Clonal complexes of carbapenem-resistant Klebsiella pneumoniae recovered from community sewage.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) are life-threatening multidrug-resistant bacteria. In this study, CR-Kp strains isolated from sewage treatment plants (STPs) (n = 12) were tested for carbapenemase genes (blaKPC, blaNDM, blaIMP, blaVIM and blaOXA-48) and had their sequence types (ST) and clonal complexes (CCs) defined. A collection of clinical CR-Kp strains recovered in local hospitals was added to phylogenetic analyses along with sewage strains in order to infer clonality among CR-Kp strains. A total of 154 CR-Kp strains were isolated from raw sewage [55.8% (86/154)], treated sewage [25.3% (39/154)] and from water body downstream from STPs [18.8% (29/154)]. No CR-Kp strain was isolated from upstream water samples. blaKPC or blaNDM were detected in 143 (92.8%) strains. The occurrence of blaKPC-or-NDM CR-Kp strains was positively associated with the number of hospitalized patients in the areas serviced by STPs. Eleven STs were detected in CR-Kp strains, most of them belonging to the clinically relevant CC11 [ST11 (n = 13-28.2%) and ST340 (n = 7-15.2%)]. CCs 11, 15, 17, 147 and 2703 are shared by clinical and sewage CR-Kp strains. In conclusion, sewage harbors clinically relevant clones of CR-Kp that resist sewage treatments, contaminating water bodies downstream from STPs.

Environmental spreading of clinically relevant carbapenem-resistant gram-negative bacilli: the occurrence of blaKPC-or-NDM strains relates to local hospital activities.

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.

Insertion sequences disrupting mgrB in carbapenem-resistant Klebsiella pneumoniae strains in Brazil.

OBJECTIVES: This study aimed to characterise insertional mutations disturbing themgrB gene in carbapenem-resistant Klebsiella pneumoniae (CRKp). METHODS: A total of 118 clinical CRKp isolates were surveyed for polymyxin resistance and insertion sequence (IS) elements disruptingmgrB. RESULTS: Of the 118 isolates, 78 (66.1%) displayed polymyxin resistance, of which 54% (42/78) hadmgrB::IS inserts. Sequencing analyses showed 13 insertion sites in mgrB. mgrB::ISSen4(IS3) was observed for the first time in CRKp. CONCLUSIONS: Ten different IS elements disruptedmgrB, with a predominance (76%) of IS5 sequences.

Identification, molecular characterization, and structural analysis of the blaNDM-1 gene/enzyme from NDM-1-producing Klebsiella pneumoniae isolates.

NDM-1 comprises a carbapenemase that was first detected in 2008 in New Delhi, India. Since then, NDM-1-producing Klebsiella pneumoniae strains have been reported in many countries and usually associated with intra and inter-hospital dissemination, along with travel-related epidemiological links. In South America, Brazil represents the largest reservoir of NMD-1-producing K. pneumoniae. Here, we focused on the detection and molecular/structural characterization of the blaNDM-1 resistance gene/enzyme from 24 K. pneumoniae clinical isolates in the Midwest region of Brazil. Antimicrobial susceptibility assays showed that all isolates are resistant to carbapenems. Molecular typing of the isolates revealed seven clonal groups among the K. pneumoniae isolates, which may indicate intra or inter-hospital dissemination. Moreover, the blaNDM-1 gene was detected in all 24 K. pneumoniae isolates and the full blaNDM-1 gene was cloned. Bioinformatics analysis showed that the NDM-1 enzyme sequence found in our isolates is highly conserved when compared to other NDM-1 enzymes. In addition, molecular docking studies indicate that the NDM-1 identified binds to different carbapenems through hydrogen and zinc coordination bonds. In summary, we present the molecular characterization of NDM-1-producing K. pneumoniae strains isolated from different hospitals, also providing atomic level insights into molecular complexes NDM-1/carbapenem antibiotics.

Genomic Comparison among Lethal Invasive Strains of Streptococcus pyogenes Serotype M1.

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a human pathogen that causes diverse human diseases including streptococcal toxic shock syndrome (STSS). A GAS outbreak occurred in Brasilia, Brazil, during the second half of the year 2011, causing 26 deaths. Whole genome sequencing was performed using Illumina platform. The sequences were assembled and genes were predicted for comparative analysis with emm type 1 strains: MGAS5005 and M1 GAS. Genomics comparison revealed one of the invasive strains that differ from others isolates and from emm 1 reference genomes. Also, the new invasive strain showed differences in the content of virulence factors compared to other isolated in the same outbreak. The evolution of contemporary GAS strains is strongly associated with horizontal gene transfer. This is the first genomic study of a Streptococcal emm 1 outbreak in Brazil, and revealed the rapid bacterial evolution leading to new clones. The emergence of new invasive strains can be a consequence of the injudicious use of antibiotics in Brazil during the past decades.

Virulence Markers and Phylogenetic Analysis of Escherichia coli Strains with Hybrid EAEC/UPEC Genotypes Recovered from Sporadic Cases of Extraintestinal Infections.

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 combinations for controlling colistin-resistant Enterobacter cloacae.

Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and ß-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin-chloramphenicol for infections caused by colistin-resistant E. cloacae.

Characterization of Tn3000, a Transposon Responsible for blaNDM-1 Dissemination among Enterobacteriaceae in Brazil, Nepal, Morocco, and India.

In Enterobacteriaceae, the blaNDM 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 blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK 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 blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world.

Antibiofilm peptides increase the susceptibility of carbapenemase-producing Klebsiella pneumoniae clinical isolates to ß-lactam antibiotics.

Multidrug-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 ß-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 ß-lactams, including meropenem, represents a promising strategy to treat infections caused by KpC isolates.

Controlling resistant bacteria with a novel class of ß-lactamase inhibitor peptides: from rational design to in vivo analyses.

Peptide rational design was used here to guide the creation of two novel short ß-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 ß-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 ß-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 ß-lactamase-based resistance, a remarkable clinical challenge.