Hypervirulent and hypermucoviscous extended-spectrum ß-lactamase-producing Klebsiella pneumoniae and Klebsiella variicola in Chile.

Convergence of virulence and antibiotic-resistance has been reported in Klebsiella pneumoniae, but not in Klebsiella variicola. We, hereby, report the detection and genomic characterization of hypervirulent and hypermucoviscous K. pneumoniae and K.variicola recovered in Chile from health-care associated infections, which displayed resistance to broad-spectrum cephalosporins. One hundred forty-six K. pneumoniae complex isolates were screened by hypermucoviscosity by the "string test." Two hypermucoid isolates, one hypermucoviscous K. pneumoniae (hmKp) and one K. variicola (hmKv), were further investigated by whole-genome sequencing. In vivo virulence was analyzed by the Galleria mellonella killing assay. In silico analysis of hmKp UCO-494 and hmKv UCO-495 revealed the presence of multiple antibiotic-resistance genes, such as blaCTX-M-1, blaDHA-1 and blaLEN-25 among others clinically relevant resistance determinants, including mutations in a two-component regulatory system related to colistin resistance. These genetic features confer a multidrug-resistant (MDR) phenotype in both strains. Moreover, virulome in silico analysis confirmed the presence of the aerobactin gene iutA, in addition to yersiniabactin and/or colicin V encoding genes, which are normally associated to high virulence in humans. Furthermore, both isolates were able to kill G. mellonella and displayed higher virulence in comparison with the control strain. In summary, the convergence of virulence and the MDR-phenotype in K. pneumoniae complex members is reported for the first time in Chile, denoting a clinical problem that deserves special attention and continuous surveillance in South America.

Molecular diversity of Staphylococcus aureus and the role of milking equipment adherences or biofilm as a source for bulk tank milk contamination.

Staphylococcus aureus is one of the most frequent pathogens causing intramammary infections in dairy herds. Consequently, virulence factors, pathobiology, and epidemiology of Staphylococcus aureus strains have been widely assessed through the years. Nevertheless, not much has been described about the epidemiology of Staph. aureus strains from bulk tank milk (BTM) and adherences on milking equipment (AMES), even when these strains may play a role in the quality of milk that is intended for human consumption. The objective of this study was to assess the strain diversity of 166 Staph. aureus isolates collected from 3 consecutive BTM samples, and from AMES in contact with milk from 23 Chilean dairy farms. Isolates were analyzed and typed using pulsed-field gel electrophoresis. Diversity of strains, both within and among farms, was assessed using Simpson's index of diversity (SID). On farms where Staph. aureus was isolated from both AMES and BTM (n = 8), pulsotypes were further analyzed to evaluate the role of AMES as a potential source of Staph. aureus strains in BTM. Among all Staph. aureus analyzed by pulsed-field gel electrophoresis, a total of 42 pulsotypes (19 main pulsotypes and 23 subtypes) were identified. Among dairy farms, strain diversity was highly heterogeneous (SID = 0.99). Within dairy farms, Staph. aureus strain diversity was variable (SID = 0 to 1), and 18 dairy operations (81.8%) had one pulsotype that was shared between at least 2 successive BTM samples. In those farms where Staph. aureus was isolated in both AMES and BTM (n = 8), 7 (87.5%) showed a clonal distribution of Staph. aureus strains between these 2 types of samples. The overlapping of certain Staph. aureus strains among dairy farms may point out common sources of Staph. aureus among otherwise epidemiologically unrelated farms. Indistinguishable Staph. aureus strains between AMES and BTM across dairy farms suggest that Staph. aureus-containing AMES may represent a source for BTM contamination, thus affecting milk quality. Our study highlights the role of viable Staph. aureus in AMES as a source for BTM contamination on dairy farms, and also describes the overlapping and presence of specific BTM and AMES pulsotypes among farms.

Occurrence of antibiotics and bacterial resistance in wastewater and sea water from the Antarctic.

