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Wildlife has recently been pinpointed as one of the drivers of dissemination of genes conferring resistances to clinically important antimicrobials. The presence of both extended-spectrum beta-lactamase- (ESBL) and carbapenemase-encoding genes has notably been reported in wild birds, that can act as sentinels of antimicrobial resistance (AMR) contamination but also as long-distance spreaders in case of migratory birds. Here, 424 wild birds brought to a rescue center in France were sampled over a 6-month period. These birds encompassed 62 different sedentary or migratory species. A further 16 wild mammals present in the center were also investigated. No carbapenemase-producer was found, but we identified a surprisingly high proportion (24.1%) of ESBL-positive isolates. A total of 144 non-duplicate isolates were collected, including Escherichia coli (n = 88), Enterobacter cloacae (n = 51), and Citrobacter freundii (n = 5), of which 123 carried the blaCTX–M–9 gene. PFGE, phylogroup, and MLST revealed the presence of a limited number of ESBL-positive clones circulating in these animals, all presenting multiple associated resistances. Next-generation sequencing on a subset of isolates, followed by Southern blot hybridization, showed the wide dissemination of an IncHI2/ST1 plasmid carrying the blaCTX–M–9, blaSHV–12 and mcr-9 genes. In all, our results undoubtedly reflect cross transmissions of ESC-resistance (ESC-R) Enterobacteriaceae within the rescue center – similarly to nosocomial spreads observed at hospital, rather than the true bacterial flora of birds. We also showed that the spread of ESC-R in this rescue center did not only rely on clonal but also on a highly successful plasmidic transmission. Since most animals are intended to get back to nature after a few days or weeks, this is obviously an issue with regard to ESBL dissemination in natural environments.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

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.

Wildlife has recently been pinpointed as one of the drivers of dissemination of genes conferring resistances to clinically important antimicrobials. The presence of both extended-spectrum beta-lactamase- (ESBL) and carbapenemase-encoding genes has notably been reported in wild birds, that can act as sentinels of antimicrobial resistance (AMR) contamination but also as long-distance spreaders in case of migratory birds. Here, 424 wild birds brought to a rescue center in France were sampled over a 6-month period. These birds encompassed 62 different sedentary or migratory species. A further 16 wild mammals present in the center were also investigated. No carbapenemase-producer was found, but we identified a surprisingly high proportion (24.1%) of ESBL-positive isolates. A total of 144 non-duplicate isolates were collected, including Escherichia coli (n = 88), Enterobacter cloacae (n = 51), and Citrobacter freundii (n = 5), of which 123 carried the bla CTX–M–9 gene. PFGE, phylogroup, and MLST revealed the presence of a limited number of ESBL-positive clones circulating in these animals, all presenting multiple associated resistances. Next-generation sequencing on a subset of isolates, followed by Southern blot hybridization, showed the wide dissemination of an IncHI2/ST1 plasmid carrying the bla CTX–M–9, bla SHV–12 and mcr-9 genes. In all, our results undoubtedly reflect cross transmissions of ESC-resistance (ESC-R) Enterobacteriaceae within the rescue center – similarly to nosocomial spreads observed at hospital, rather than the true bacterial flora of birds. We also showed that the spread of ESC-R in this rescue center did not only rely on clonal but also on a highly successful plasmidic transmission. Since most animals are intended to get back to nature after a few days or weeks, this is obviously an issue with regard to ESBL dissemination in natural environments. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacteriaceae (CRE) has been considered a serious global threat, but carbapenem resistance remains relatively uncommon in E. cloacae, especially in China. The aim of this study was to characterize carbapenem-resistant E. cloacae (CR-ECL) isolates from 2012 to 2016 in Southwest China. Our study revealed that 20 (15.2%) of the 132 CR-ECL isolates obtained from patients were identified as NDM-1, with most isolates carrying the IncFIIA plasmids. Notably, we initially observed that the E. cloacae strain co-harbored NDM-1 and IMP-8 carbapenemases simultaneously. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (blaNDM-1-bleMBL-trpF-tat) are associated with the dissemination of NDM-1, while IS26, intI1, and tniC could be involved in the spread of IMP-8. Molecular epidemiology studies showed the nosocomial outbreak caused by NDM-1-producing E. cloacae ST88. Transferring from another hospital and previous carbapenem exposure were identified as independent risk factors for the acquisition of NDM-1-producing E. cloacae. These findings emphasize the need for intensive surveillance and precautions to monitor the further spread of NDM-1 in China.

Carbapenem-resistant Enterobacteriaceae (CRE) has been considered a serious global threat, but carbapenem resistance remains relatively uncommon in E. cloacae, especially in China. The aim of this study was to characterize carbapenem-resistant E. cloacae (CR-ECL) isolates from 2012 to 2016 in Southwest China. Our study revealed that 20 (15.2%) of the 132 CR-ECL isolates obtained from patients were identified as NDM-1, with most isolates carrying the IncFIIA plasmids. Notably, we initially observed that the E. cloacae strain co-harbored NDM-1 and IMP-8 carbapenemases simultaneously. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (blaNDM-1-bleMBL-trpF-tat) are associated with the dissemination of NDM-1, while IS26, intI1, and tniC could be involved in the spread of IMP-8. Molecular epidemiology studies showed the nosocomial outbreak caused by NDM-1-producing E. cloacae ST88. Transferring from another hospital and previous carbapenem exposure were identified as independent risk factors for the acquisition of NDM-1-producing E. cloacae. These findings emphasize the need for intensive surveillance and precautions to monitor the further spread of NDM-1 in China. [ABSTRACT FROM AUTHOR]

Wildlife has recently been pinpointed as one of the drivers of dissemination of genes conferring resistances to clinically important antimicrobials. The presence of both extended-spectrum beta-lactamase- (ESBL) and carbapenemase-encoding genes has notably been reported in wild birds, that can act as sentinels of antimicrobial resistance (AMR) contamination but also as long-distance spreaders in case of migratory birds. Here, 424 wild birds brought to a rescue center in France were sampled over a 6-month period. These birds encompassed 62 different sedentary or migratory species. A further 16 wild mammals present in the center were also investigated. No carbapenemase-producer was found, but we identified a surprisingly high proportion (24.1%) of ESBL-positive isolates. A total of 144 non-duplicate isolates were collected, including Escherichia coli ( n = 88), Enterobacter cloacae ( n = 51), and Citrobacter freundii ( n = 5), of which 123 carried the bla CTX-M- 9 gene. PFGE, phylogroup, and MLST revealed the presence of a limited number of ESBL-positive clones circulating in these animals, all presenting multiple associated resistances. Next-generation sequencing on a subset of isolates, followed by Southern blot hybridization, showed the wide dissemination of an IncHI2/ST1 plasmid carrying the bla CTX-M- 9 , bla SHV- 12 and mcr-9 genes. In all, our results undoubtedly reflect cross transmissions of ESC-resistance (ESC-R) Enterobacteriaceae within the rescue center - similarly to nosocomial spreads observed at hospital, rather than the true bacterial flora of birds. We also showed that the spread of ESC-R in this rescue center did not only rely on clonal but also on a highly successful plasmidic transmission. Since most animals are intended to get back to nature after a few days or weeks, this is obviously an issue with regard to ESBL dissemination in natural environments., (Copyright © 2020 Haenni, Métayer, Jarry, Drapeau, Puech, Madec and Keck.)