Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

A mutation in the glycosyltransferase gene lafB causes daptomycin hypersusceptibility in Enterococcus faecium.

OBJECTIVES: To verify dissemination of daptomycin-non-susceptible Enterococcus faecium in a hospital where daptomycin was not in use and to understand the evolutionary pathways connecting daptomycin hypersusceptibility to non-susceptibility. METHODS: Clonality of 26 E. faecium was assessed by PFGE and the STs of these isolates were determined. The most daptomycin-susceptible isolate was evolved in vitro by stepwise daptomycin selection, generating isolates for genome comparisons. RESULTS: The spread of a high-risk daptomycin-non-susceptible VRE clone was detected, as was the occurrence of an unusual daptomycin-hypersusceptible strain (HBSJRP18). To determine the basis for daptomycin hypersusceptibility, we evolved HBSJRP18 in vitro and identified candidate genetic alterations potentially related to daptomycin susceptibility. Both lafB, encoding glycosyltransferase, which is putatively involved in lipoteichoic acid (LTA) biosynthesis, and dak, encoding a dihydroxyacetone kinase likely involved in fatty acid metabolism, were mutated in multiple independent experiments. Trans-complementation showed that the lafB polymorphism naturally occurring in HBSJRP18 caused its daptomycin hypersusceptibility. Fourier-transform infrared spectroscopy identified differences between the extracted LTA spectra from the hypersusceptible isolate and its revertant, as well as other non-susceptible variants, supporting a role for LafB in E. faecium LTA biosynthesis. Zeta potential difference was detected in one evolved dak mutant derivative. While much more susceptible to daptomycin, HBSJRP18 showed enhanced growth in the presence of piperacillin, suggesting that this, or another cell wall-targeting antibiotic, may have selected for the daptomycin-hypersusceptible phenotype. CONCLUSIONS: Our findings provide new information on the basis for daptomycin susceptibility in E. faecium, with implications for limiting the development and spread of daptomycin resistance.

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)2 and Its Serum Degradation Product against Multidrug-Resistant Bacteria.

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)2, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller-Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)2 were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys12/Lys13-(p-BthTX-I)2), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)2, and des-Lys12/Lys13-(p-BthTX-I)2 were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys12/Lys13-(p-BthTX-I)2 and (p-BthTX-I)2 degraded Staphylococcus epidermidis biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic S. epidermidis in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)2 are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.

Synthesis, Characterization, and Study of the Antimicrobial Potential of Dimeric Peptides Derived from the C-Terminal Region of Lys49 Phospholipase A2 Homologs.

Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys49 PLA2-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)2K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)2K can be applied to other molecules originating from the C-terminal region of PLA2-like Lys49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)2K and (p-ACL)2K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)2K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)2K peptide were shown to apply to other peptides derived from Lys49 PLA2-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.

Global diversity of enterococci and description of 18 novel species.

Enterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species, Enterococcus faecalis and E. faecium uniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool of Enterococcus-adapted genes from which known facile gene exchangers such as E. faecalis and E. faecium may draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition. E. faecalis and E. faecium were isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus, potential threats to human health, and new insights into its evolution.

Clonal relationships determined by multilocus sequence typing among enteropathogenic Escherichia coli isolated in Brazil.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

The role of the vacB gene in the pathogenesis of Brucella abortus.

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

High-Quality Draft Genome Sequence of the Multidrug-Resistant Clinical Isolate Enterococcus faecium VRE16.

Specific lineages of the commensal bacterium Enterococcus faecium belonging to CC17, especially ST412, have been isolated from patients in several hospitals worldwide and harbor antibiotic resistance genes and virulence factors. Here, we report a high-quality draft genome sequence and highlight features of E. faecium VRE16, a representative of this ST.

Clonal complexes of Staphylococcus aureus: all mixed and together.

In this manuscript, we show that the most important clonal complexes of Staphylococcus aureus, CC1, CC5, CC8, CC15 and CC97, are now all connected by eburst when run on the Multi-locus sequence typing (mlst) database. The seven loci suggested for the mlst scheme of S. aureus are not enough to separate the most important clones such as New York/Japan and Brazilian Epidemic Clone (BEC). They now all belong to the same clonal complex and this may be the time to think about a new way to discriminate them.

Draft Genome Sequence of Methicillin-Resistant Staphylococcus aureus Strain SA16, Representative of an Endemic Clone from a Brazilian Hospital.

Here we report the draft genome sequence of a bloodstream isolate of methicillin-resistant Staphylococcus aureus strain SA16. Strain SA16 is a sequence type 5 (ST5)-staphylococcal cassette chromosome mec type II (SCCmec II) clone and was the most prevalent isolate at a Brazilian hospital during the second half of 2009.

Multilocus sequence typing of uropathogenic ESBL-producing Escherichia coli isolated in a Brazilian community.

In the present study, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction detection of three resistance genes were combined to characterize seven uropathogenic E. coli isolated from outpatients. Selected portions of seven housekeeping and three antibiotic-resistance genes of the isolates were sequenced. The seven isolates were classified into four different sequence types (STs) by MLST and five PGFE types. Three isolates had a novel allelic profile representing a new ST designated as ST528 and showed the same PFGE and resistance genes. Two isolates, both characterized as ST359, were differentiated by PFGE and shared only one of the antibiotic-resistance genes studied. Comparison of MLST results with those of PFGE and resistance genes demonstrated that Escherichia coli had acquired different antibiotic-resistance genes and DNA rearrangements, causing alterations in PFGE patterns but maintaining the same ST. Furthermore, this article also reports the first detection of a CTX-M-2 ESBL E. coli and SHV-5 in a Brazilian community.

Sequence analysis of Enterococcus faecium strain 10/96A (VanD4), the original vancomycin-resistant E. faecium strain in Brazil.

Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil.

Occurrence of insertion sequences within the genomes and Tn1546-like elements of glycopeptide-resistant enterococci isolated in Brazil, and identification of a novel element, ISEfa5.

Insertion sequences (IS) occur widely within the Tn1546-like elements responsible for VanA glycopeptide resistance in enterococci from several countries. As such insertions can be used as epidemiological markers and for studying horizontal transfer of gene clusters, we investigated the distribution of IS6770, IS1542, IS1216V, IS1476, and IS1251 elements in 26 VanA Enterococcus faecium and 21 VanA Enterococcus faecalis from Brazil. PCR, using genomic DNA as a template, indicated that most of the isolates contained IS6770 (97%), IS1216V (87%) and IS1476 (72%) elements. IS1251 was also detected, but at a higher frequency in E. faecium (80%) than in E. faecalis (14%). None of the isolates harboured IS1542. Only two of 47 isolates had IS elements within their Tn1546-like elements; one possessed IS1251 between vanS and vanH, as reported in the United States; another possessed a novel IS element, designated ISEfa5, located between vanX and van Y. This novel element was found in the genomic DNA of 25 (96%) E. faecium and II (52%) E. faecalis. In stability studies, no IS-mediated changes were detected in the Tn1546-like elements of 25 vancomycin-resistant enterococci (VRE) monitored over 11 months. These results suggest that the occurrence of IS in Brazilian isolates is similar to that reported in American isolates, but that these elements occur rarely within the vanA gene clusters. As patterns of IS carriage did not correlate with the PFGE type of the VRE, the prevalence of IS elements in genomic DNA of VRE is not a useful epidemiological marker. However, the presence of IS-modified Tn1546-like elements, which appear to be rare in Brazil, could be a useful molecular marker in local epidemiological studies to monitor the evolution and horizontal transmission of VanA elements.