The potential presence of introduced antibiotics in the aquatic environment is a hot topic of concern, particularly in the Antarctic, a highly vulnerable area protected under the Madrid protocol. The increasing presence of human population, especially during summer, might led to the appearance of pharmaceuticals in wastewater. The previous discovery of Escherichia coli strains resistant to antibiotics in sea water and wastewater collected in King George Island motivated our investigation on antibiotics occurrence in these samples. The application of a multi-residue LCMS/MS method for 20 antibiotics, revealed the presence of 8 compounds in treated wastewater, mainly the quinolones ciprofloxacin and norfloxacin (92% and 54% of the samples analyzed, average concentrations 0.89 µg/L and 0.75 µg/L, respectively) and the macrolides azithromycin and clarithromycin (15% positive samples, and average concentrations near 0.4 µg/L), and erythromycin (38% positive samples, average concentration 0.003 µg/L). Metronidazole and clindamycin were found in one sample, at 0.17 and 0.1 µg/L, respectively; and trimethoprim in two samples, at 0.001 µg/L. Analysis of sea water collected near the outfall of the wastewater discharges also showed the sporadic presence of 3 antibiotics (ciprofloxacin, clindamycin, trimethoprim) at low ng/L level, illustrating the impact of pharmaceuticals consumption and the poor removal of these compounds in conventional WWTPs. The most widespread antibiotic in sea water was ciprofloxacin, which was found in 15 out of 34 sea water samples analyzed, at concentrations ranging from 4 to 218 ng/L. Bacteria resistance was observed for some antibiotics identified in the samples (e.g. trimetropim and nalidixic acid -a first generation quinolone). However, resistance to some groups of antibiotics could not be correlated to their presence in the water samples due to analytical limitations (penicillins, tetraciclines). On the contrary, for some groups of antibiotics detected in samples (macrolides), the antibacterial activity against E. Coli was not investigated because these antibiotics do not include this bacterial species in their spectrum of activity. Our preliminary data demonstrate that antibiotics occurrence in the Antarctic aquatic environment is an issue that needs to be properly addressed. Periodical monitoring of water samples and the implementation of additional treatments in the WWTPs are recommended as a first step to prevent potential problems related to the presence of antibiotics and other emerging contaminants in the near future in Antarctica.

Metagenomic study of bacterial microbiota in persistent endodontic infections using Next-generation sequencing.

AIM: To determine the bacterial microbiota in root canals associated with persistent apical periodontitis and their relationship with the clinical characteristics of patients using next-generation sequencing (NGS). METHODOLOGY: Bacterial samples from root canals associated with teeth having persistent apical periodontitis were taken from 24 patients undergoing root canal retreatment. Bacterial DNA was extracted, and V3-V4 variable regions of the 16S rRNA gene were amplified. The amplification was deep sequenced by Illumina technology to establish the metagenetic relationships among the bacterial species identified. The composition and diversity of microbial communities in the root canal and their relationships with clinical features were analysed. Parametric and nonparametric tests were used to analyse differences between patient characteristics and microbial data. RESULTS: A total of 86 different operational taxonomic units (OTUs) were identified and Good's nonparametric coverage estimator method indicated that 99.9 ± 0.00001% diversity was recovered per sample. The largest number of bacteria belonged to the phylum Proteobacteria. According to the medical history from the American Society of Anesthesiologists (ASA) Classification System, ASA II-III had higher richness estimates and distinct phylogenetic relationships compared to ASA I individuals (P < 0.05). Periapical index (PAI) score 5 was associated with increased microbiota diversity in comparison to PAI score 4, and this index was reduced in symptomatic patients. CONCLUSIONS: Based on the findings of this study, it is possible to suggest a close relationship between several clinical features and greater microbiota diversity with persistent endodontic infections. This work provides a better understanding on how microbial communities interact with their host and vice versa.

Synthesis of Copper Nanowires and Their Antimicrobial Activity on Strains Isolated Persistent Endodontic Infections.

Copper nanowires, Cu-NWs may have a good antimicrobial effect in endodontic treatment. The objective of this work was to synthesize, characterize and evaluate the antibacterial activity of Cu-NWs on strains obtained from human root canal. A wide distribution of Cu-NWs diameters from 30 to 90 nm was obtained with lengths ranging from 5 to 40 µm. Structural analysis of Cu-NWs showed crystalline planes, which corresponded to Cu, with preferential growth in the direction [110]. The geometric mean MICs was of 289.30 µg/mL, with a MIC50 of 256 µg/mL and a MIC90 of 512 µg/mL for Cu-NWs. Cellular viability of 28 a biofilm tends to decrease the longer it is exposed to Cu-NWs. Synthesized and characterized Cu-NWs have a good antimicrobial effect against clinical strains used in the present study and has a potential to be used for disinfection of the root canal system.