (one isolate), SHV (eleven isolates), and Tem (eight isolates). The nucleotide sequences of these three tested genes were blasted against sequences deposited in the NCBI nucleotides database. The amplicon sequence of CTX-M showed 99.28% identity to Ctx-M15 and it was deposited in Genbank under the accession number KJ127475.1. The amplicon sequences for the two other genes, SHV and Tem, showed 99.40% and 99.52% identit to SHV5 and TEM1, respectively and they were deposited in GenBank under the numbers EF653399.1 for SHV and MF095058.1 for TEM. Thirdly, detection of plasmids; the variable number of bands may be plasmids with different molecular weights per isolate which was detected in 99% of A.baumannii isolates. Besides, one isolate; 1% has not harbored any plasmid. The 86, ciprofloxacin-resistant isolates carried qnrA (66.27%; 57/86), qnrS (70.93%; 61/86), aac (6’)-Ib-cr (52.32%; 45/86), oqxA (73.25%; 63/86) and oqxB (39.53%; 34/86), while qepA and qnrB were undetected in these isolates. Although 14 isolates were susceptible to ciprofloxacin, some resistant genes were detected in these isolates, these included qnrA (7/14), qnrS (7/14), aac (6’)-Ib-cr (3/14), oqxA (12/14), and oqxB (11/14). Four different isolates were selected for sequencing qnrS, acc(6,)-ib-cr,oqxA, and oqxB genes. The blast results revealed that the oqxA and oqxB sequences are not identified in A.baumannii but they were identified previously in Klebsiella aerogenes strain NCTC9793 and Klebsiella pneumoniae, respectively with corresponding identities of 99.78% and 99.77%. On the other hand, the sequence of qnrS, and acc(6,)-ib-cr showed homology to those of A.baumannii deposited in GeneBank database with identity ranged from 97.98% to 98.28%. Co-occurrence of multiple resistance genes was detected for PAFQR and ESBLs genes in tested A.baumannii isolates.
Studying the correlation between resistance to CIP and resistance to other tested antimicrobial agents among A. baumannii test isolates revealed significant associations between resistance to CIP and resistance to gentamicin, gatifloxacin, and levofloxacin but not to the other tested antimicrobial agents. While studying the correlation between FQ acquired resistance genes by the tested A. baumannii isolates and:
(i) resistance to CIP revealed significant correlations (p < 0.05) between resistance to CIP and acquired CTX-M, SHV, aac(6’)-Ib-cr and OqxB genes but not for the remaining acquired FQ resistance genes,
(ii) MIC values of CIP showed a significant correlation between acquired CTX-M gene but not for other acquired genes and the MIC values of CIP aginst the tested isolates,
(iii) type of specimens revealed a significant correlation (p < 0.05) between acquired OqxB and the type of specimens from which tested A. baumannii isolates were recovered, where prevalence of OqxB recorded ≥50% among A. baumannii isolates recovered from blood, CVC, nasal, skin, UTC and wouds specimens. Other acquired resistance genes showed insignificant correlations.
Combination of CIP or LEV with other antibiotics including, ampicillin, ceftriaxone, amikacin, or doxycycline was tested on 34 selected A. baumannii isolates (2 PDR plus 32 XDR) which have been shown to be genotypically non-clonal isolates. This was carried out using the standard protocols of calculating fractional inhibitory concentrations (ƩFICs). Results showed synergism in 23.5%, 17.65%, 32.35% and 17.65% of the tested isolates for the corresponding combination of CIP with, ampicillin, ceftriaxone, amikacin, and doxycycline, respectively and corresponding percentages of 26.47%, 8.28%, 14.71% and 26.47% of the tested isolates for the combination of LEV. On the other hand combination of FQs with vancomycin had nearly no affect. FQs combination with non-antibiotics on susceptibility of the selected resistant A.baumannii isolates revealed that CPZ when combined with CIP or LEV at 200 µg/ml increased the number of susceptible isolates to the two antibiotics by 44.12% and 94.12%, respectively. Propranolol (PR 0.5 mg /ml) decreased the number of resistant isolates by 50% and 85.29% when combined with CIP or LEV, respectively. Interestingly, PR (1 mg/ml) and sodium diclofenac (DIC 4 mg/ml) completely abolished FQ resistance for all the tested isolates when each was used in combination with either CIP or LEV.