Molecular characterization, genotyping and combination susceptibility testing for colistin, rifampin and fosfomycin of Acinetobacter baumannii

Other Title(s)

التوصيف الجزيئي و التنميط الجيني و الجمع في اختبارات الحساسية بين الكوليستين و الريفامبين و الفوسفوميسين على الأسينيتوباكتر بوماني

Dissertant

al-Khatib, Sana Ghalib

Thesis advisor

Farraj, Muhammad A.

University

Birzeit University

Faculty

Faculty of Science

University Country

Palestine (West Bank)

Degree

Master

Degree Date

2022

English Abstract

A total of 29 clinical isolates of Acinetobacter baumannii were collected from Palestine Medical Complex in Ramallah and Al-Makassed Charitable Foundation in Jerusalem.

All the isolates were multidrug resistant including resistance to carbapenems.

The minimal inhibitory concentration (MIC) for all isolates was determined for colistin, rifampin and fosfomycin.

In addition, combination of colistin-rifampin and colistin-fosfomycin were tested using the checkerboard method to determine the possible presence of synergy.

Furthermore, the mechanism of resistance for carbapenems was evaluated by Polymerase Chain Reaction (PCR).

Genes encoding for beta lactamases (carbapenemases) from Ambler group A, B, C, and D were selected and tested by PCR.

The following beta lactamase genes were tested; blaKPC-2 (group A), blaNDM-1 (group B), and b/aOXA-23, b/aOXA-24, blaOXA-48, b/aOXA-51, and blaOXA-58 (Group D).

Biofilm formation was also determined after growing the isolates in tubes and stained with crystal violet and confirmed by repeating the experiment and staining with safranine.

The epidemiologic genotyping of the A.

baumannii isolates was determined by the Random Amplified Polymorphic DNA polymerase chain reaction (RAPD-PCR) fingerprinting method.

The MIC for colistin was < 2 ug/mL in 89.7% (26/29) indicating that these isolates were susceptible to colistin.

However, an MIC of >4 ug/mL was found in 10.3% of the isolates (3/29).

The combination of colistin-rifampin showed the presence of synergy in 5 isolates where the Fractional Inhibitory Index (FIC) for the combination was <0.5, other isolates showed additive effect that could help relief the selective pressure and prevent the evolution of colistin resistance.

This synergistic effect has resulted in the reduction of the MICs of colistin (MIC>4 became 0.25) and rifampin (MIC>4 became <1) when combined with each other’s where resistant isolates turned to sensitive.

As for fosfomycin, the MICs were >64 ug/mL and none of the isolates tested were susceptible to fosfomycin.

However, the MIC results showed that most of isolates became fosfomycin susceptible with an MIC of <4 ug/ml after combination with colistin (MIC >64 became <4).

There was one isolate only that remained resistant to fosfomycin after combination with colistin at MIC >64.

It is also interesting to note that the MIC result showed that one of the isolate that was colistin resistance (MIC=32ug/ml) became sensitive to colistin when combined with fosfomycin (MIC was reduced to 1 ug/ml).

There was no synergy detected in colistin -fosfomycin combination.

However, the effect was additive (FIC is 0.5-4).

Since the MICs for fosfomycin were significantly lowered, it may be useful to use it in combination with colistin to treat infections caused by A.

baumannii which may prevent evolution of resistance to colistin due to selective pressure.

The mechanisms of carbapenem resistance in A.

baumannii are the acquisition of Class D beta lactamases OXa-23, OXA51 and OXa-48, followed by Class B metallo beta lactamase NDM-1 then Class A KPC-2.

All of the clinical isolates carried the genes that encoded for OXA-23 and OXA-51 at rate of 100% and this is too close to literature.

OXA-48 was present in 93.1% of the isolates, a finding that is also very close to that reported in literature.

However, OXA-24 and OX-58 were not found in our research, but was found in low rates in literature.

NDM-1 and KPC-2 was found in high rates in our research (79.3% and 48.3%), but in low rates in literature.

We found out that OXA-23 is the most prominent OXA-type in our study, followed by OXA-48, NDM-1 and KPC-2, respectively.

Biofilm was formed by 65.5% of the A.

baumannii isolates tested.

It was interesting to note that all A.

baumannii isolates that formed a biofilm are positive for blaNDM-1 and most of them has the blaKPC-2 gene .In the absence of blaNDM-1 , no biofilm was formed where the presence of blaNDM-1 gene in isolates that formed biofilm has a rate of 100% (19/19) .The presence of blaKPC-2 gene in isolates that formed biofilm has a rate of 63.2% (12/19).

Biofilm formation could be correlated with the presence of blaNDM-1.

The results of the RAPD-PCR were put in 9 different classes based on the size and number of these bands.

Most of the isolates (58.6%, 17/29) were located in one main classe (I), the remaining isolates were placed in separate classes.

It is of interest to note that 58.8% (10/17) of the isolates in class I pattern came from the intensive care unit (ICU).

Our data showed that four main bands of 2500, 1500, 1000 and 600 were observed in the gel, only 5 isolates had one different band than these mentioned above (650, 500, 400,250 bp).

The band of 2500 bp and 1000 bp have been seen in 93.1% (27/29) of the isolates, the band of 1500 bp has the highest rate of 96.6% (28/29) among the isolates and the band of 600 bp has a rate of 75.9% (22/29).

The band of 500bp has a rate of 6.9% (2/29) while bands 400, 650 and 250 bp each has a rate of 3.4% (1/29).

In conclusion, A.

baumannii is an organism that causes many infections and claims the lives of many patients.

It has evolved to become multidrug resistant and can only be treated with colistin.

The emergence of colistin resistant isolates has been observed in this research and reported in literature.

It is of utmost importance to implement measures to prevent the emergence of colistin resistance in this pathogen.

We recommend based on our finding to use combination therapy such as rifampin which proved to be synergistic when combined with colistin.

Combination with fosfomycin is also recommended where reduction in MIC was observed.

Main Subjects

Science

No. of Pages

128

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-1429291