Accepted Proposal for WGS, Synergy, Newer antimicrobials, SQU

Proposed research outline must include at least the following sub-sections:

1. INTRODUCTION

Urinary tract infections (UTIs) are among the most frequent infections in clinical practice
worldwide requiring a considerable number of antibiotic prescriptions. It is a common
complaint among patients presenting to General Practitioners, Family Medicine, Emergency
Medicine, Medicine, Surgery, Obstetrics & Gynaecology and Paediatrics. About 60% of
women and 12% of men will have at least one UTI during their lifetime, thus accounting for a
significant burden of empiric antibiotic usage worldwide. ⁴

The rapid pace of emergence of extended spectrum beta-lactamase producing organisms
(ESBL), AmpC and carbapenem resistant organisms has made it difficult for the clinicians to
keep up to date with the resistance patterns. ^5-7

Prescribing effective empiric antibiotic therapy can be quite a challenging task in these days
of escalating AMR often leading to inappropriate antimicrobial use.⁸ A rigorous study of
epidemiology, demographics, etiology, antimicrobial resistance, virulence, and evaluation of
newer drugs by the Microbiology Department is the need of the hour to enable appropriate
antimicrobial prescribing and optimal patient care.

UTIs are caused predominantly by the extraintestinal Escherichia coli commonly known as
uropathogenic E. coli (UPEC).
⁹
E. coli fall into four major phylogenetic groups; A, B1, B2
and D.
¹⁰
Clermont et al have described a simpler phylogenetic analysis which targets three
genetic markers chuA (required for haeme transport), yjaA (unknown function) genes and a
DNA fragment TSPE4.C2 ¹¹ The majority of virulent E. coli strains belong to phylogenetic
groups B2 and D as described by Johnson and Stell.
¹²The major circulating UPEC clone, ST
131 is replete with toxins, adhesins, toxins and siderophores which aid in overcoming host
defences. ¹³ Intimin like adhesins binds to the bladder epithelial cell lining.
¹⁴ Cytotoxic
necrotizing and haemolytic factors assist in both spread and persistence of bacteria in the
urinary tract.

The galloping antimicrobial resistance of UPEC strains is a grave public health concern. The
strains are becoming more adept at acquiring resistance to antimicrobials through mobile
genetic elements like plasmids, transposons and integrons.
While a large number of studies have focused on E. coli there is a paucity of similar studies
on other uropathogens like Klebsiella pneumoniae, Tribe Proteae, Pseudomomas aeruginosa,
E. faecalis and Staphylococcus aureus and S. saprophyticus.

Few studies have characterized the virulence properties and antibiotic resistance of
uropathogens associated with UTIs in adults and children from the Middle East. In this study,
we will investigate the prevalence of virulence genes, antimicrobial resistance and
phylogenetic groups of pathogens isolated from patients with UTIs, along with assessment of
newer drugs against XDR and PDR Gram negative bacilli isolated from UTI.

1.1. LITERATURE REVIEW

Antimicrobial resistance (AMR) is being increasingly referred to as “the silent tsunami facing
modern medicine”.
¹⁵
This unprecedented upsurge in AMR is breeding a new generation of
‘superbug microbes’ that are virtually untreatable with the existing antimicrobials.
¹⁶ AMR
has become a grave global public health challenge partly due to a lack of newly developed
antibiotics and an over reliance on existing drugs. According to the World Health
Organization (WHO) this may lead to a global catastrophe leaving many people harmed or
dying from simple infections that have become complicated (WHO, Antimicrobial
Resistance, 2017).
¹⁷ The impacts of burgeoning antimicrobial resistance are wide ranging and
costly, not only financially, but also in terms of global health, food sustainability and security,
environmental wellbeing, and socio-economic development.¹⁸

AMR infections are estimated to cause 700,000 deaths each year globally.
¹⁹ O’Neill (2016)
have found that due to the rising drug resistance for pathogens it is estimated that the burden
of deaths from AMR could balloon to 10 million lives each year by 2050. O’Neill (2016)
claimed that on this basis, by 2050, the death could be one person every three seconds.
²⁰

