Identification of Vibrio metschnikovii and Vibrio injensis Isolated from Leachate Ponds: Characterization of Their Antibiotic Resistance and Virulence-Associated Genes.

This study aimed to evaluate the antibiotic resistance of 22 environmental Vibrio metschnikovii isolates and 1 Vibrio injensis isolate from landfill leachates in southwestern Colombia. Isolates were identified by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF), and 16S ribosomal RNA gene sequencing. Analysis of the susceptibility to six antibacterial agents by the Kirby-Bauer method showed susceptibility of all the isolates to ciprofloxacin and imipenem. We recorded resistance to beta-lactams and aminoglycosides, but no multidrug resistance was observed. The genome of one of the isolates was sequenced to determine the pathogenic potential of V. injensis. Genes associated with virulence were identified, including for flagellar synthesis, biofilm formation, and hemolysins, among others. These results demonstrate that landfill leachates are potential reservoirs of antibiotic-resistant and pathogenic bacteria and highlight the importance of monitoring Vibrio species in different aquatic environments.

Beta-Lactam-Resistant Enterobacterales Isolated from Landfill Leachates.

Antibiotic resistance is one of the main challenges worldwide due to the high morbidity and mortality caused by infections produced by resistant bacteria. In Colombia, this problem has been studied mainly from the clinical perspective; however, it is scarcely studied in the leachates produced in landfills. The objective of this study was to detect, identify and determine the antibiotic sensitivity profile of Enterobacterales isolated from a leachate treatment plant located in Cali, Colombia. Detection was performed using selective culture media, bacterial identification using Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF, bioMérieux) and by sequencing the gene coding for the 16S ribosomal RNA subunit when discrepancies were observed between phenotypic characteristics and MALDI-TOF. Antibiotic sensitivity profiling was determined using the automated VITEK®2 system (bioMérieux). Twenty-one isolates were obtained, of which Klebsiella pneumoniae was the most frequent (23.8%), and 34% of the isolates showed decreased sensitivity to beta-lactam antibiotics such as cefoxitin, ampicillin/sulbactam and piperacillin/tazobactam. These findings suggest that leachates from landfills could be a reservoir of pathogenic bacteria carrying antibiotic resistance determinants, so periodic microbiological characterization of these effluents should be performed, promoting the One Health approach.

Phage Therapy in the Era of Multidrug Resistance in Bacteria: A Systematic Review.

Bacteriophages offer an alternative for the treatment of multidrug-resistant bacterial diseases as their mechanism of action differs from that of antibiotics. However, their application in the clinical field is limited to specific cases of patients with few or no other alternative therapies. This systematic review assesses the effectiveness and safety of phage therapy against multidrug-resistant bacteria through the evaluation of studies published over the past decade. To that end, a bibliographic search was carried out in the PubMed, Science Direct, and Google Scholar databases. Of the 1500 studies found, 27 met the inclusion criteria, with a total of 165 treated patients. Treatment effectiveness, defined as the reduction in or elimination of the bacterial load, was 85%. Except for two patients who died from causes unrelated to phage therapy, no serious adverse events were reported. This shows that phage therapy could be an alternative treatment for patients with infections associated with multidrug-resistant bacteria. However, owing to the phage specificity required for the treatment of various bacterial strains, this therapy must be personalized in terms of bacteriophage type, route of administration, and dosage.

Molecular Characterization of KPC-2-Producing Enterobacter cloacae Complex Isolates from Cali, Colombia.

The Enterobacter cloacae complex is an emerging opportunistic pathogen whose increased resistance to carbapenems is considered a public health problem. This is due to the loss of efficacy of beta-lactam antibiotics, which are used as the first treatment option in the management of infections caused by Gram-negative bacteria. The objective of this study was to perform the molecular characterization of 28 isolates of the E. cloacae complex resistant to cephalosporins and carbapenems isolated between 2011 and 2018 from five hospitals located in the municipality of Santiago de Cali, Colombia. Molecular detection of blaKPC, blaVIM, blaNDM and blaOXA-48-like genes was performed on these isolates and the genetic relationship between the isolates was assessed using multilocus sequence typing (MLST). Forty-three percent of the isolates carried the blaKPC-2 gene variant. MLST showed high genetic diversity among isolates, the most frequent being the sequence type ST510 with a frequency of 50%. The identification of the genes involved in carbapenem resistance and dispersing genotypes is an important step toward the development of effective prevention and epidemiological surveillance strategies in Colombian hospitals.

Overexpression of mfpA Gene Increases Ciprofloxacin Resistance in Mycobacterium smegmatis.

Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, but FQ-resistant mutants can be selected rapidly. Although mutations in the DNA gyrase are the principal cause of this resistance, pentapeptide proteins have been found to confer low-level FQ resistance in Gram-negative bacteria. MfpA is a pentapeptide repeat protein conserved in mycobacterial chromosomes, where it is adjacent to a group of four highly conserved genes termed a conservon. We wished to characterize the transcriptional regulation of the mfpA gene and relate its expression to ciprofloxacin resistance in M. smegmatis. Reverse transcription PCR showed that mfpA gene is part of an operon containing the conservon genes. Using a transcriptional fusion, we showed that a promoter was located 5' to the mfpEA operon. We determined the promoter activity under different growth conditions and found that the expression of the operon increases slightly in late growth phases in basic pH and in subinhibitory concentrations of ciprofloxacin. Finally, by cloning the mfpA gene in an inducible vector, we showed that induced expression of mfpA increases the ciprofloxacin Minimal Inhibitory Concentration. These results confirm that increased expression of the mfpA gene, which is part of the mfpEA operon, increases ciprofloxacin resistance in M. smegmatis.

Epidemiología molecular de aislados clínicos de Klebsiella pneumoniae productores de carbapenemasas tipo KPC provenientes de dos hospitales públicos en los estados Carabobo y Zulia, Venezuela / Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolated from patients in two public hospitals in Carabobo and Zulia states, Venezuela

Klebsiella pneumoniae productora de carbapenemasas tipo KPC es uno de los principales agentes causante de infecciones nosocomiales a nivel mundial. En Venezuela se han identificado aislados de esta bacteria, sin embargo, se conoce poco sobre su dispersión. El objetivo de este estudio fue realizar la epidemiología molecular de aislados de K. pneumoniae productores de KPC provenientes de dos hospitales públicos ubicados en los estados Carabobo y Zulia. Se seleccionaron 32 aislados de K. pneumoniae clasificados fenotípicamente como productores de KPC, se les realizó la detección del gen bla KPC así como su ubicación en el transposón Tn4401 a través de PCR. El producto de PCR del gen blaKPC se secuenció para identificar los alelos circulantes. El análisis genotípico se realizó empleando las técnicas de amplificación por PCR de las secuencias repetidas extragénicas palindrómicas (rep-PCR) y la secuenciación de múltiples loci (MLST). Mediante ensayos de conjugación, se determinó si los genes bla KPC se encontraban en moléculas plasmídicas.Los resultados indican que los 32 aislados contenían la variante del gen bla KPC-2 asociada a la isoforma Tn4401 b y se distribuyeron en 9 secuencias tipo (ST), siendo una de ellas nueva. Los ensayos de conjugación indican que el 87,5% de los aislados tienen al gen bla KPC en plásmidos movilizables. En estos hospitales el gen bla KPC-2 se está dispersando a través de plásmidos que llevan al transposón Tn4401 b. Las ST más comunes pertenecen a los Complejos Clonales CC258 y CC147, que desempeñan un papel importante en la dispersión de la resistencia a carbapenemes a nivel mundial.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria ( K. pneumoniae carbapenemase ) are the most important causative agents of nosocomial infections worldwide. These isolates have been identified in Venezuela, but little is known about their local spread. The aim of this study was to perform molecular epidemiology of KPC-producing K. pneumoniae isolated from two public hospitals in the Carabobo and Zulia states of Venezuela. Thirty-two K. pneumoniaei solates, phenotypically classified as KPC producers were subjected to PCR to detect the presence of bla KPC genes and their location within transposon Tn4401 , and the bla KPC product was sequenced to identify the KPC allele. Genotypic analysis was performed using repeated extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, a conjugation assay determined whether the bla KPC genes were carried on transferable plasmids. The results indicate that the 32 isolates contained the bla KPC-2 variant associated with isoform Tn4401 b, and were distributed in nine sequence types (ST), one of which was new. Conjugation assays indicate that 87.5% of the isolates contain the gene bla KPC on mobilizable plasmids. In these hospitals, the bla KPC-2 gene is spreading through the plasmids carrying the transposon Tn4401 b. The most common ST belongs to Clonal Complexes CC258 and CC147, which play an important role in the dispersion of resistance to carbapenems worldwide.

Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolated from patients in two public hospitals in Carabobo and Zulia states, Venezuela. / Epidemiología molecular de aislados clínicos de Klebsiella pneumoniae productores de carbapenemasas tipo KPC provenientes de dos hospitales públicos en los estados Carabobo y Zulia, Venezuela.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria (K. pneumonia carbapenemase) are the most important causative agents of nosocomial infections worldwide. These isolates have been identified in Venezuela, but little is known about their local spread. The aim of this study was to perform molecular epidemiology of KPC-producing K. pneumoniae isolated from two public hospitals in the Carabobo and Zulia states of Venezuela. Thirty-two K. pneumoniaei solates, phenotypically classified as KPC producers were subjected to PCR to detect the presence of blaKPC genes and their location within transposon Tn4401, and the blaKPC product was sequenced to identify the KPC allele. Genotypic analysis was performed using repeated extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, a conjugation assay determined whether the blaKPC genes were carried on transferable plasmids. The results indicate that the 32 isolates contained the blaKPC-2 variant associated with isoform Tn4401b, and were distributed in nine sequence types (ST), one of which was new. Conjugation assays indicate that 87.5% of the isolates contain the gene blaKPC on mobilizable plasmids. In these hospitals, the blaKPC-2 gene is spreading through the plasmids carrying the transposon Tn4401b. The most common ST belongs to Clonal Complexes CC258 and CC147, which play an important role in the dispersion of resistance to carbapenems worldwide.

