Acinetobacter sp. Ver3 is a polyextremophilic strain characterized by a high tolerance to radiation and pro-oxidants. The Ver3 genome comprises the sodB and sodC genes encoding an iron (AV3SodB) and a copper/zinc superoxide dismutase (AV3SodC), respectively; however, the specific role(s) of these genes has remained elusive. We show that the expression of sodB remained unaltered in different oxidative stress conditions whereas sodC was up-regulated in the presence of blue light. Besides, we studied the changes in the in vitro activity of each SOD enzyme in response to diverse agents and solved the crystal structure of AV3SodB at 1.34 Å, one of the highest resolutions achieved for a SOD. Cell fractionation studies interestingly revealed that AV3SodB is located in the cytosol whereas AV3SodC is also found in the periplasm. Consistently, a bioinformatic analysis of the genomes of 53 Acinetobacter species pointed out the presence of at least one SOD type in each compartment, suggesting that these enzymes are separately required to cope with oxidative stress. Surprisingly, AV3SodC was found in an active state also in outer membrane vesicles, probably exerting a protective role. Overall, our multidisciplinary approach highlights the relevance of SOD enzymes when Acinetobacter spp. are confronted with oxidizing agents.

Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii being the species of greatest worldwide concern due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Colonisation of this environment is associated with a multitude of bacterial factors, and the molecular features that promote environmental persistence in abiotic surfaces, including intrinsic desiccation resistance, biofilm formation and motility, have been previously addressed. On the contrary, mechanisms enabling Acinetobacter spp. survival when faced against other biological competitors are starting to be characterised. Among them, secretion systems (SS) of different types, such as the T5bSS (Contact-dependent inhibition systems) and the T6SS, confer adaptive advantages against bacterial aggressors. Regarding mechanisms of defence against bacteriophages, such as toxin-antitoxin, restriction-modification, Crispr-Cas and CBASS, among others, have been identified but remain poorly characterised. In view of this, we aimed to summarise the present knowledge on defence mechanisms that enable niche establishment in members of the Acinetobacter genus. Different proposals are also described for the use of some components of these systems as molecular tools to treat Acinetobacter infections.

Acinetobacter sp . Ver3 is a polyextremophilic strain characterized by a high tolerance to radiation and pro-oxidants. The Ver3 genome comprises the sodB and sodC genes encoding an iron (AV3SodB) and a copper/zinc superoxide dismutase (AV3SodC), respectively; however, the specific role(s) of these genes has remained elusive. We show that the expression of sodB remained unaltered in different oxidative stress conditions whereas sodC was up-regulated in the presence of blue light. Besides, we studied the changes in the in vitro activity of each SOD enzyme in response to diverse agents and solved the crystal structure of AV3SodB at 1.34 Å, one of the highest resolutions achieved for a SOD. Cell fractionation studies interestingly revealed that AV3SodB is located in the cytosol whereas AV3SodC is also found in the periplasm. Consistently, a bioinformatic analysis of the genomes of 53 Acinetobacter species pointed out the presence of at least one SOD type in each compartment, suggesting that these enzymes are separately required to cope with oxidative stress. Surprisingly, AV3SodC was found in an active state also in outer membrane vesicles, probably exerting a protective role. Overall, our multidisciplinary approach highlights the relevance of SOD enzymes when Acinetobacter spp. are confronted with oxidizing agents. ### Competing Interest Statement The authors have declared no competing interest.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Acinetobacter baumannii represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we con-ducted whole-genome sequencing analysis of the Acinetobacter sp. NCIMB8209 col-lection strain, isolated in 1943 from the aerobic degradation (retting) of desert gu-ayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to A. baumannii, a result supported by the identification of a chromosomal blaOXA-51-like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary A. baumannii clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. More -over, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward Galleria mellonella and Caenorhabditis elegans. Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary A. baumannii clinical strains represents an intrinsic characteristic of the species. IMPORTANCE Acinetobacter baumannii is an ESKAPE (Enterococcus faecium, Staphylo-coccus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aerugi-nosa, and Enterobacter species) opportunistic pathogen, with poorly defined natural habitats/reservoirs outside the clinical setting. A. baumannii arose from the Acineto-bacter calcoaceticus-A. baumannii complex as the result of a population bottleneck, followed by a recent population expansion from a few clinically relevant clones en-dowed with an arsenal of resistance and virulence genes. Still, the identification of virulence traits and the evolutionary paths leading to a pathogenic lifestyle has re-mained elusive, and thus, the study of nonclinical ("environmental") A. baumannii isolates is necessary. We conducted here comparative genomic and virulence studies on A. baumannii NCMBI8209 isolated in 1943 from the microbiota responsible for the decomposition of guayule, and therefore well differentiated both temporally and epidemiologically from the multidrug-resistant strains that are predominant nowa-days. Our work provides insights on the adaptive strategies used by A. baumannii to escape from host defenses and may help the adoption of measures aimed to limit its further dissemination.

