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Genomic characterization and synergy testing of various antibiotic combinations by checkerboard

Genomic characterization and synergy testing of various antibiotic combinations by checkerboard, time-kill and E-test based methods for carbapenem resistant Gram negative bacteria isolated from hospital acquired infection patients.
Hospital acquired (nosocomial) infections are responsible for significant proportion of mortality and morbidity throughout the world. Infection acquired during the hospital stay are generally called hospital acquired infections, initially known as infection arising after 48hrs of hospitaladmission 1,2. The source of nosocomial infections includes objects, people,food water and air with in the hospital 3. A vast majority of these infections are caused by multidrug resistant bacteria, therefore limiting the therapeutic options.
Gram-negative bacilli (GNB), though species may vary in their prevalence, are claimed as the predominant etiological agents in causingthe four most frequent types of hospital-acquired infections namely Bloodstreaminfection, surgical site infection, pneumonia, urinary tract infection. The bacterial disease burden in India is among the highest in the world 4, possibly not due to lack of life- saving antibiotics but often limiting their clinical utility, therefore, enhancing the morbidity and mortality. As a marker of disease burden, pneumonia causes an estimated 410,000 deaths in India each year 5, and is the leading cause of mortality in thechildren 6.
There is a drastic change in the infection control practices, health care and antibiotic use and resistance might have influenced the frequency of these GNB. Drug selection pressure is the single most factors in the evolution of drug resistance in bacteria. Antimicrobial resistance is of global concern with significant impact in developing countries where the infectious disease burden is high and cost constraints for the development of new drugs to tackle the menace. In spite of rapid advances in genomics and high throughput screening technologies, there exists a long intermission in the evolvement of new drug molecules.

PhD Synopsis 2018
Therefore, proper control and management of common and most serious infectious diseases have been critically compromised by the spread of resistance mechanisms among GNB.
The magnitude of drug resistance is directly proportional to the use of particular antibiotic in the community. Indiscriminate/inappropriate use of antibiotics even in situations where they are not expected to improve the patient’s condition, such as, the common cold and uncomplicated cases of diarrhea (which are appropriately treated with oral rehydration therapy) is another contributing practice for the spread of drug resistance. The reasons for selective pressure induced by the drugare multi factorial and involve both human and animal use. Although drug resistance is primarily a medical concern, the factors that influence the spread of resistance are ecological, epidemiological, cultural, social, and economic.
In most of the developing and developed countries antibiotic resistance has become a less priority when Compared with many other infectious diseases. Resistance against antibiotics are at high levels in certain places in India but the problem has remained largely unknown because of relatively few studies were published. However, the issue came to the focus when the New Delhi -Metallo Beta Lactamase-1 first reported in 2009.
Carbapenems are a class of beta- lactam antibiotics primarily intended to use for the treatment of infections caused by GNB. The most commonly used carbapenems are imipenem, meropenem, ertapenems and doripenems. These carbapenems have broad spectrum of activity and highly potent against multidrug resistant GNB. Because of their high efficacy, they are often used as last line agents in life threatening infections. These antibiotics are stable to undergo inactivation by beta-lactamases including the extended spectrum beta-lactamases (ESBL) and AMPc produced by most of the Gram negative pathogens. 7 However, resistance to carbapenems developed by Gram negative bacteria has created a global panic thus, further limiting the antibiotic arsenal. 8