The emergence of the family of carbapenemase enzymes such as KPC, NDM-1, IMP, VIM,
OXA-48 is undoubtedly one of the most significant health challenges of the century.
Carbapenem resistant organisms (CRO) are usually hospital acquired pathogens and are
associated with high mortality (>60%) and morbidity as published in many studies [9, 10].
Mechanisms of carbapenem resistance are heterogeneous. It can be mediated by
carbapenemase production or via production of extended-spectrum β-lactamases (ESBLs)
and/or AmpC cephalosporinases (AmpC) combined with altered membrane permeability
(non-CP-CRE.²¹ CRO are an increasing concern worldwide. The risk factors for CRO comes
from KPC variants, the zinc-dependent metallo beta-lactamases (VIM, IMP, and NDM types)
and OXA like (OXA-48, OXA 181, OXA-232, OXA-23) which inactivate many clinically
relevant beta lactams.²² Moreover, CRO show resistance to many important antibiotics, such
as aminoglycosides and fluoroquinolones. ²²

3. METHODOLOGY

This study will be conducted in the Department of Microbiology and Immunology in the
Sultan Qaboos University (SQU), Muscat, Oman.
3.1 Study duration: January 2024 to January 2026.
3.2 Study Design: Prospective
3.3 Sample size: The sample size will be measured as follows: prevalence of antimicrobial
resistance in Gram negative bacilli in Oman ranges from 17.3% to 37.6%. Considering
prevalence as 30% and level of confidence as 95%, with an error margin of 5%, on either side
of the estimate, the population size was 2000 with positivity as 30% the optimum sample
size for the study was obtained as 273 (Raosoft calculator and Wayne et al, 7th ed). We
however plan to include a minimum 330 Gram negative bacilli for our study. Raosoft
calculator was used for calculating the sample size. http://www.raosoft.com/samplesize.html
3.4 Study Group
Consecutive UTI patients infected with XDR and PDR Gram negative bacilli will be
recruited in the study. Demographic and clinical outcome data will be collected from the
patient’s electronic medical record.
3.5 Risk factors for acquiring XDR and PDR Gram negative bacilli:
The risk factors will include age, sex, status at culture collection, microbiologic data,
antibiotics administered, treatment duration and site of infection. The risk factors will be
collected from the patient’s electronic medical record.
3.6 Collection and processing of samples
Appropriate urine samples (mid-stream, clean catch samples, catheter samples, percutaneous
nephrostomy, suprapubic catheter, suprapubic aspirate etc) will be collected with proper
aseptic precautions and transported rapidly in cool storage boxes (4oC) to the microbiology
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laboratory for analysis. One aliquot will be separated for routine culture and sensitivity. All
samples will be processed according to the Clinical and Laboratory Standards Institute
guideline (CLSI 2020) ²⁹ using the routine semi-quantitative culture method . ³⁰ Phenotypic
characterization will initially be performed by MALDI-TOF (Bruker diagnostics) and BD
Phoenix automated system (Becton Dickinson Diagnostic systems, Spark, MD, USA) XDR
and PDR isolates will be characterised genotypically to carbapenemase level by Cepheid
Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). Antimicrobial susceptibility testing
will be performed by Phoenix automated system as per CLSI2020, M100 30th ed.
All strains will be preserved and stored at -40oC in sterile CryoBeads (Mast Diagnostics, UK)
till further analysis.

3.7 Methodology at a glance:

Detailed Methodology
3.8.1 Genomic DNA Extraction for Whole genome sequencing:

Bacterial genome sequencing will be performed using the Ion Torrent PGM (Life
Technologies, Carlsbad, CA) at Central Public Health laboratory. The bacterial genomic
DNA will be extracted from pure bacterial cultures grown overnight on non-selective media.
DSP Virus/Pathogen Mini Kit (Qiagen) will be used on automated nucleic extractor
(QIAsymphony SP/AS, Qiagen) for extracting DNA. The concentration and purity of DNA
will be checked using Qubit Fluorometric Quantification (Thermo).

3.8.2 Analysis of WGS data
The obtained sequence data will be analyzed using the freely available bioinformatics tools in
the Centre for Genomic Epidemiology website. ResFinder, MLST, PlasmidFinder and
CSIPhylogeny tools will be used to investigate the acquired antimicrobial resistance genes,
multilocus sequence typing (MLST), plasmids and bacterial relatedness respectively. The
CSIPhylogeny tool identifies the variations between the obtained sequence data (FASTA
files) by identifying or calling and filtering high-quality SNPs (z-score higher than 1.96 for
all SNPs) and eventually forming a phylogenetic tree using FastTree algorithm. In addition,
the CARD bioinformatics website will be used to look for the resistance genes and their
mechanisms of action.

