A long-term survey of Serratia spp. bloodstream infections revealed an increase of antimicrobial resistance involving adult population.

Serratia spp. is a well-recognized pathogen in neonates; however, limited data are available in adults. We studied microbiological and clinical characteristics of Serratia spp. causing bloodstream infections (BSI) in our institution (January 2005-July 2020). Overall, 141 BSI episodes affecting 139 patients were identified and medical records reviewed. Antimicrobial susceptibility was recovered from our informatics system and 118 isolates from 116 patients were available for further microbiological studies. Whole genome sequencing (WGS) was completed in 107 isolates. Incidence of Serratia BSI was 0.3/1000 overall admissions (range 0.12-0.60), with maximum prevalence (27 episodes, 19.1%) during 2017-2018. Relevant patients' clinical characteristics were 71.9% ≥60 years (n = 100), with high comorbidity rates (49%, ≥2), 23 (74.2%) of them died within 1 month of the BSI episode. WGS identified all isolates as Serratia marcescens when Kraken bioinformatics taxonomic tool was used despite some which were identified as Serratia nematodiphila (32/118) or Serratia ureilytica (5/118) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nevertheless, when using MASH distance, Serratia nevei (63/107), S. ureilytica (38/107), and S. marcescens (6/107) were assigned. Carbapenemase (blaVIM-1) and extended-spectrum ß-lactases (ESBL) (blaSHV-12) genes were found in seven and three isolates, respectively, one of them expressing both genes. The worldwide-disseminated IncL/M scaffold plasmid was identified in six VIM producers. Four genotypes were established based on their virulence factors and resistome. Serratia spp. emerged as a relevant nosocomial pathogen causing BSI in elderly patients in our hospital, particularly in recent years with a remarkable increase in antibiotic resistance. ESBL and carbapenemases production related to plasmid dissemination are particularly noteworthy.IMPORTANCESerratia spp. is the third most frequent pathogen involved in outbreaks at neonatal facilities and is primarily associated with bacteremia episodes. In this study, we characterized all causing bloodstream infection (BSI) in patients admitted to our hospital during a 16-year period (2005-2020). Despite having no neonatal intensive care unit in our hospital, this study revealed that Serratia spp. is a relevant pathogen causing BSI in elderly patients with high comorbidity rates. A significant increase of antimicrobial resistance was detected over time, particularly in 2020 and coinciding with the coronavirus disease (COVID-19) pandemic and nosocomial spread of multidrug-resistant Serratia spp. isolates. extended-spectrum ß-lactases and carbapenemases genes associated with plasmid dissemination, typically detected in other Enterobacterales species, were also identified, reinforcing the role of Serratia spp. in the antimicrobial resistance landscape. Additionally, this work highlights the need to reclassify the species of Serratia, since discrepancies were observed in the identification when using different tools.

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks.

BACKGROUND: Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds. METHODS: Widespread Tn1546-carrying plasmids of different families RepA_N (n = 5), Inc18 (n = 4) and/or pHTß (n = 1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison. RESULTS: All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. CONCLUSIONS: The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

Population biology of intestinal enterococcus isolates from hospitalized and nonhospitalized individuals in different age groups.

The diversity of enterococcal populations from fecal samples from hospitalized (n = 133) and nonhospitalized individuals (n = 173) of different age groups (group I, ages 0 to 19 years; group II, ages 20 to 59 years; group III, ages ≥60 years) was analyzed. Enterococci were recovered at similar rates from hospitalized and nonhospitalized persons (77.44% to 79.77%) of all age groups (75.0% to 82.61%). Enterococcus faecalis and Enterococcus faecium were predominant, although seven other Enterococcus species were identified. E. faecalis and E. faecium (including ampicillin-resistant E. faecium) colonization rates in nonhospitalized persons were age independent. For inpatients, E. faecalis colonization rates were age independent, but E. faecium colonization rates (particularly the rates of ampicillin-resistant E. faecium colonization) significantly increased with age. The population structure of E. faecium and E. faecalis was determined by superimposing goeBURST and Bayesian analysis of the population structure (BAPS). Most E. faecium sequence types (STs; 150 isolates belonging to 75 STs) were linked to BAPS groups 1 (22.0%), 2 (31.3%), and 3 (36.7%). A positive association between hospital isolates and BAPS subgroups 2.1a and 3.3a (which included major ampicillin-resistant E. faecium human lineages) and between community-based ampicillin-resistant E. faecium isolates and BAPS subgroups 1.2 and 3.3b was found. Most E. faecalis isolates (130 isolates belonging to 58 STs) were grouped into 3 BAPS groups, BAPS groups 1 (36.9%), 2 (40.0%), and 3 (23.1%), with each one comprising widespread lineages. No positive associations with age or hospitalization were established. The diversity and dynamics of enterococcal populations in the fecal microbiota of healthy humans are largely unexplored, with the available knowledge being fragmented and contradictory. The study offers a novel and comprehensive analysis of enterococcal population landscapes and suggests that E. faecium populations from hospitalized patients and from community-based individuals differ, with a predominance of certain clonal lineages, often in association with elderly individuals, occurring in the hospital setting.

The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs.