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PhD Synopsis 2018
Resistance to carbapenems may be acquired by following mechanisms
? Production of beta lactamases(carbapenemases) that hydrolysescarbapenems.
? Changes in outer membrane porins that block the entry of these antibiotics.
? Active pumping of the antibiotics out of the cell using complex efflux pumps.
Among these, production of carbapenemase appears to be the most widespread cause of carbapenem resistance. Carbapenemase have the ability to hydrolyze pencillins, cephalosporins, monobactams and carbapenems therefore, limiting the treatment options.
Based on hydrolytic mechanism at the active site three major classes are present. They are of class A, B and D beta-lactamases. class B enzymes are Metallo-Beta-Lactamases (MBL) that contain zinc in the active site. WhileclassA have serine at the active site. An increase in the prevalence of carbapenem resistance mediated by acquired MBL has been reported at high frequency, as the MBL genes reside in mobile gene cassetteintegrons.
The important Class B belongs to the SIM,GIM, SPM, VIM, and IMP families. class A include IMI, SME,NMC,GES and KPC families. While class D carbapenemases consist of OXA-type beta-lactamases frequently detected in Acinetobacterbaumannii 9.
Generally, in clinical microbiology laboratory, screening and surveillance of carbapenemase producing microorganisms is of high priority for the selection of restorative schemes and application of infection prevention measures. Therefore, any GNB with carbapenem resistant or intermediate sensitive should routinely be tested for carbapenemase production by standard methods.
The aim of the study is to identify the carbapenem resistant GNB from clinical samples and also to find the most suitable synergistic antibiotic combination that can be applied in clinical practice. We proposed to use a combination of phenotypic and genotypic methods for screening of carbapenemase production. Similarly, different methods such as, Checkerboard, Time-Kill and E-test have been chosen to identify the synergistic antibiotic combination for each of these resistant GNB.
PhD Synopsis 2018
Multidrug resistant Gram negative bacilli cause significant proportion of life threatening infections, therefore increasing the mortality and morbidity due to limited therapeutic options. Although antibiotics like colistin, tigecycline are often used for treatment of these infections, monotherapy may lead to clinical failure because of various factors. Hence, clinicians often prefer combination of drugs over monotherapy for the proper control of such infections. However, there is a great paucity of information about the synergistic antibiotic combinations with respect to the diverse resistance mechanisms of frequently encountered GNB.Also, little is known about the prevalence of carbapenem resistant GNB in the Indian literature. Therefore, we anticipate that, this study may provide the mechanistic insights in understanding the carbapenem resistance and also authentication of a sophisticated methodology for selection of an appropriate antibiotic combination would be highly beneficial in treating multidrug resistant Gram negative bacterial infections.
1. To estimate the prevalence of carbapenem resistant Gram negative bacilli from clinical isolates in our hospital.
2. To know the mechanism of resistance to carbapenems by phenotypic and genotypic characterization.
3. To identify the synergistic antibiotic combination using time kill assay, checkerboard and E-test and also to compare these methods for their optimal clinical utility to identify the most suitable combination against resistant microorganisms.
4. To select the most appropriate test method for immediate clinical application.

PhD Synopsis 2018
Materials and methods
Bacterial isolates
A minimum of 100 carbapenem resistant Gram negative bacilli will be isolated and identified from various clinical samples received in the department of microbiology. The carbapenem resistant organisms included will be Klebsiella pneumonia, Acinetobacterbaumannii, Pseudomonas aeruginosa, E. coli, and Enterobacter species.
The antibiotics included for synergy testing will be colistin, tigecycline, rifampicin, imipenem, meropenem, ciprofloxacin, cefaperazonesulbactum, amikacin (Sigma Aldrich).
Antibiotic sensitivity testing
Antibiotic susceptibility testing of Gram negative bacilli will be performed by both disc diffusion (Kirby-baur) methods as well as by automated VITEK 2 system. Antimicrobial sensitivity will be determined by measuring the diameter of zone of inhibition according to Clinical and Laboratory Standards Institute (CLSI) recommendations. The isolates which show resistant or intermediate sensitive for imipenems (zone diameter of 16-21mm) will be selected and tested for carbapenemase production.
Tests for carbapenemase screening
Phenotypic methods
GNB isolated from clinical samples will be screened for carbapenemaseproduction by using the following methods
Modified hodge test
Modified hodge test will be performed by making a lawn culture of ATCC Ecoli 25922 and applyingmeropenem disc in the center of the agar plate. The test organisms will be streaked
PhD Synopsis 2018
from the edge of the antibiotic disk to the edge of the plate. After the incubation, development of cloverleaf-like indentation near the disc is the indication for carbapenemase production 10.
EDTA disc synergy test
It is used for phenotypic detection of MBL prodection in clinical isolates. Imipenemdisc will be applied at the centre of the plate with lawn culture of test organism and a filter paper disc soaked in 0.5 M EDTA will be placed at 10 mm distance.EDTA is a poly amino carboxylic acid which binds to zinc metal ionsand inactivate the MBL. After overnight incubation at 37ºC, the appearance of zone betweenthe two discswill be interpreted as positive for MBL detection 11.
Genotypic methods
All the bacterial isolates that are positive for carbapenemase production by phenotypic methods will be further subjected for genomic characterizationfor the detection of various carbapenem resistance genes such as; blaIMP, blaVIM, blaOXA, blaNDM and blaKPC by real time polymerase chain reaction (RT-PCR).
Tests to identify synergistic combination
The synergistic antibiotic combination for the carbapenemase producing GNB mentioned will be identified by using the following methods.
? Checkerboard dilution assay
? Time kill assay
? E (epsilometer)- test
Based on the results obtained in the above tests, the synergistic/additive/antagonistic interaction of the antibiotic combination will be identified by using fractional inhibitory concentration (FIC) methodology. FIC is selected because of its reliability, accuracy, economy, and adaptability to automated and semi-automated platforms and easy to perform.