The polyextremophilic strain Acinetobacter sp. Ver3 isolated from high‐altitude Andean lakes exhibits elevated tolerance to UV‐B radiation and to pro‐oxidants, a feature that has been correlated to its unusually high catalase activity. The Ver3 genome sequence analysis revealed the presence of two genes coding for monofunctional catalases: AV3KatE1 and AV3KatE2, the latter harboring an N‐terminal signal peptide. We show herein that AV3KatE1 displays one of the highest catalytic activities reported so far and is constitutively expressed at relatively high amounts in the cytosol, acting as the main protecting catalase against H2O2 and UV‐B radiation. The second catalase, AV3KatE2, is a periplasmic enzyme strongly induced by both peroxide and UV, conferring supplementary protection against pro‐oxidants. The N‐terminal signal present in AV3KatE2 was required not only for transport to the periplasm via the twin‐arginine translocation pathway, but also for proper folding and subsequent catalytic activity. The analysis of catalase distribution among 114 Acinetobacter complete genomes revealed a great variability in the catalase classes, with A. baumannii clinical isolates exhibiting higher numbers of isoenzymes and the most variable profiles.

Several Acinetobacter spp. act as opportunistic pathogens causing healthcare-associated infections worldwide, and in this respect their ability to resist antimicrobial compounds has certainly boosted up their global propagation. Acinetobacter clinical strains have demonstrated a remarkable ability to evolve and become resistant to almost all available drugs in the antimicrobial arsenal, including the last-resort carbapenem β-lactams. The dissemination of antimicrobial resistant genes (ARG), heavy metals-detoxification systems and other traits such as virulence factors is facilitated by mobile genetic elements (MGE) through horizontal gene transfer. Among them, plasmids have been shown to play a critical role in this genus. Despite the continuous increase of Acinetobacter plasmid sequences present in databases, there are no reports describing the basic traits carried by these MGE. To fill this gap, a broad analysis of the Acinetobacter plasmidome was performed. A search for Acinetobacter complete plasmids indicated that 905 sequences have been deposited in the NCBI-GenBank public database, of which 492 are harbored by Acinetobacter baumannii strains. Plasmid-classification schemes based on Rep proteins homology have so far described 23 different groups for A. baumannii (GR1-23), and 16 Acinetobacter Rep3 Groups (AR3G1-16) for the complete genus. Acinetobacter plasmids size ranges from 1.3 to 400 kb. Interestingly, widespread plasmids which are < 20 kb make up 56% of the total present in members of this genus. This led to the proposal of Acinetobacter plasmid assignation to two groups according to their size (< 20 kb and > 20 kb). Usually, smaller plasmids are not self-transmissible, and thereby employ alternative mechanisms of dissemination. For instance, a subgroup of < 20 kb-plasmids belonging to the pRAY-family, lack a rep gene, but encode a relaxase enabling their mobilization by conjugative plasmids. Other subgroup, including small GR2 Acinetobacter plasmids, does not encode a relaxase gene. However, they could still be mobilized by conjugative plasmids which recognize an oriT region carried by these small plasmids. Also, these < 20 kb-plasmids usually carry accessory genes bordered by XerC/D-recombinases recognition sites which have been hypothesized to mediate plasmid plasticity. Conversely, many cases of larger plasmids are self-transmissible and might encode virulence factors and their regulators, thus controlling strain pathogenicity. The ARGs carried by the > 20 kb-plasmids are usually encoded within other MGEs such as transposons, or as part of integrons. It has been recently noted that some of the > 20 kb-plasmids are derived from excised phages, and thus dubbed as phage-like plasmids. All in all, the plethora of plasmids found in strains of this genus and the multiple strategies promoting their evolution and dissemination have certainly contributed to survival of the Acinetobacter members in different habitats, including the clinical environment.

CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.

Acinetobacter baumannii (Aba) is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among Aba strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR Aba strains from Argentinean hospitals, assigned to the CC104O/CC15P clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource β-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a bla OXA-58 gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn10 element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the Galleria mellonella infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104O/CC15P strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of A. baumannii to the clinical setting.

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two radically different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOB P111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOB Q1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii as the species of greatest concern worldwide due to its multi-drug resistance and recent appearance of hyper-virulent strains in the clinical setting. Acinetobacter colonization of the environment and the host is associated with a multitude of factors which remain poorly characterized. Among them, the secretion systems (SS) encoded by Acinetobacter species confer adaptive advantages depending on the niche occupied. Different SS have been characterized in this group of microorganisms, including T6SS used by several Acinetobacter species to outcompete other bacteria and in some A. baumannii strains for Galleria mellonella colonization. Therefore, to better understand the distribution of the T6SS in this genus we carried out an in-depth comparative genomic analysis of the T6SS in 191 sequenced strains. To this end, we analyzed the gene content, sequence similarity, synteny and operon structure of each T6SS loci. The presence of a single conserved T6SS-main cluster (T6SS-1), with two different genetic organizations, was detected in the genomes of several ecologically diverse species. Furthermore, a second main cluster (T6SS-2) was detected in a subgroup of 3 species of environmental origin. Detailed analysis also showed an impressive genetic versatility in T6SS-associated islands, carrying VgrG, PAAR and putative toxin-encoding genes. This in silico study represents the first detailed intra-species comparative analysis of T6SS-associated genes in the Acinetobacter genus, that should contribute to the future experimental characterization of T6SS proteins and effectors.

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.

Acinetobacter baumannii represents nowadays an important nosocomial opportunistic pathogen whose reservoirs outside the clinical setting are obscure. Here, we traced the origins of the collection strain A. baumannii DSM30011 to an isolate first reported in 1944, obtained from the enriched microbiota responsible of the aerobic decomposition of the resinous desert shrub guayule. Whole-genome sequencing and phylogenetic analysis based on core genes confirmed DSM30011 affiliation to A. baumannii. Comparative studies with 32 complete A. baumannii genomes revealed the presence of 12 unique accessory chromosomal regions in DSM30011 including five encompassing phage-related genes, five containing toxin genes of the type-6 secretion system, and one with an atypical CRISPRs/cas cluster. No antimicrobial resistance islands were identified in DSM30011 agreeing with a general antimicrobial susceptibility phenotype including folate synthesis inhibitors. The marginal ampicillin resistance of DSM30011 most likely derived from chromosomal ADC-type ampC and blaOXA-51-type genes. Searching for catabolic pathways genes revealed several clusters involved in the degradation of plant defenses including woody tissues and a previously unreported atu locus responsible of aliphatic terpenes degradation, thus suggesting that resinous plants may provide an effective niche for this organism. DSM30011 also harbored most genes and regulatory mechanisms linked to persistence and virulence in pathogenic Acinetobacter species. This strain thus revealed important clues into the genomic diversity, virulence potential, and niche ranges of the preantibiotic era A. baumannii population, and may provide an useful tool for our understanding of the processes that led to the recent evolution of this species toward an opportunistic pathogen of humans.

This article refers to:Microbiological features and clinical impact of the type VI secretion system (T6SS) in Acinetobacter baumannii isolates causing bacteremia

ABSTRACT Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6′-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different transporters is used: the maltose-PTS transporter MalT, or the MdxEFG-MsmX ABC transporter. MdxEFG-MsmX takes up longer maltodextrins as well as complex molecules, such as panose and isopanose.

Acinetobacter baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections. Its success in the hospital environment obeys to multiple causes, among them, the ability to resist antimicrobial compounds. It can rapidly evolve Multidrug Resistance (MDR) when confronted with antibiotic therapy and in particular, the emerging resistance to last-resort carbapenems represents a major concern worldwide. The most frequent cause of carbapenem resistance in A. baumannii is represented nowadays by the acquired Carbapenem-Hydrolyzing Class D β-Lactamases (CHDL) of the OXA-23, OXA-40 and OXA-58 groups, with the respective blaOXA genes generally embedded in distinct genetic structures carried by plasmids.

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain.

Background: Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens.Results: An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an "enterococcal gene core" of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens.Conclusion: Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.