Isoprenoids, a diverse group of compounds derived from the five-carbon building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), are essential for survival in all organisms. Animals synthesize their isoprenoids from mevalonic acid (MVA), whereas most pathogenic bacteria and the malaria parasites utilize a completely different pathway for IPP and DMAPP synthesis, the methylerythritol phosphate (MEP) pathway. Plants use both pathways for the synthesis of isoprenoid precursors. The recent elucidation of the MEP pathway has opened the possibility to develop new strategies against microbial pathogens. Novel immunotherapeutic agents can be developed based on the MEP pathway intermediates known to activate the proliferation of human V-delta-9 V-gamma-2 T-cells after infection by many pathogenic bacteria and protozoa. Moreover, the design of specific inhibitors of MEP pathway enzymes (which are highly conserved but show no homology to mammalian proteins) should result in herbicides and drugs with broad-spectrum antimicrobial activity without mechanism-based toxicity to humans. A good example is the cure of bacterial infections and malaria with fosmidomycin, a highly stable inhibitor of the MEP pathway. The use of plants as test systems has led to the identification of additional inhibitors such as ketoclomazone. Biochemical, genetic and crystallographic approaches with the MEP pathway enzymes are now starting to characterize the inhibition kinetics and identify which residues play a structural or catalytic role. Current efforts should eventually contribute to an effective drug designed to fight against microbial pathogens that show resistance to currently available agents.

Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid Synthesis.

The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described transketolase-like enzyme. To identify key residues for DXS activity, we compared the amino acid sequence of Escherichia coli DXS with that of E. coli and yeast transketolase (TK). Alignment showed a previously undetected conserved region containing an invariant histidine residue that has been described to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These findings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity.

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening.

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.

Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain.

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase alpha-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a(2) position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degrees C and 42 degrees C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase beta-subunit.

Up-regulation of genes encoding novel extracellular proteins during fruit set in pea.

The transition from the carpel of the flower to a developing fruit is a poorly characterized process despite its agricultural importance. We have identified two genes, GIC19 and GIC4, which are expressed after induction of pea (Pisum sativum L.) fruit set either by exogenous gibberellins or by pollination. GIC19 expression is temporally and spatially regulated, with transcripts mainly found in growing carpels and young fruit. Similar to GIC19, GIC4 expression is developmentally regulated during carpel and fruit development. However, GIC4 transcripts are found in other growing tissues throughout the plant. Analysis of their sequences and localization of fusion proteins with GFP indicate that both GIC19 and GIC4 are extracellular proteins. While GIC19 is a small proline-rich protein with no overall homology to other reported proteins. GIC4 belongs to a novel family of proteins. Our results reinforce a model of gibberellin mode of action during pea fruit set and development involving enhanced synthesis of extracellular proteins and secretory activity to provide materials and energy for cell growth.

Cutting edge: human gamma delta T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis.

Activation of V gamma 9/V delta 2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C:-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the V gamma 9/V delta 2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate gamma delta T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.

Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli.

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest. However, only the first steps of this pathway have been determined. Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway. The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway. Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

Prenylation of the floral transcription factor APETALA1 modulates its function.

The Arabidopsis MADS box transcription factor APETALA1 (AP1) was identified as a substrate for farnesyltransferase and shown to be farnesylated efficiently both in vitro and in vivo. AP1 regulates the transition from inflorescence shoot to floral meristems and the development of sepals and petals. AP1 fused to green fluorescent protein (GFP) retained transcription factor activity and directed the expected terminal flower phenotype when ectopically expressed in transgenic Arabidopsis. However, ap1mS, a farnesyl cysteine-acceptor mutant of AP1, as well as the GFP-ap1mS fusion protein failed to direct the development of compound terminal flowers but instead induced novel phenotypes when ectopically expressed in Arabidopsis. Similarly, compound terminal flowers did not develop in era1-2 transformants that ectopically expressed AP1. Together, the results demonstrate that AP1 is a target of farnesyltransferase and suggest that farnesylation alters the function and perhaps specificity of the transcription factor.

Functional requirement of plant farnesyltransferase during development in Arabidopsis.

Arabidopsis era1 was identified as an abscisic acid-hypersensitive mutant caused by disruptions or deletions of the gene for the beta subunit (AtFTB) of farnesyltransferase (FTase). The heterodimeric enzyme catalyzes the covalent attachment of the 15-carbon farnesyl diphosphate to the C terminus of regulatory proteins and is essential for growth in yeast. The first disruption of FTB in a multicellular context revealed several developmental and growth regulatory processes that require the function of FTase. The lack of FTase activity in the Arabidopsis era1-2 FTB deletion mutant resulted in enlarged meristems and organs, supernumerary organs in floral whorls, arrested development of axillary meristems, late flowering, and homeotic transformations of flowers. Complementation of era1-2 with LeFTB, the tomato gene for the beta subunit of FTase, restored a normal phenotype and confirmed that the lesion is in AtFTB alone. The effect of this lesion on control of meristem size and on developmental processes suggests the involvement of regulatory proteins that require farnesylation for their function. At least three distinct processes that require the function of FTase were identified: regulation of cellular differentiation in the meristems, meristem maintenance, and regulation of flower development. Together, these results provide a basis for future studies on the involvement of FTase in specific developmental processes and for structure-function analysis of FTase in vivo.

Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase.

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.

Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli.

An alternative mevalonate-independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.

Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein.

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.

The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

Immunolocalization of lipoxygenase in pea (Pisum sativum L.) carpels.

Polyclonal antibodies against a part of pea (Pisum sativum L.) LOXG protein have been raised to study the pattern of distribution of related lipoxygenases in pea carpels. The antiserum recognized three lipoxygenase polypeptides in carpels. One of them became undetectable 24 hours after fruit development induction, suggesting that it may correspond to the protein derived from loxg cDNA. Immunolocalization experiments showed that lipoxygenase protein was present only in pod tissues: it was abundant in the mesocarp and, from the day of anthesis, in the endocarp layers. Lipoxygenase distribution is regulated throughout development. The association of lipoxygenase with cells in which processes of expansion and growth will potentially take place support a role in pod growth and development.