PhD Synopsis 2018
Checkerboard assay
Checkerboard assays (CB) utilizesstandard powers (sigma erlich) antimicrobial combinations at different concentrations inthe micro-broth (100µl) method. For interpretation of the result, fractional inhibitory concentration index (FIC) is used. FIC is calculated by comparing the minimum inhibitory concentration (MIC) value of each agent alone with the combination-derived MIC. FIC ? 0.5 are considered synergistic; FIC in the ?0.5 to? 2 is considered indifferent and ?2 as antagonistic 12.

Time kill assay
Time kill assay (TKA) is astandard reference method for the determination of synergy between antimicrobial agents. It determines the actual reduction in the viable count of the organisms after exposure to the drug combination compared to the most active single agent at different time intervals. It is performed by adding the standard inoculum in the broths containing the individual antimicrobial agents and its combinations. Sub-culturing is done from the broth containing antimicrobials at different time intervals and the number of colonies will be counted. Synergy is then defined as a 2-log10 decrease in colony count at 24 hrs. by the combinations compared with that by the most active single agent 12.
E-strips containing gradient of antimicrobial agents have been used to determine the synergistic combination. The different versions of E- test methods are;
? E-test cross method
? E- test fixed ratio method
? E- strip agar method

PhD Synopsis 2018
E-test cross method
According to this method, two E- strips, each with different antibiotic will be placed on the agar plate perpendicular to each other, intersecting at the MIC value for each antimicrobial when tested alone. After incubation for 18 hrs., the zone of inhibition is read and the FIC is calculated and interpreted as described for CB assay.
E-test fixed ratio method
In this method synergy will be performed by placing the first E- strip (drug 1) on the inoculated agar plate for 60 min and is then removed. A second strip (drug 2) is then placed in the same
place where the first strip has been kept and incubated for 24h.The FIC is calculated and interpreted as described for CB assay.
E-strip agar method
In this method, incorporate the active antibiotic (drug1) into the agar medium at a concentration specified and place the different antibiotic (drug 2) E-strips on the plates (four strips/plate) after inoculating the organism and incubate overnight. The interpretation of E-test synergy is based on the FIC calculation similar to that of CB method 12.
Statistical analysis
Statistical analysis will be performed by using graph pad prism software version 6.0. Data will be summarized by Mean ± SDfor continuous normal data and Median ± IQR for continuous non normal data/ordinal data. The comparison between the groups will be done by oneway analysis of variants test and followed by posthoc multiple comparison tests for continuous normal data and kruskalwuallis test and followed by posthoc multiple comparison test for continuous non normal data. All p values less than 0.05 will be considered as statistical significant.

PhD Synopsis 2018
It is anticipated that, the phenotypic and genotypic methods proposed will identify the prevalence of carbapenem resistance in frequently encountered Gram negative bacilli and also optimal antibiotic combination that can be applied in regular clinical practice. Critical analysis of the data obtained may provide an opportunity for framing proper infection control and antibiotic policies in order to expand the life span of last line of antibiotics.
Outcome and significance
Completion of the proposed research plan is anticipated to yield information on the clinical utility and precision of the methodologies to identify the most appropriate synergistic antibiotic combination. This will further enable to screen more of such novel antibiotic combinations that a
readily available for effective management of Gram negative bacterial infections. It will also
help in finding out the distribution of specific carbapenem resistance genes among different bacterial species.
Better understanding the dynamics of synergy testing of various antimicrobial agents will help directly in the treatment of the patients and to reduce the mortality and indirectly to prevent the emergence of Multi drug resistance(MDR) pathogens and nosocomial spread of these pathogens in a hospital care setup.


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