dMultidrug-resistant carbapenemase-producing Klebsiella pneumoniae (KpC) strains are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe the antimicrobial and antibiofilm activities of synthetic peptides DJK-5, DJK-6, and 1018 against five KpC isolates. Using static microplate assays, it was observed that the concentration required to prevent biofilm formation by these clinical isolates was below the MIC for planktonic cells. More-sophisticated flow cell experiments confirmed the antibiofilm activity of the peptides against 2-day-old biofilms of different KpC isolates, and in some cases, the peptides induced significant biofilm cell death. Clinically relevant combinations of DJK-6 and ␤-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KpC strain1825971. Interestingly, peptide DJK-6 was able to enhance, at least 16-fold, the ability of meropenem to eradicate preformed biofilms formed by this strain. Using peptide DJK-6 to potentiate the activity of ␤-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates. Klebsiella pneumoniae is a Gram-negative, encapsulated, opportunistic pathogen of the family Enterobacteriaceae (1). It has become a significant cause of infections, especially pneumonia, but also meningitis, sepsis, and urinary tract, device-associated, and surgical wound site infections (2-7). The most severe clinical cases of such infections are often associated with K. pneumoniae strains that produce a plasmid-borne carbapenemase (KpC).The rate of fatality from KpC-producing K. pneumoniae is high (Ͼ50%) among patients with bloodstream infection (2,8,9).The treatment of these infections is difficult since KpC strains are resistant to all ␤-lactam antibiotics, including carbapenems, which are typically used as a last resort (10). In addition, bacteria harboring carbapenemase genes are often resistant to other antibiotics. As a consequence of this multidrug resistance, failure in treatment contributes to increased rates of morbidity and mortality (9,11,12). Tigecycline, aminoglycosides, and the polymyxins (polymyxin B and colistin) are currently the best treatment options for carbapenemase-producing strains (13). However, these strains also have the potential to develop resistance to these antibiotics (14-16).The treatment of infections caused by multidrug-resistant bacteria is a challenge, and the ability of bacterial pathogens to form biofilms can further complicate this scenario. Biofilms, a distinct microbial lifestyle of bacteria, are surface-associated multicellular communities of bacteria encased in a protective polymeric matrix that can include polysaccharides, proteins, and DNA (17). They represent a substantial problem in the clinic, as they can lead to the development of chronic long-lived infections on body surfaces, including the lung, skin, hea...

Virulence genes from different E. coli pathotypes are blended in hybrid strains. E. coli strains with hybrid enteroaggregative/uropathogenic (EAEC/UPEC) genotypes have sporadically emerged causing outbreaks of extraintestinal infections, however their association with routine infections is yet underappreciated. We assessed 258 isolates of E. coli recovered from 86 consecutive cases of extraintestinal infections seeking EAEC and hybrid genotype (EAEC/UPEC) strains. Extensive virulence genotyping was carried out to detect 21 virulence genes, including molecular predictors of EAEC and UPEC strains. Phylogenetic groups and sequence types (STs) were identified, as well as it was performed phylogenetic analyses in order to evaluate whether hybrid EAEC/UPEC strains belonged to intestinal or extraintestinal lineages of E. coli. Adhesion assays were performed to evaluate the biofilm formation by hybrid strains in human urine and cell culture medium (DMEM). Molecular predictors of UPEC were detected in more than 70% of the strains (chuA in 85% and fyuA in 78%). Otherwise, molecular predictors of EAEC (aatA and aggR) were detected in only 3.4% (9/258) of the strains and always along with the UPEC predictor fyuA. Additionally, the pyelonephritis-associated pilus (pap) gene was also detected in all of the hybrid EAEC/UPEC strains. EAEC/UPEC strains were recovered from two cases of community-onset urinary tract infections (UTI) and from a case of bacteremia. Analyses revealed that hybrid EAEC/UPEC strains were phylogenetically positioned in two different clades. Two representative strains, each recovered from UTI and bacteremia, were positioned into a characteristic UPEC clade marked by strains belonging to phylogenetic group D and ST3 (Warwick ST 69). Another hybrid EAEC/UPEC strain was classified as phylogroup A-ST478 and positioned in a commensal clade. Hybrid EAEC/UPEC strains formed biofilms at modest, but perceptible levels either in DMEM or in urine samples. We showed that different lineages of E. coli, at least phylogenetic group A and D, can acquire and gather EAEC and UPEC virulence genes promoting the emergence of hybrid EAEC/UPEC strains.

Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. The peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro-inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. The peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.

kIn Enterobacteriaceae, the bla NDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the bla NDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFII K and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the bla NDM-1 gene may be transmitted by Tn3000 in different parts of the world.

No abstract

Peptide rational design was used here to guide the creation of two novel short β-lactamase inhibitors, here named dBLIP-1 and -2, with length of five amino acid residues. Molecular modeling associated with peptide synthesis improved bactericidal efficacy in addition to amoxicillin, ampicillin and cefotaxime. Docked structures were consistent with calorimetric analyses against bacterial β-lactamases. These two compounds were further tested in mice. Whereas commercial antibiotics alone failed to cure mice infected with Staphylococcus aureus and Escherichia coli expressing β-lactamases, infection was cleared when treated with antibiotics in combination with dBLIPs, clearly suggesting that peptides were able to neutralize bacterial resistance. Moreover, immunological assays were also performed showing that dBLIPs were unable to modify mammalian immune response in both models, reducing the risks of collateral effects. In summary, the unusual peptides here described provide leads to overcome β-lactamase-based resistance, a remarkable clinical challenge.

Background Aquatic matrices impacted by sewage may shelter carbapenem-resistant (CR) Gram-negative bacilli (GNB) harboring resistance genes of public health concern. In this study, sewage treatment plants (STPs) servicing well-defined catchment areas were surveyed for the presence of CR-GNB bearing carbapenemase genes (blaKPC or blaNDM). Results A total of 325 CR-GNB were recovered from raw (RS) and treated (TS) sewage samples as well as from water body spots upstream (UW) and downstream (DW) from STPs. Klebsiella-Enterobacter (KE) group amounted to 116 isolates (35.7%). CR-KE isolates were recovered from TS, DW (35.7%) and RS samples (44.2%) (p = 0.001); but not from UW samples. KE isolates represented 65.8% of all blaKPC or blaNDM positive strains. The frequency of blaKPC-or-NDM strains was positively associated with the occurrence of district hospitals located near STPs, as well as with the number of hospitalizations and of sewer connections serviced by the STPs. blaKPC-or-NDM strains were recovered from ST samples in 7 out of 14 STPs, including four tertiary-level STPs; and from 6 out of 13 DW spots whose RS samples also had blaKPC-or-NDM strains. Conclusions Clinically relevant GNB bearing blaKPC-or-NDM resist sewage treatments and spread into environmental aquatic matrices mainly from STPs impacted by hospital activities.

No abstract