Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

Meropenem plus Ceftaroline Is Active against Enterococcus faecalis in an In Vitro Pharmacodynamic Model Using Humanized Dosing Simulations.

The standard of care for serious Enterococcus faecalis infections is ampicillin plus ceftriaxone. Ampicillin's inconvenient dosing schedule, drug instability, allergy potential, along with ceftriaxone's high risk for Clostridioides difficile infection and its promotion of vancomycin-resistant enterococci (VRE), led our team to explore alternative options. This work aimed to understand the role of carbapenems in combination with cephalosporins in these infections. We selected two ampicillin and penicillin susceptible E. faecalis strains (AMP-MIC 0.5-2 µg/mL; PCN-MIC 2 µg/mL) and simulated human therapeutic dosing regimens in a 48-h in vitro pharmacodynamic model (IVPD) with ampicillin (2g q4h), ertapenem (1g q24h), meropenem (2g q8h), ceftriaxone (2g q12h), and ceftaroline (600 mg q8h). As expected, ampicillin plus ceftriaxone demonstrated enhanced activity compared with ampicillin monotherapy with no MIC increases in either isolate. Meropenem and ceftaroline demonstrated significant kill against both isolates, with no regrowth or MIC increases occurring. Meropenem plus ceftriaxone also demonstrated significant kill, and while no MIC increases were identified for meropenem, there was minor regrowth and larger standard deviations. Ertapenem combined with either ceftriaxone or ceftaroline enhanced activity at 24 h, but at 48 h, regrowth occurred, and ertapenem MIC increases were noted. Meropenem-based combination therapy against E. faecalis may provide clinicians with another regimen to treat severe E. faecalis infections. Meropenem plus ceftaroline was as active as the standard of care treatment (ampicillin plus ceftriaxone) and may serve as an alternative for serious E. faecalis infections. Further studies are warranted to determine the clinical efficacy.

Ready-to-eat dairy products as a source of multidrug-resistant Enterococcus strains: Phenotypic and genotypic characteristics.

The enterococci are ubiquitous bacteria able to colonize the human and animal gastrointestinal tracts and fresh and fermented food products. Their highly plastic genome allows Enterococcus spp. to gain resistance to multiple antibiotics, making infections with these organisms difficult to treat. Food-borne enterococci could be carriers of antibiotic resistance determinants. The goal of this work was to study the characteristics of Enterococcus spp. in fermented milk products from Poland and their antibiotic resistance gene profiles. A total of 189 strains were isolated from 182 dairy products out of 320 samples tested. The predominant species were Enterococcus faecium (53.4%) and Enterococcus faecalis (34.4%). Isolates were resistant to streptomycin (29.1%), erythromycin (14.3%), tetracycline (11.6%), rifampicin (8.7%), and tigecycline (8.1%). We also detected 2 vancomycin-resistant and 3 linezolid-resistant strains; however, no vanA or vanB genes were identified. A total of 57 high-level aminoglycoside resistance strains (30.2%) were identified, most of which have the ant(6')-Ia gene, followed by the aac(6')-Ie-aph(2″)-Ia and aph(3″)-IIIa genes. Resistance to tetracycline was most often conferred by tetM and tetL genes. Macrolide resistance was most frequently encoded by ermB and ermA genes. Conjugative mobile genetic element (transposon Tn916-Tn1545) was identified in 15.3% of the strains, including 96.3% of strains harboring the tetM gene. This study found that enterococci are widely present in retail ready-to-eat dairy products in Poland. Many isolated strains are antibiotic resistant and carry transferable resistance genes, which represent a potential source of transmission of multidrug-resistant bacteria to humans.

The Enterococcus: a Model of Adaptability to Its Environment.

The genus Enterococcus comprises a ubiquitous group of Gram-positive bacteria that are of great relevance to human health for their role as major causative agents of health care-associated infections. The enterococci are resilient and versatile species able to survive under harsh conditions, making them well adapted to the health care environment. Two species cause the majority of enterococcal infections: Enterococcus faecalis and Enterococcus faecium Both species demonstrate intrinsic resistance to common antibiotics, such as virtually all cephalosporins, aminoglycosides, clindamycin, and trimethoprim-sulfamethoxazole. Additionally, a remarkably plastic genome allows these two species to readily acquire resistance to further antibiotics, such as high-level aminoglycoside resistance, high-level ampicillin resistance, and vancomycin resistance, either through mutation or by horizontal transfer of genetic elements conferring resistance determinants.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

Structural and Regulatory Changes in PBP4 Trigger Decreased ß-Lactam Susceptibility in Enterococcus faecalis.

Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 -35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to ß-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to "suppress" infections in prosthetic joints and offer novel insights into the interaction of ß-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of ß-Lactam and Vancomycin Resistance in Enterococcus faecium.

The transfer of DNA between Enterococcus faecium strains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes.

Genome Sequence of the Multiantibiotic-Resistant Enterococcus faecium Strain C68 and Insights on the pLRM23 Colonization Plasmid.

Enterococcus faecium infections are a rising concern in hospital settings. Vancomycin-resistant enterococci colonize the gastrointestinal tract and replace nonresistant strains, complicating the treatment of debilitated patients. Here, we present a polished genome of the multiantibiotic-resistant strain C68, which was obtained as a clinical isolate and is a useful experimental strain.

Draft Genome Sequence of Vancomycin-Susceptible, Ampicillin-Intermediate Enterococcus faecium Strain D344RRF.

Enterococcus faecium is an important nosocomial pathogen, causing a substantial health burden due to high resistance to antibiotics and its ability to colonize the gastrointestinal tract. Here, we present the draft genome of vancomycin-susceptible, ampicillin-intermediate strain D344RRF, a rifampicin/fusidic acid-resistant and commonly used laboratory strain, which is useful in studying the transfer of antibiotic resistance.

Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium.

UNLABELLED: The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. IMPORTANCE: ß-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to ß-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to ß-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a resistant phenotype. The present work identifies a novel, previously uncharacterized, protein that interacts with Pbp5, whose expression increases in conjunction with stimuli that increase resistance to cephalosporins, and that confers increased resistance to cephalosporins when overexpressed. P5AP may represent a promising new target, inhibition of which could restore cephalosporin susceptibility to E. faecium.

Evolution of early development in dipterans: reverse-engineering the gap gene network in the moth midge Clogmia albipunctata (Psychodidae).

Understanding the developmental and evolutionary dynamics of regulatory networks is essential if we are to explain the non-random distribution of phenotypes among the diversity of organismic forms. Here, we present a comparative analysis of one of the best understood developmental gene regulatory networks today: the gap gene network involved in early patterning of insect embryos. We use gene circuit models, which are fitted to quantitative spatio-temporal gene expression data for the four trunk gap genes hunchback (hb), Krüppel (Kr), giant (gt), and knirps (kni)/knirps-like (knl) in the moth midge Clogmia albipunctata, and compare them to equivalent reverse-engineered circuits from our reference species, the vinegar fly Drosophila melanogaster. In contrast to the single network structure we find for D. melanogaster, our models predict four alternative networks for C. albipunctata. These networks share a core structure, which includes the central regulatory feedback between hb and knl. Other interactions are only partially determined, as they differ between our four network structures. Nevertheless, our models make testable predictions and enable us to gain specific insights into gap gene regulation in C. albipunctata. They suggest a less central role for Kr in C. albipunctata than in D. melanogaster, and show that the mechanisms causing an anterior shift of gap domains over time are largely conserved between the two species, although shift dynamics differ. The set of C. albipunctata gene circuit models presented here will be used as the starting point for data-constrained in silico evolutionary simulations to study patterning transitions in the early development of dipteran species.

Fungal virulence in a lepidopteran model is an emergent property with deterministic features.

UNLABELLED: ABSTRACT Virulence has been proposed to be an emergent property, which by definition implies that it is not reducible to its components, but this application of a philosophical concept to the host-microbe interaction has not been experimentally tested. The goals of our study were to analyze the correlation of the phenotype with the ability to cause disease and to determine the dynamics of an experimental cryptococcal infection in Galleria mellonella and Acanthamoeba castellanii. By studying the outcome of infection as host death, we showed that the dynamics of virulence in the G. mellonella/Cryptococcus neoformans interaction follow a predictable pattern. We also found that the experimental temperature and not the presence of virulence factors was a critical parameter defining the pathogenic potential of cryptococcal species. Our results established that cryptococcal species not considered pathogenic could be pathogens given suitable conditions. Our results support the idea that virulence is an emergent property that cannot be easily predicted by a reductionist approach and yet it behaves as a deterministic system in a lepidopteran cryptococcal infection. These findings provide a road map for evaluating whether host-microbe interactions in other systems are chaotic, deterministic, or stochastic, including those with public health importance. IMPORTANCE: Virulence is a complex phenotype that cannot be easily studied by analyzing its individual components in isolation. By studying the outcome of infection as the death of the host, we found that a given microbial phenotype does not necessarily correlate with its ability to cause disease and that the presence of so-called virulence factors does not predict pathogenicity, consistent with the notion that virulence is an emergent property. This paper reports that the dynamics of virulence in Galleria mellonella larvae infected with the fungus Cryptococcus neoformans follows a predictable pattern. Establishing that virulence is an emergent property is important because it implies that it is not reducible to its components, and consequently, this phenomenon needs to be studied by a holistic approach.

