The wide, often improper, use of antibiotics over the past few decades has led to the onset of bacterial resistance to antibiotics, which is becoming a global emergency (Tran et al., 2019). Infections caused by multidrug-resistant (MDR) bacteria have a relevant epidemiological value as both nosocomial and community-acquired infections, but also a clinical significance due to the morbidity and mortality that are highly associated with MDR (Colomb-Cotinat et al., 2016).

In solid organ transplantation (SOT), the incidence of bacteraemia due to Gram-negative bacteria, particularly enteric bacteria such as Klebsiella pneumoniae (KP), has significantly increased over time (Singh et al., 2004; Macesic et al., 2018). In endemic areas, the incidence of 1%-18% of carbapenem-resistant- (CR-) KP infections in SOT recipients is similar when comparing liver, kidney, lung, and heart transplants, with mortality reaching 60% (Pouch and Satlin, 2017). A multicentre study on transplanted adult patients who were carrying a CR-Enterobacteriaceae infection (CR-EI), showed a survival rate of 72%, with a median interval of 50 days since CREI to exitus one year after liver transplant (LT) (Huprikar et al., 2016). In comparison, the survival rate of the overall LT patients during the same time was higher than 88% (Adam et al., 2018; Kwong et al., 2020). The majority of these studies focused on adult LT recipients (Hand and Patel, 2016; Ferrarese et al., 2018) while in a paediatric population they are starting to appear (Del Chierico et al., 2018; Sun et al., 2021).

In paediatric age patients, biliary atresia (BA) represents the main indication for LT (Adam et al., 2018; Kwong et al., 2020). Most patients with BA undergo Kasai’s portoenterostomy after diagnosis, which improves survival, although it is burdened by complications such as ascending cholangitis (Cazares et al., 2017). Patients with BA are characterized by specific gut microbiota, likely due to an alteration of the bile flow with an inverse microbial richness proportional to the degree of cholestasis and a reduced abundance of Enterobacter (Tessier et al., 2020). Baek et al. showed that the most prevalent pathogens implicated in cholangitis-related infections are Enterobacteriaceae family members as Escherichia coli, Enterobacter cloacae, and KP (Baek et al., 2020). The gut microbiota represents a large reservoir of MDR bacteria (Gargiullo et al., 2019). In this environment, MDR bacteria can transfer their resistance genes to commensals increasing the pathogenic potential of the microbiota (Baron et al., 2018). This is particularly relevant for frail patients, especially when antibiotic therapies may select MDR bacteria already present in their gut microbiota (Rice, 2008; Biliński et al., 2016). Nevertheless, an eubiotic gut microbiota guarantees protection from gut colonization by MDR microbes through colonization resistance mechanisms (Ubeda et al., 2013), by competing bacteria for space and trophic resources, or by generation of bactericidal factors (Kim et al., 2017). MDR opportunistic pathogens may translocate across the intestinal barrier or contaminate skin and other body sites through catheter or intravenous lines (Lübbert et al., 2014), causing infections and even sepsis (van Schaik, 2015). Moreover, SOT recipients colonized by MDR bacteria may serve as important reservoirs of nosocomial transmission across hospital settings (Pereira and Uhlemann, 2016). Therefore, the monitoring of commensal microbiota alteration during antibiotic (ATB) treatment can assist in preventing and in treating adverse events occurring during antibiotic therapies, especially in SOT recipients.

In this context, this pilot study aimed to estimate the impact of MDR colonization on a small cohort of paediatric LT patients. Clinical evaluation, MDR bacteria monitoring, and gut microbiota profiling were evaluated to design optimal prevention and therapy strategies for these patients.

The MDR screening program carried out at our Centre allowed us to perform early identification of MDR bacteria for either infected or colonized patients, thus avoiding the infection spreading. Moreover, this approach allowed for the implementation of “ad hoc” intervention strategies, especially for patients coming from European countries without any previous microbiological information, which represent most of the patients (71.4%). Furthermore, through this screening, we were able to distinguish MDR microbe “carrier” patients before transplant from those who contracted the infection from the graft and/or in the period following the transplant. This information is useful especially in split liver and multi-organ transplants from a single donor for which the early intercommunication amongst recipient centers about the possible status of the organ’s carrier may be helpful for patient management following transplant (Mularoni et al., 2015; Errico et al., 2019). Awareness of the high vulnerability of these patients, especially in the early stages, leads to the wide use of antibiotics, but in MDR-colonized patients, it is not yet well established whether it is necessary to empirically resort to targeted antibiotics or use standard prophylactic therapy.

In 2018, the group for the ‘Study of Infection in Transplantation of the Spanish Society of Infectious Diseases and Clinical Microbiology and Spanish Network for Research in Infectious Diseases’ did not recommend a surgical prophylaxis regimen different from the local standard for adult recipients colonized with CP-Enterobacteriaceae before LT (Aguado et al., 2018). Moreover, previous studies have been conducted only for MDR infection in LT during outbreaks, over limited time periods, or with a retrospective modality (Birgand et al., 2013; Feldman et al., 2013; Lübbert et al., 2014; Sun et al., 2021).

