الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
S.aureus is a main cause of community and hospital-acquired infections. The
pathogenicity of S.aureus is a sophisticated process including a varied range of virulence
factors which are expressed through various phases of infection via a web of virulence
regulators.
Molecular basis of pathogenicity of S.aureus encompasses expression of an array of
accessory genes located on agr locus. Several reports suggested that the role of agr in
human infection is sophisticated, enhanced by the isolation of agr defective strains from
clinical samples. agr Dysfunctional (defective) strains usually have a higher ability to form
biofilm and are more fit in vitro because of the huge metabolic burden of having an active
agr system. agr Dysfunction has been associated with persistent bacteremia, reduced
susceptibility to vancomycin and thrombin-induced platelet microbicidal protein.
The aim of the present study was to study the association of accessory gene regulator
alleles, vancomycin susceptibility and biofilm formation among Staphylococcus aureus
isolated from clinical and nasal carrier specimens.
A total of 150 S.aureus isolates were included in this study, 100 S.aureus isolates
from clinical samples, collected from patients attending the Microbiology department of
the Medical Research Institute, Alexandria, Egypt during the period from December 2017
to November 2018. These isolates were isolated from different types of clinical samples,
pus 51 (34%), wound 36(24%), blood 13 (8.7%) and 50 S.aureus isolates (33.3%) were
collected from nasal carriers by nasal swab.
The study showed the following results:
❖ Susceptibility of the 150 S.aureus isolates to different classes of antibiotics was
determined by disc diffusion method. The highest level of resistance was encountered
for β-lactams as Penicillin (98.7%), Ampicillin (90%), Cefoxitin (83.3%), and for
Fucidin (57.3%), Gentamicin (56.7%), Doxycycline (50%) and Erythromycin(32%).
meanwhile the highest level of sensitivity was encountered for Linezolid (100%),
Rifampicin and Tigecycline (86.7%), Chloramphenicol (78.7%), co-trimoxazole
(77.3%), Ciprofloxacin (62.7%) and Clindamycin (59.3%).
❖ Out of the 150 S.aureus isolates included in the current study 125(83.3%) were MRSA.
and 25 isolates (16.7%) were MSSA. Ninety-nine (99 %) of the clinical isolates were
MRSA compared to only 52 %(22/50) of the nasal carrier isolates.
❖ Susceptibility to vancomycin was determined by both the broth Microdilution
technique and the agar dilution technique; 145(96.7%) out of 150 S.aureus isolates
were vancomycin sensitive with MIC of ≤ 1 μg/ml while 5(3.3%) isolates were
inhibited in presence of concentration of ≥2-4 μg/ml. According to CLSI definition,
these 5 isolates were assumed to be hVISA or VISA. By using agar dilution technique
the results exhibited none of the S.aureus isolates grew on 2μg/ml, 3μg/ml, 4μg/ml nor
6 μg/ml vancomycin agar, therefore none of the isolates were VISA, hVISA or sVISA.
No VRSA was detected among the studied isolates.
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80
❖ Evaluation of agr functionality among the 150 S.aureus isolates was done via agr
CAMP test. Eighty four (84%) of the clinical samples were negative for agr
functionality compared to 44% (22/50) of samples isolated from nasal carriers. This
difference was found to be statistically significant. (P = 0.001)
❖ There has been a highly significant difference between the agr functionality in the
MRSA and MSSA isolates (P = 0.001), as 76% (95/125) MRSA isolates were agr
dysfunctional compared to 44% (11/25) of MSSA isolates.
❖ The sensitivity of agr non functioning S.aureus isolates to both ciprofloxacin and
rifampicin was higher (64.3%,89.3%) than agr-functioning isolates (56.2%, 68%), yet
a statistically significant difference was found only with rifampicin (P=0.046)
compared to ciprofloxacin (P=0.733).
❖ In vitro biofilm formation capability was determined via microtiter tissue culture
plates. Among the 150 S.aureus tested isolates 27 (18%), 35(23.3%), 55(36.7%),
33(22.0%) were strong, moderate, weak and non-biofilm producers respectively.
Eighty-eight (88%) of the clinical samples were positive for biofilm formation
compared to only 58%( 29/50) of samples isolated from nasal carriers were positive.
This difference was found to be statistically significant. (P= 0.001)
❖ There was a statistically significant association between agr functionality and biofilm
formation (P<0.001). As, out of 106 S.aureus isolates with non- agr functioning 98
isolates (92.5%) were biofilm producers. Meanwhile, out of 44 S.aureus isolates with
functioning agr 19 isolates (43.2%) were biofilm producers.
❖ By detection of biofilm genes icaA and icaD by PCR; 95 (63.3%) isolates were
positive for icaD gene only, while 42 (28%) isolates were positive for icaA + icaD
genes. On the other hand, none of the isolates were positive for the icaA gene only and
13 (8.6%) isolates (8MRSA and 5MSSA) were negative for both genes.