Global warming will bring new fungal diseases for mammals.

Fungi are major pathogens of plants, other fungi, rotifers, insects, and amphibians, but relatively few cause disease in mammals. Fungi became important human pathogens only in the late 20th century, primarily in hosts with impaired immunity as a consequence of medical interventions or HIV infection. The relatively high resistance of mammals has been attributed to a combination of a complex immune system and endothermy. Mammals maintain high body temperatures relative to environmental temperatures, creating a thermally restrictive ambient for the majority of fungi. According to this view, protection given by endothermy requires a temperature gradient between those of mammals and the environment. We hypothesize that global warming will increase the prevalence of fungal diseases in mammals by two mechanisms: (i) increasing the geographic range of currently pathogenic species and (ii) selecting for adaptive thermotolerance for species with significant pathogenic potential but currently not pathogenic by virtue of being restricted by mammalian temperatures.

A systematic analysis of the gap gene system in the moth midge Clogmia albipunctata.

The segmentation gene hierarchy of Drosophila melanogaster represents one of the best understood of the gene networks that generate pattern during embryogenesis. Some components of this network are ancient, while other parts of the network have evolved within the higher Diptera. To further understand the evolution of this gene network, we are studying the role of gap genes in a representative of a basally diverging dipteran lineage, the moth midge Clogmia albipunctata. We have isolated orthologues of all of the Drosophila trunk gap genes from Clogmia, and determined their domains of expression during the blastoderm stage of development, in relation to one another, and in relation to the expression of even-skipped (Calb-eve), a component of the pair-rule system that is directly regulated by the gap genes in Drosophila. We find that hunchback (Calb-hb), Krüppel (Calb-Kr), knirps (Calb-knl), giant (Calb-gt) and tailless (Calb-tll) are all expressed in patterns consistent with a gap segmentation role during blastoderm formation, but huckebein (Calb-hkb) is not. In the anterior half of the embryo, the relative positions of the gap gene expression domains in relation to one another, and in relation to the eve stripes, are rather well conserved. In the posterior half of the embryo, there are significant differences. Posteriorly, Calb-gt is expressed only transiently and very weakly, in a domain that overlaps entirely with that of Calb-knl. At late blastoderm stages, none of the candidate genes we have tested is expressed in the region between the posterior Calb-knl domain and Calb-tll. It is likely that the regulation of Calb-eve expression in this posterior region depends on combinations of gap gene factors that differ from those utilised for the same stripes in Drosophila. Both the gap and the pair-rule patterns of gene expression are dynamic in Clogmia, as they are in Drosophila, shifting anteriorly as blastoderm development proceeds.

Cloning and functional characterization of the Anopheles albimanus DMT1/NRAMP homolog: implications in iron metabolism in mosquitoes.

In addition to its wide role in metabolism, iron in insects has been implicated in vitellogenesis and the immune response. The NRAMP family comprises a well-conserved family of divalent cation transporters in metazoans. To gain insight on the role of NRAMP in Anopheles albimanus, we cloned a cDNA encoding a 571-residue protein (AnaNRAMP) with the structural features defining the NRAMP family. AnaNRAMP mRNA induced (59)Fe(2+) incorporation when injected into Xenopus oocytes. Western blot analysis revealed that AnaNRAMP is expressed in the head, midgut and at high levels in Malpighian tubules of unfed female mosquito. Upon blood feeding, AnaNRAMP levels were reduced in the midgut whereas they increased in the Malpighian tubules. Using immuno-localization by transmission electron microscopy, AnaNRAMP was localized in the membrane of the intra-cellular concretions or spherites of the Malpighian tubule principal cells. Taken together, our results suggest an important role of AnaNRAMP in iron transport and indicate a role of the mosquito Malpighian tubule as an important organ for iron homeostasis.