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Quantification of the intestinal load of a targeted set of resistance genes to Monitor Antibiotic Resistance in paediatric transplant patients

Periodic Reporting for period 1 - qMAR (Quantification of the intestinal load of a targeted set of resistance genes to Monitor Antibiotic Resistance in paediatric transplant patients)

Reporting period: 2018-09-01 to 2020-08-31

The gut microbiome protects against colonization by Multi-Drug Resistant Organisms (MDROs) and infections, among other things. The overuse of antibiotics could result in disequilibrium of this microbiome by driving the increase in MDROs, possibly leading to their extra-intestinal spread. Since paediatric transplant patients are usually heavily reliant on antibiotics, qMAR’s main objective is to evaluate the usefulness of qPCR for monitoring the intestinal Relative Loads (RLs) of antibiotic resistance genes over time to be used as a biomarker for Personalized Medicine among these patients. qMAR’s objectives are to:

Collect non-invasive samples from paediatric transplant patients
Track and quantify antibiotic resistance genes over time using qPCR
Determine associations between clinical interventions, RLs of resistance genes, and clinical outcome
Disseminate and exploit project results
Develop and implement a career development plan
Rectal swabs routinely obtained from paediatric patients were used to quantify the total amount of bacterial in the gut using qPCR directed towards the 16SrDNA gene that is present in all bacteria. At the same time, qPCRs directed towards the genes of resistance endemic at the La Paz University Hospital (HULP) were performed (blaCTX-M-1-Family and blaOXA-1 genes that break down β-lactams, and blaOXA-48 and blaVIM that are break down carbapenems). The intestinal RLs of these genes were normalized to the total bacterial load using the ∆∆Ct method.
The RLs of blaOXA-48 and blaVIM were higher among Transplant (Tx) patients as compared to Non-Transplant (non-Tx) patients (p<0.05). The highest RLs were obtained from the Liver Tx ward, where large amounts of antibiotics are given to the patients and the most MDRO detected was Klebsiella pneumoniae (68.5%). The hospital’s records were screened in order to determine whether the same organism detected in the rectal swab was isolated from an extra-intestinal site within 5 days of the respective swab. In total, 17 (33.3%) Tx patients and 15 (21.4%) non-Tx patients have had the same resistance gene(s) and organism detected in the rectal swab and in an extra-intestinal sample. Of the extra-intestinal isolates, 68.42% caused infections while 31.58% were colonisations. Extra-intestinal K. pneumoniae isolates were recovered from the hospital’s bacterial collection and clonality analysis showed that in 12 out of 13 cases the same clone was detected intra- and extra-intestinally. The RLs of the rectal swabs for these patients ranged from 1.2% to 6.9% of the total intestinal bacterial population, demonstrating how even a small intestinal disequilibrium can allow MDROs to spread (Enterobacteriaceae form <1% in normal gut microbiomes).
Receiver Operating Characteristic (ROC) analysis was used in order to divide the RLs into low-risk and high-risk groups for extra-intestinal spread of MDROs. Antibiotic consumption data of paediatric Tx patients was also collected and it showed a significant correlation between consumption of non-carbapenem β-lactams in the previous 3 months and the high-risk groups for extra-intestinal spread of MDROs having blaCTX-M-1-Family and blaOXA-1 (p<0.05). Moreover, when plotting antibiotic consumption in relation to the RLs over time for each patient, a clear relation was observed between the maintenance of high intestinal RLs and antibiotic consumption.

Our data demonstrates the usefulness of the tool developed through qMAR that allows for the tracking of the intestinal RLs of resistance genes over time. This can be predictive of extra-intestinal spread of MDROs that can cause infections and/or play a role in transmission. Monitoring the RLs could allow for healthcare workers and infection control personnel to take pre-emptive measures to minimize the risk on the patient and the spread of MDROs in the hospital.
Several dissemination activities were performed during qMAR that include:

Five seminars given to researchers and healthcare professionals at HULP
Three public outreach events
Poster presented at the SEIMC Conference
REA cluster meeting and monitoring mission
Abstract published in the ECCMID 2020 abstract book
Project-special section in the IdiPAZ website
Press release by IdiPAZ
Using #qMAR, @EU_Commission @MSCActions, @IdipazScience, and @Micro_LaPaz on Twitter
Posting qMAR information on Linked-In
qMAR project featured on the Innovation Radar

Also, 3 manuscripts are prepared and currently under review by the co-authors (to be published in open-access journals):

Quantifying the Intestinal Load of Genes of Antibiotic Resistance among two Paediatric Patient Populations: Not all “Positives” are Equal
The effect of Antibiotic Consumption on the Intestinal Loads of Resistance Genes and Extra-Intestinal Dissemination of Multi-Drug Resistant Organisms among Paediatric Liver Transplant Patients
Relative Quantification of the Intestinal Loads of Serratia spp. among Neonates during an Outbreak

Finally, the results have been exploited by several groups within HULP where the data generated from qMAR was used to discuss infection control practices among paediatric liver Tx patients.
The main innovation of qMAR is using pre-existing technology to develop a fast, cheap, accurate, and easy-to implement tool that is able to incorporate any number of resistance genes that are endemic in any hospital. Moreover, the link demonstrated between antibiotic consumption, high RLs, and extra-intestinal spread of MDROs shows how tracking the RLs over time can be a useful biomarker that allows healthcare personnel to react in real-time to minimize adverse effects on the patients. This innovative process was demonstrated in the flexibility of its application to various settings where we used its rationale in tracking an outbreak caused by Serratia marcescens in the hospital’s neonatal unit, and normalizing the Ct values obtained for the SARS-CoV-2 RT-PCR testing during the COVID-19 pandemic.
qMAR’s tool can create a new market for diagnostic kits designed to track the RLs of resistance genes instead of simply detecting their presence or absence. Moreover, it can impact policy makers where they could use it to better assess infection control measures and patient outcome. To further the usefulness and the rapid implementation of qMAR’s tool, the process has been validated, extensively tested, and evaluated with healthcare professionals at HULP in order to make it as market-ready as possible. On a societal level, the tool developed in qMAR can be used to improve upon patient outcomes, and directly affect their own quality of life and that of their families.
qMAR has greatly impacted the researcher’s career where he was able to receive hands-on training in cutting-edge technologies, was awarded a 3-year contract after qMAR, was awarded a JPI-AMR Network 2020 grant as a principal investigator, and collaborated in two national research projects. Finally, the researcher impacted HULP through the various seminars and meetings performed, and the collaborations that he forged.
qMAR project outlook