A cluster of KPC-2 and VIM-2-producing Klebsiella pneumoniae ST833 isolates from the pediatric service of a Venezuelan Hospital.

BACKGROUND: Klebsiella pneumoniae is a bacterial pathogen that has developed resistance to multiple antibiotics and is a major cause of nosocomial infections worldwide. Carbapenemase-producing Klebsiella pneumoniae have been isolated in many hospitals in Venezuela, but they have not been well-studied. The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae isolates from the pediatric service of a hospital located in Anzoategui State, in the eastern part of Venezuela. METHODS: Nineteen Klebsiella pneumoniae strains isolated in the hospital from April to July 2014 were evaluated phenotypically and molecularly for the presence of carbapenemases blaKPC, blaIMP and blaVIM. Molecular epidemiology was performed with Repetitive Extragenic Palindromic-PCR (REP-PCR) and Multilocus Sequence Typing (MLST). They were also studied for phenotypic and molecular resistance to a quaternary ammonium compound (QAC) disinfectant. RESULTS: All 19 isolates contained both bla VIM-2 and bla KPC-2 genes, and the bla KPC-2 gene was associated with Tn4401b. All isolates were phenotypically sensitive to QACs and contained qacΔE and addA2 genes typical of class 1 integrons. Analysis by REP-PCR and MLST showed that all isolates had identical profiles characteristic of sequence type ST833. CONCLUSION: All 19 strains are bla VIM-2 and bla KPC-2-producing ST833 K. pneumoniae sensitive to QACs. This analysis may help to understand the routes of dissemination and confirms that QAC disinfectants can be used to help control their spread.

Aislamiento e identificación de cepas del género Bifidobacterium presentes en productos lácteos fermentados tipo yogur / Isolation and identification of strains belonging to the Bifidobacterium genus found in fermented yoghurt type milk products

La identificación de microorganismos probióticos del género Bifidobacterium es de gran importancia por su uso como suplemento que favorece la salud del consumidor. En Venezuela son pocos los estudios sobre caracterización microbiológica de estas bacterias y no existen métodos oficiales para su estudio en alimentos. Esta investigación reporta la estandarización de técnicas microbiológicas y moleculares para el aislamiento e identificación de bifidobacterias aisladas de dos productos tipo yogur, I con probiótico y II sin probiótico. Se analizaron 10 muestras de cada yogur, una por semana, aislando 3 colonias por muestra. Los resultados mostraron que de los 60 aislados analizados, 27 colonias del Yogur I y 11 del Yogur II concordaron con las características de bifidobacterias. Se comparó el crecimiento bacteriano en dos medios de cultivo (MRS-m, RCA), sembrando por profundidad en placas y en tubos Miller-Pricket, obteniéndose mejores resultados con el medio MRS-m y las siembras por profundidad en tubos. De las extracciones de ADN se obtuvieron los patrones de ERIC-PCR y REP-PCR, determinándose que 34 aislados eran clones indistinguibles, mostrando el patrón de B. lactis utilizado como control positivo. Esta metodología puede ser utilizada por la industria y los entes encargados del control de la calidad de los productos probióticos.

The identification of probiotic microorganisms belonging to the Bifidobacterium genus is very important due to their use as supplements favorable for consumer’s health. In Venezuela there have been few studies of the microbiological characterization of these bacteria and there are no official methods for their study in food. This investigation reports the standardization of microbiological and molecular techniques for the isolation and identification of bifidobacteria isolated from two yoghurt type products: I with probiotic and II without probiotic. Ten samples from each yoghurt type product were analyzed, one per week, and 3 colonies were isolated per sample. Results showed that of the 60 isolates analyzed, 27 colonies of Yoghurt I and 11 of Yoghurt II coincided with the characteristics of bifidobacteria. Bacterial growth was compared in two culture media (MRS-m, RCA), inoculating in-depth in plates and Miller-Pricket tubes; the best results were obtained with MRS-m medium and in-depth inoculations in tubes. By DNA extraction we obtained ERIC-PCR and REP-PCR patterns, determining that 34 isolates were indistinguishable clones, showing the same pattern of the B. lactis used as positive control. This methodology can be used by the industry and the institutions in charge of quality control of probiotic products.