Abstract
Species of the Enterobacter cloacae complex (ECC) represent an increasing cause of hospital-acquired infections and commonly exhibit multiple antibiotic resistances. In order to identify genes that may play a role in its ability to colonize the host, we used the transposon-sequencing (Tn-seq) approach. To this end, a high-density random transposon insertion library was obtained from E. cloacae subsp. cloacae ATCC 13047, which was used to analyze the fitness of ca. 300,000 mutants in Galleria mellonella colonization model. Following massively parallel sequencing, we identified 624 genes that seemed essential for the optimal growth and/or the fitness within the host. Moreover, 63 genes where mutations resulted in positive selection were found, while 576 genes potentially involved in the in vivo fitness were observed. These findings pointed out the role of some transcriptional regulators, type VI secretion system, and surface-associated proteins in the in vivo fitness of E. cloacae ATCC 13047. We then selected eight genes based on their high positive or negative fold changes (FCs) and tested the corresponding deletion mutants for their virulence and ability to cope with stresses. Thereby, we showed that ECL_02247 (encoding the NAD-dependent epimerase/dehydratase) and ECL_04444 (coding for a surface antigen-like protein) may correspond to new virulence factors, and that the regulator ECL_00056 was involved in in vivo fitness. In addition, bacterial cells lacking the flagellum-specific ATP synthase FliI (ECL_03223) and the hypothetical protein ECL_01421 were affected for mobility and resistance to H2O2, respectively. All these results yield valuable information regarding genes important for infection process and stress response of E. cloacae ATCC 13047 and participate to a better understanding of the opportunistic traits in this bacterial pathogen.
Highlights
Species of the Enterobacter cloacae complex (ECC) are widely encountered in the environment and are part of the intestinal microbiota of both humans and animals (Sanders and Sanders, 1997)
We constructed a library of ca. 300,000 mutants by Tn insertions in the E. cloacae ATCC 13047 strain, which was used to colonize larvae of G. mellonella in order to identify fitnessassociated genes
Among the 5241 genes annotated in the E. cloacae ATCC 13047 chromosome, 624 (11.9%) were found to lack transposon insertion in at least one of the two conditions tested (Supplementary Tables S3, S4)
Summary
Species of the Enterobacter cloacae complex (ECC) are widely encountered in the environment and are part of the intestinal microbiota of both humans and animals (Sanders and Sanders, 1997). As for other Gram-negative bacteria, cellular elements such as the type III secretion system (T3SS), T4SS, T6SS, fimbriae, and pili seem to be involved in the development of infection or colonization within the host (Zeng et al, 2003; Bingle et al, 2008; Schwarz et al, 2010; Mulder et al, 2012; Liu et al, 2013; Piqué et al, 2015) These virulence genes are often associated with pathogenicity islands that may be acquired by horizontal gene transfer (Dobrindt et al, 2004; Juhas et al, 2009). Clusters belonging to E. cloacae (Cluster 3) and E. hormaechei (Clusters 6 and 8) are the most frequent clusters of ECC isolated from ICU patients and Enterobacter bugandensis (belonging to Cluster 9) appears highly virulent (Dalben et al, 2008; Mezzatesta et al, 2012; Pati et al, 2018)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
