The nucleoid protein HU positively regulates the expression of type VI secretion systems in Enterobacter cloacae.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

El método de inactivación de carbapenemas modificado (mCIM): herramienta altamente sensible y específica de bajo costo para evaluar la producción y no producción de carbapenemasas en bacilos gramnegativos / The Modified Carbapenem Inactivation Method (mCIM): Highly sensitive and specific low-cost tool to assess carbapenemase-producing and non-producing in Gram-negative bacilli

Introduction: Carbapenemase-producing Gram-negative bacilli is a worldwide problem, which represent a health concern, because most of its resistance mechanisms are encoded by plasmids, therefore easily transmissible in hospital settings. Many methods have been proposed to detect such resistance, but screening is still challenging, recently the modified carbapenem inactivation method has shown promising results, however more studies need to be performed. Hence, this study aimed to evaluate the performance of the mCIM in carbapenem-resistant Enterobacteriaceae and nonfermenting Gram-negative bacilli isolates. Materials and methods: From a microbial collection with molecular characterization of carbapenemase genes previously conducted, 100 Gram-negative bacilli isolates were selected, fifty-two non-carbapenemase producing and 48 carbapenemase-producing isolates. The mCIM was performed according to the CLSI guidelines, and to assess the validity of the method sensitivity and specificity were calculated.Results: The sensitivity of the mCIM observed in this study was 96% (46/48) and the specificity was 96,2% (50/52). Most of the Gram-negative bacilli carrying a carbapenemase gene were mCIM-positive, moreover, in carbapenem-resistant isolates that do not produce a carbapenemase (Enterobacteriaceae and non-fermenters) the results of the mCIM were negatives. Conclusion: Overall the mCIM provides a low-cost alternative for the screening of carbapenemase-producing Gram-negative bacilli. Our findings highlight that mCIM is a sensitive and specific method to assess carbapenemase-producing in Gram negative bacilli non-fermenters and Enterobacteriaceae as Enterobacter cloacae.

Introducción: Los bacilos Gram negativos productores de carbapenemasas representan un problema de salud pública mundial. Estos mecanismos de resistencia se encuentran codificados en su mayoría en plásmidos, por lo que son fácilmente transmisibles en el entorno hospitalario. Se han propuesto muchos métodos para detectar la presencia de carbapenemasas; sin embargo, la detección sigue siendo un desafío. Recientemente, el método de inactivación de carbapenémicos modificado (mCIM) ha mostrado resultados prometedores; sin embargo, es necesario realizar más estudios. Este estudio tuvo como objetivo evaluar el rendimiento del mCIM en Enterobacterias y bacilos Gram negativos no fermentadores resistentes a carbapenémicos. Materiales y métodos: A partir de una colección microbiana con previa caracterización molecular de genes que codifican carbapenemasas, se seleccionaron 100 aislados de bacilos Gram negativos, cincuenta y dos aislados no productores de carbapenemasas y 48 productores de carbapenemasas. El mCIM se realizó de acuerdo con las directrices del CLSI, y para evaluar la validez del método se calcularon la sensibilidad y la especificidad. Resultados: La sensibilidad del mCIM observada en este estudio fue del 96% (46/48) y la especificidad del 96,2% (50/52). La mayoría de los bacilos Gram negativos portadores de carbapenemasas fueron mCIM positivos, además, en los aislados resistentes a carbapenémicos no productores de carbapenemasas (Enterobacterias y bacilos Gram negativos no fermentadores) los resultados de mCIM fueron negativos. Conclusión: En general, el mCIM proporciona una alternativa de bajo costo para la detección de carbapenemasas en bacilos gramnegativos. Nuestros hallazgos destacan el mCIM como un método sensible y específico para evaluar la producción de carbapenemasas en bacilos Gram negativos no fermentadores y Enterobacterias como Enterobacter cloacae.

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

Inhibition of Wild Enterobacter cloacae Biofilm Formation by Nanostructured Graphene- and Hexagonal Boron Nitride-Coated Surfaces.