❖ Among the 95 only icaD gene positive isolates: 15 (15.8%), 22 (23.2%), 33 (34.7%),
25 (26.3%) were: strong, moderate, weak, non-biofilm formation respectively. While,
among the 42 icaA + icaD genes positive isolates: 10 (23.8%), 11 (26.2%), 21 (50.0%),
0 (0%) were: strong, moderate, weak, non-biofilm formation respectively. (P=0.001)
❖ The thirteen (8.6%) isolates (8MRSA and 5MSSA) were negative for both genes. All 5
MSSA (100%) isolates were non biofilm forming compared to only 3(37.5%) of
8MRSA isolates negative for both icaA or icaD genes were non biofilm forming, and
the rest 5 MRSA isolates 2(25%)were moderate 2(25%)were strong and 1(12.5%) was
weak biofilm producers.
❖ There was a statistically significant association between icaD gene and agr dysfunction
(P<0.001). Among the 95 icaD gene positive isolates: 27(28.4%) were agr functioning
and 68(71.6%) agr non-functioning; also, among the 42 icaA + icaD genes positive
isolates: 9(21.4%) were agr functioning and 33(78.6%) agr non-functioning; on the
other hand, only out of 13 isolates negative for either icaA, icaD 5 MRSA (38.5%)
isolates were agr non-functioning and 8(61.5%) were agr functioning ( 3 MRSA + 5
MSSA).
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81
❖ Distribution of the 150 S.aureus isolates according to agr typing was carried out using
multiplex PCR. ; 81 isolates agrI, 18 isolates agrII, 30 isolates agrIII: 5 isolates agrIV
and 16 non-typeable agr isolates.
❖ Regarding the relation of agr alleles of the150 S.aureus isolates to the source of
specimen, type of infection, resistance to methicillin, agr functionality and biofilm
production the results revealed :-
a- agr group I was predominant in all clinical and healthy specimens. There has been
no significant association between agr types and the source of samples whether
clinical or from nasal carriers (P = 0.174)
b- The agr group I was higher in blood samples (53.8%) than others. agr-specific
group II was relatively higher in wound swabs (16.7%), agr-specific group III was
higher in pus specimens (31.4%), and agr-specific group IV was found only among
5.9% of S.aureus isolated from pus specimens. However, these differences were
not statistically significant(P=0.053).
c- A statistically significant association of agrIII with MRSA was found as it was
only present in 24% of the 125 MRSA isolates compared to 0.0% of the 25 MSSA
isolates (P=0.006).
d- There was a highly statistically significant association between agrII and strong
biofilm formation as 66.7% of agrII formed strong biofilm(p=0.005). Similarly,
there was a highly statistically significant association between agrIV and weak
biofilm formation as 80% of agrIV isolates formed weak biofilm (P=0.001).
e- There was statistically significant difference in the distribution of agr alleles with
respect to agr functionality. agr dysfunction was detected in 16/18 isolates belong
to agr II, on the otherhand agr functioning S.aureus isolates was detected in 3/5
isolates belong to agr IV .
Conclusion
1. High resistance to different classes of antibiotics was detected among S.aureus
isolates.
2. A high percentage of MRSA (83.3%) among the different types of clinical samples
with a high prevalence among pus and blood samples (100 %).
3. No VISA, hVISA, sVISA or VRSA was detected among the 150 S.aureus isolates
included in this study.
4. The percentage of agr dysfunction isolates and the ability to form biofilm were
higher in clinical samples than in nasal swabs isolates, and more prevalent in MRSA
than in MSSA.
5. IcaD gene was the most prevalent biofilm formation gene detected. The formation
of the biofilm in MSSA depends on ica genes, while in MRSA a biofilm can be
formed in absence of both icaA & icaD genes.
6. agrI was the most predominant allele followed by agrIII and agrII, while agr IV
was the least found.
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82
7. The agrII allele was more common among clinical samples (14%) than nasal carrier
samples (8%) additionally, it was statistically significant associated with strong
biofilm formation (P=0.001) and with agr dysfunction (P=0.030).
Recommendations
1. Further studies to compare agr CAMP test and other methods as the Vesicle Lysis
Test (VLT) and quantitative reverse-transcriptase real-time PCR (qRT-PCR) for
identification of agr functionality.
2. The biofilm formation capability of some isolate in the absence of icaABCD genes
highlights the significance of further genetic researches of ica independent biofilm
formation mechanisms especially in MRSA isolates.
3. Larger-scale studies to detect prevalence of virulence genes in agr-deficient isolates
compared to S.aureus isolates that carried agr gene.
4. Further studies assessing the association between agr dysfunction and antibiotic
resistance, especially vancomycin resistance, are imperative.
5. Further studies evaluating non typable agr isolates by sequencing and detection of
the different genes regulating agr are warranted.
6. Large scale studies targeting S.aureus isolated from bacteraemia evaluating the
association between agr functionality and the different virulence factors.
7. Additional studies are needed to explore the feasibility of agr system as a new target
for antimicrobial agents.