3.8.3 Identification of virulence genes and antibiotic resistance genes
BLAST will be used to align denovo assembled genome sequences against the NCBI
Bacterial Antimicrobial Resistance Reference Gene Database
(http://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047),ResFinder
(http://www.genomicepidemiology.org70), and the Pasteur database of virulence genes
(http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Antibiotic resistance genes investigated
included carbapenemases and other ß-lactamase genes.

3.8.4 Molecular Typing by MLST

The MLST database at the Center of Genomic Epidemiology will be used to identify the
sequence types (ST) of the study isolates using WGS data
(https://cge.cbs.dtu.dk/services/MLST/).

eBURST

The program eBURST v 3.0

3.9 Susceptibility to newer antimicrobial agents
The susceptibilities of isolated strains to new antibiotics will be performed by determining the minimum inhibitory concentration (MIC) using microbroth dilution techniques (CLSI guideline) 30 This study aims to evaluate the efficacy of the following antimicrobial agents:
Fosfomycin, colistin, doripenem, tigecycline, plazomicin, ceftolazane + tazobactam, ceftaroline + avibactam, meropenem + vaborbactam, ceftazidime + avibactam, eravacycline, cefiderocol by E tests (Liofilchem and Biomerieux)

• The antimicrobial combinations which will be tested for synergy are the following
• Plazomicin+tigecycline
• Plazomycin+meropenem
• Eravacycline+Plazomicin
• Eravacycline + meropenem
• Cefiderocol+plazomicin
• Cefiderocol+ meropenem

3.10 Antimicrobial synergy testing
The in vitro synergistic bactericidal effects of the selected antibiotics against XDR and PDR
strains will be assessed by Time kill assay, Checkerboard assay and E-test.

3.10.1 Time kill Assay (TKA)
Time kill Assay (TKA) will be performed using 96-well microtiter plate according to a
published technique. 31Antimicrobials will be tested alone and in combination with
concentrations ranging from 1, 0.5, 0.25. MICs will be selected along with other ratios based
on checkerboard results. Control experiments without antimicrobial agents will be conducted
simultaneously with the TK assay.

The vials containing cation-adjusted Mueller-Hinton broth, antimicrobials, and the tested
organisms at an initial density of 106 CFU/ml (10 ml volume) will be incubated at 35 ± 2 °C in
ambient air. Aliquots will be removed at times 0, 6, and 24 h and serially diluted in 0.85%
sodium chloride solution and plated on Mueller-Hinton agar plates for viable-colony
counting.

Time-kill curve will be created by plotting bacterial cell counts for control, antibiotic alone or
in combination with other antibiotics over estimated time. Results will be interpreted as
follow:

– Synergy:if a reduction in colony count by ≥ 2 log10 CFU/ml at 6 or 24 h occurs compared
with the most active single agent

Addition

-Antagonism: if an increase of ≥2 log10 CFU/ml occurs in the combination versus the most
active single agent.

-Indifference: : if an increase or decrease of < 2 log10 in colony counting with an antibiotic
combination versus individual antimicrobials.

-Additivity:If FICI > 0.5 to ≤1

3.10.2 Checkerboard Assay (CB)
Broth microdilution checkerboard technique (BMC) will be performed to assess MICs of
single or of combined antibiotics as recommended by CLSI.
The interactions will then calculate by using the formula:

§ FICI= FICA+FICB ,where:

–    FICI = Fractional Inhibitory Concentration Index

–    FICA = MIC_A in combination/ MIC_A alone

–    FICA = MIC_B in combination/ MIC_B alone

FICI will be interpreted as follows:

–    Synergy: If FICI 0.5

–    Partial synergism: If FICI > 0.5 to <1 [3]

–    Indifference: If FICI ≥ 1 to 4

–    Antagonism: If FICI > 4

3.10.3 E-test methodsMinimum inhibitory concentration value of assayed antibiotics will be tested against all
isolates using an E-test (BioMérieux, France). E-tests will be performed and interpreted
according to the manufacturer’s instructions. The Synergy testing will be performed in three
different methods:

I) Method 1 Method (Cross Methods)
Mueller-Hinton agar (MHA) plate is inoculated with 0.5 McFarland matched inoculum, to
which E-test strips are placed one over the other at 90° angle crossing at the MICs of the
individual agent of the organism determined earlier. After incubation for 18 h, the zone of
inhibition is read.