The prophylaxis of paediatric patients affected by MDR bacteria with targeted antibiotics still represents an open avenue and it is important to increase consciousness of the risks of over-treatment. For this reason, different ATB prophylactic regimens in homogeneous groups of MDR bacteria affected paediatric patients need to be assessed. Our multi-disciplinary approach allowed us to evaluate the reduction of antibiotic mismanagement during the transplant waiting list. Moreover, we also evaluated the impact of post-operative complications without affecting the rates of morbidity and mortality (up to 12 months) of our patients’ compared to non-MDR patients, especially in the first phases after transplant, where intense immunosuppression is required. Moreover, in our cohort, post-transplant complications including reoperation or surgery complications, rejection incidence, and infections, were similar in both groups, both in the immediate post-transplant and in the medium and long follow-up period. This evidence suggests that MDR microbe ‘targeted ATB’ prophylaxis associated with LT prevents neither the onset of complications related to the transplantation itself, nor the risk of bacterial translocation from the gastrointestinal tract, nor the post-LT hospitalization length. In the case of sepsis, we used a combination of antibiotics rather than a monotherapy with a good outcome, as recommended by scientific literature (Bassetti et al., 2018), suggesting that targeted-ATB therapy represents a valid choice in the case of effective treatment and not for prophylaxis. Of course, the use of other therapies (antibiotic, antiviral, and antifungal) in the post-transplant period may potentially affect the outcome but, since the complications are similar in the two groups, we consider additional therapies as part of the normal management of transplant patients.

In our previous study, performed in a single case study, we proposed microbiota profiling in the clinical management of these transplant patients. Our evidence opened a new point of view that may guide clinicians on the choice of the appropriate antibiotic strategies and monitor the MDR colonization and microbiota eubiotic reversion after MDR colonization and ATB treatment insults (Del Chierico et al., 2018). The identification of high-risk patients by microbial signatures may allow for tailored interventions and an improvement in LT outcomes (Annavajhala et al., 2019).

Our results demonstrate a faster increase in microbiota richness in the medium term after standard ATB therapy, suggesting a more rapid restoration of gut microbiota eubiosis compared to the ATB targeted therapy. Moreover, the standard treatment produced a positive effect in the increment of the beneficial Actinobacteria and Bacteroidetes and the reduction of potentially harmful Proteobacteria compared to the ATB targeted treatment, even in the medium- and long-term. In particular, the microbiota of patients treated by standard ATB therapy was enriched in beneficial microorganisms like Lactobacillus spp., Bulleidia, and Dialister, which are natural producers of antimicrobials and short chain fatty acids (SCFAs) (Umu et al., 2017). On the contrary, the microbiota of the patients treated by targeted therapy was enriched in the highly immunogenic Erysipelotrichaceae family (Kaakoush, 2015), in the inflammatory polysaccharide producer, such as Ruminococcus gnavus, and in the opportunistic pathogen as Klebsiella spp. (Martin and Bachman, 2018). Furthermore, both treatments reduced the abundance of Enterobacteriaceae at the end of the follow up, but the standard therapy seemed to anticipate this effect at 6 months, while the targeted one was affected only at 12 months. In addition, in standard ATB prophylaxis Klebsiella spp. was completely eradicated, while in targeted ATB prophylaxis a slight and progressive reduction of it was observed, even maintaining positivity until 12 months.

Despite the small number of patients – due to the fact that hepatic transplantation in paediatric age patients is less common than in adults and the presence of MDR bacteria in these patients, which, fortunately, does not cover a wide range of cases – this study offers an innovative discussion of the management of paediatric MDR patient candidates to LT, by addressing multiple aspects of this problem. To the best of our knowledge and despite the limitations outlined below, this is the first study analyzing MDR bacteria colonization and microbiota profiling in a paediatric population undergoing LT, comparing two antibiotic prophylactic regimens (standard vs targeted) in the management of these patients. In our case series, colonization with MDR bacteria did not represent a contraindication for LT; the presence of MDR bacteria at the time of transplant did not compromise the prognosis and almost all patients had progressive decontamination, mainly after being discharged. Early MDR microbe identification and active surveillance may allow clinicians to establish a specific strategy of prevention and targeted treatment in case of complications, avoiding broad-spectrum antibiotic overuse. The routine use of a ‘targeted ATB’ therapy during transplantation does not seem to bring benefits either from a clinical or microbiological point of view.

These gut microbiota profiling results are noteworthy in the description of the impact of each therapy on this complex ecosystem, especially in the presence of CR-KP colonization. In particular, standard therapy was effective in early decontamination of CR-KP, restoring gut microbiota eubiosis, beneficial bacteria growth, and the spontaneous reduction of Klebsiella spp.

Based upon the results of this study, we suggest that for MDR microbe colonized patients it would be appropriate to use a standard protocol of prophylaxis for LT management, reserving the use of targeted antibiotics only in case of serious complications.

The dataset presented in this study can be found online: https://www.ncbi.nlm.nih.gov/PRJNA741471.

The protocol was approved by the Bambino Gesù Children’s Hospital Ethics Committee (Protocol Number 1538). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Conceptualization, GT. and LP. Methodology, SR and FC. Formal analysis, FC. Investigation, SC, FC, SR, MC, PB, AP, and MS. Resources, LP. Data curation, SC and FC. Writing-original draft preparation, SC and FC. Writing—review and editing, GT, LP, MS, SC, and FC. All authors contributed to the article and approved the submitted version.

This work was supported by the Ministry of Health (Ricerca Corrente 201802G004314 assigned to LP).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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