Although biofilm formation is a very effective mechanism to sustain bacterial life, it is detrimental in medical and industrial sectors. Current strategies to control biofilm proliferation are typically based on biocides, which exhibit a negative environmental impact. In the search for environmentally friendly solutions, nanotechnology opens the possibility to control the interaction between biological systems and colonized surfaces by introducing nanostructured coatings that have the potential to affect bacterial adhesion by modifying surface properties at the same scale. In this work, we present a study on the performance of graphene and hexagonal boron nitride coatings (h-BN) to reduce biofilm formation. In contraposition to planktonic state, we focused on evaluating the efficiency of graphene and h-BN at the irreversible stage of biofilm formation, where most of the biocide solutions have a poor performance. A wild Enterobacter cloacae strain was isolated, from fouling found in a natural environment, and used in these experiments. According to our results, graphene and h-BN coatings modify surface energy and electrostatic interactions with biological systems. This nanoscale modification determines a significant reduction in biofilm formation at its irreversible stage. No bactericidal effects were found, suggesting both coatings offer a biocompatible solution for biofilm and fouling control in a wide range of applications.

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

BACKGROUND: Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. METHODS: Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. RESULTS: A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. CONCLUSIONS: There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

Contaminación in vitro de cepillos dentales / In vitro contamination of toothbrushes

Se deierminó que los cepillos dentales mantienen viables y pueden transmitir 3 microorganismos frecuentemente implicados en infecciones orales. Los microorganismos fueron inoculados in vitro en cepillos mantenidos a temperatura ambiente y sometidos a subcultivo desde las 3 horas hasta los 16 días. Inóculos de aproximadamente 5 millones de bacterias/ml de un importante periodontopático, el Actinobacillus actinomycetemcomitans; de un entérico oportunista, el Enterobacter cloacae y una dosis infectiva 50, ID50 de un herpesvirus oral, el Herpes simplex virus tipo 1, (HSV-1) fueron colocados sobre cerdas de los cepillos. A. actinomycetemcomitans y el HSV-1 resultaron viables hasta por 72 horas. E. cloacae fué viable hasta por 16 dias, el tiempo máximo de subcultivo planteado en este estudio (Tabla 1). La viabilidad bacteriana se demostró por subcultivo de los microorganismos en TSBV y la identidad de los microorganismos se determinó por la morfología de la colonia, la catalasa, el MUG, la reacción en cadena de la polimerasa especie/especifico para A. actinomycetemcomitans y pruebas bioquímicas rápidas como subcultivo en McConkey y asimilación de sustratos para E. cloacae. La viabilidad viral se estableció por aparición del efecto citopatogénico en mononocapas de pulmón embrionario humano del inóculo recuperado de los cepillos, pase seriado e lFA indirecta contra HSV-1. Este estudio concluye que los cepillos dentales pueden ser un reservorio y además transmitir importantes patógenos orales entre familiares o individuos.

This study determined that toothbrushes could maintain viable and perhaps transmit to other family member 3 important oral pathogens. The toothbrushes were infected with an approximate inoculum of 5 million bacteria's per ml of Actinobacillus actinomycetemcomitans, and Enterobacter cloacae, respectively and an infective dose 50 (ID 50) of Herpes Simplex type I, (H5V-1 ). These microorganisms were placed directly on the tooth bristles at room temperature and subcultured at different times to establish individual microbial survival rates. Microorganisms were cultured at 3 hours. 24 hours, 96 hours, 5 days, 12 days, and 16 days after the initial toothbrush inoculation. A. actinomycetemcomitans, and HSV-1 resulted viable after 72 hours on toothbrushes. E. cloacae was viable as far as 16 days after the initial inoculation. The microbial viability was determined by subculture in TSBV and the identity of the microorganisms established by the bacterial colony morphology, rapid biochemical tests, and specie-specific polymerase chain reaction for A. actinomycetemcomitans. Viral viability was determined by visualization of the viral induced cytopathic effect on a cultured monolayer of embryonic lung fibroblasts from replicating HSV-1. Positive cultures were confirmed by IFA assay against HSV-L 1. In conclusion this study demonstrated in vitro that toothbrushes could act as a reservoir of microbes and maybe transmit important oral pathogens.