II) Method 2 (Fixed ratio methods)
Bacterial suspensions of investigated isolates with turbidity of 0.5 McFarland units will be
inoculate in MHA (Mueller Hinton Agar) (150 mm diameter). MICs of drug A and Drug B
will be performed along with the combination setup. For the combination setups, E-test strips
containing the study antibiotics will be added to the bacterial lawn sequentially; the first E-
Page 9 of 22
test strips (strip A) will be incubated for 1 hr at room temperature, then removed and saved as
MICA reading. The second E-test strips (strip B) will be added immediately over the imprint
of the E-test strips A. Plates will be incubated for 18 hrs at 35o C. Respective MIC
strips/scales will be used for MICs reading by placing them in each gradient’s position.

III) Method 3 (E-test MIC: MIC method)
In this method, one test strip is placed on the inoculated MHA plate and incubated at room
temperature for 1 h to allow diffusion of the agent. After 1 h, the agar is marked adjacent to
the previously determined MIC of the agent and removed. The second E-test strip is then
placed over the imprint of the previous strip such that the mark on the agar corresponds to the
MIC of the second agen . The resulting ellipse of inhibition is read after 20 h of incubation at
37°C.

Fractional inhibitory concentration index (FICI) will be calculated to assess synergistic
activity in both methods by using the following formula:

§ FICI= FICA+FICB,where:

–    FICI = Fractional Inhibitory Concentration Index

–    FICA = MIC_A in combination/ MIC_A alone

–    FICI = Fractional Inhibitory Concentration Index

–    FICA = MIC_B in combination/ MIC_B alone

FICI interpretation will be as follows:

–    Synergy : If FICI 0.5

–    Additivity : If FICI > 0.5 to ≤1

–    Indifference : If FICI ≥ 1 to 4

–    Antagonism : If FICI > 4.³²

3.12 Statistical analysis
A database of all clinical and other relevant details with real-time data entry will be created in
Microsoft Excel (Office 2019 Professional for Windows; Microsoft). The statistical analysis
will be performed using IBM Statistical Package for the Social Sciences (SPSS) Statistics for
Windows [version 23.0, Professional] (IBM Corp., Armonk, N.Y., USA). The normality of
the data will be analyzed through the Shapiro-Wilk test. For continuous variables, descriptive
statistics will be used. Categorical variables will be reported as frequencies and percentages
and appropriate statistical tests will be employed. Comparison of quantitative data between
variables will be done using 2 tailed paired t-test. Results will be graphically represented and
demonstrated.

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4. SIGNIFICANCE

Antibiotic resistance has been referred to as “the silent tsunami facing modern
medicine”. Along with the rest of the Middle-east, Oman is also facing this
unprecedented crisis in healthcare. With the exponential rise of antimicrobial
resistance, it is important to map the resistance locally, nationally, and regionally to
understand which antimicrobials are effective and which new antimicrobials should
be introduced in the national therapeutic formulary for optimal patient management.
Effective combination antimicrobial therapies may soon be the only treatment option
available. Therefore, it is essential to identify the synergistic antimicrobial
combinations that would play a pivotal role in reducing patient morbidity, mortality
and length of stay in the hospital. This will be the first study emanating from Oman,
which will focus on new synergistic antimicrobial combinations against not only the
predominant OXA 48 gene producing CRGNB but also NDM, KPC, VIM, IMP and
colistin- resistant GNB ( phoPQ, pmr AB, mgrB mutations and mcr A and B genes)

5. OUTCOME OF THE STUDY AND HOW IT WILL BENEFIT SQUH

This study will assess efficacy of newer antimicrobials as well as identify synergistic
combinations to treat extremely and pan drug resistant Gram negative bacilli causing
urinary tract infections. The knowledge emanating from this study will go a long way
in guiding clinicians to prescribe the appropriate antimicrobials- whether they should
resort to new drugs or use combination of drugs. If combination of drugs is opted for,
then the results of this study will guide in selection of the correct combination which
is likely to have synergistic outcome. This will undoubtedly lead to more effective
patient care. The circulating MLST clonal types and phylogenetic groups of these
bacteria will be identified and their movement traced within the hospital. This will
help to initiate effective infection control and preventive measures not only in SQUH
but also in the community.

6. TIMELINE/ DELIVERABLES