Lacticaseibacillus rhamnosus CRL 2244 secreted metabolites display killing and antibiotic synergistic activity against multi-drug resistant pathogens.

A growing increase in the number of serious infections caused by multidrug resistant bacteria (MDR) is challenging our society. Despite efforts to discover novel therapeutic options, few antibiotics targeting MDR have been approved by the Food and Drug Administration (FDA). Lactic acid bacteria have emerged as a promising therapeutic alternative due to their demonstrated ability to combat MDR pathogens in vitro. Our previous co-culture studies showed Lacticaseibacillus rhamnosus CRL 2244 as having a potent killing effect against carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Here we report that cell-free conditioned media (CFCM) samples obtained from Lcb. rhamnosus CRL 2244 cultures incubated at different times display antimicrobial activity against 43 different pathogens, including CRAB, methicillin-resistant Staphylococcus aureus (MRSA) and carbapenemase Klebsiella pneumoniae (KPC)-positive strains. Furthermore, transwell and ultrafiltration analyses together with physical and chemical/biochemical tests showed that Lcb. rhamnosus CRL 2244 secretes a <3 kDa metabolite(s) whose antimicrobial activity is not significantly impaired by mild changes in pH, temperature and various enzymatic treatments. Furthermore, sensitivity and time-kill assays showed that the bactericidal activity of the Lcb. rhamnosus CRL 2244 metabolite(s) enhances the activity of some current FDA approved antibiotics. We hypothesize that this observation could be due to the effects of Lcb. rhamnosus CRL 2244 metabolite(s) on cell morphology and the enhanced transcriptional expression of genes coding for the phenylacetate (PAA) and histidine catabolic Hut pathways, metal acquisition and biofilm formation, all of which are associated with bacterial virulence. Interestingly, the extracellular presence of Lcb. rhamnosus CRL 2244 induced the transcription of the gene coding for the CidA/LgrA protein, which is involved in programmed cell death in some bacteria. Overall, the findings presented in this report underscore the promising potential of the compound(s) released by Lcb. rhamnosus CRL2244 as an alternative and/or complementary option to treat infections caused by A. baumannii as well as other MDR bacterial pathogens.

Using a microbial physiologic and genetic approach to investigate how bacteria sense physical stimuli.

A laboratory exercise was designed to illustrate how physical stimuli such as temperature and light are sensed and processed by bacteria to elaborate adaptive responses. In particular, we use the well-characterized Des pathway of Bacillus subtilis to show that temperature modulates gene expression, resulting ultimately in modification of the levels of unsaturated fatty acids required to maintain proper membrane fluidity at different temperatures. In addition, we adapt recent findings concerning the modulation by light of traits related to virulence such as motility and biofilm formation in the chemotropic bacterium Acinetobacter baumannii. Beyond the theoretical background that this activity provides regarding sensing of environmental stimuli, the experimental setup includes approaches derived from classic genetics, microbiology, and biochemistry. The incorporation of these kind of teaching and training activities in middle-advanced Microbiology or Bacterial Genetics courses promotes acquisition of general and specific techniques and improves student's comprehension of scientific literature and research.

Staring at the cold sun: blue light regulation is distributed within the genus Acinetobacter.

We previously showed that the opportunistic nosocomial pathogen Acinetobacter baumannii is able to sense and respond to light via BlsA, a BLUF (Blue-Light-sensing Using FAD)-domain photoreceptor protein. Here, we extend our previous studies showing that light regulation is not restricted to A. baumannii, but rather widespread within the genus Acinetobacter. First, we found that blue light modulates motility and biofilm formation in many species of the genus, including members of the Acinetobacter calcoaceticus-A. baumannii complex. In many of these species blue light acts as a key factor guiding the decision between motility or sessility at 24°C, whereas in A. baumannii, light inhibits both motility and biofilm formation. We also show that light regulation of motility occurred not only at 24°C but also at 37°C in non-A. baumannii species, contrasting the situation of A. baumannii which only shows photoregulation at 24°C. Second, we show that Acinetobacter baylyi (strain ADP1) BLUF-photoreceptors can functionally replace in vivo the A. baumannii 17978 BlsA protein and that the pathways leading to biofilm formation are inversely regulated at 24°C between these two microorganisms. Finally, we found the presence of predicted genes coding BLUF-containing proteins in all Acinetobacter sequenced genomes, even though the copy number is variable among them. Phylogenetic analysis suggests a common origin for all BLUF domains present in members of this genus, and could distinguish well-differentiated clusters that group together BLUF homologs from different species, a situation particularly clear for members of the ACB complex. Despite a role played by these BLUF domain-containing proteins in the photoregulation observed in the members of the genus Acinetobacter is a likely scenario given our findings in A. baumannii and A. baylyi, further research will contribute to confirm this possibility.

Horizontal gene transfer and assortative recombination within the Acinetobacter baumannii clinical population provide genetic diversity at the single carO gene, encoding a major outer membrane protein channel.

We described previously the presence in Acinetobacter baumannii of a novel outer membrane (OM) protein, CarO, which functions as an L-ornithine OM channel and whose loss was concomitant with increased carbapenem resistance among clonally related nosocomial isolates of this opportunistic pathogen. Here, we describe the existence of extensive genetic diversity at the carO gene within the A. baumannii clinical population. The systematic analysis of carO sequences from A. baumannii isolates obtained from public hospitals in Argentina revealed the existence of four highly polymorphic carO variants among them. Sequence polymorphism between the different A. baumannii CarO variants was concentrated in three well-defined protein regions that superimposed mostly to predicted surface-exposed loops. Polymorphism among A. baumannii CarO variants was manifested in differential electrophoretic mobilities, antigenic properties, abilities to form stable oligomeric structures, and l-ornithine influx abilities through the A. baumannii OM under in vivo conditions. Incongruence between the phylogenies of the clinical A. baumannii isolates analyzed and those of the carO variants they harbor suggests the existence of assortative (entire-gene) carO recombinational exchange within the A. baumannii population. Exchange of carO variants possessing differential characteristics mediated by horizontal gene transfer may constitute an A. baumannii population strategy to survive radically changing environmental conditions, such as the leap from inanimate sources to human hosts and vice versa, persistence in a compromised host, and/or survival in health care facilities.

Characterization of the IgA1 protease from the Brazilian purpuric fever strain F3031 of Haemophilus influenzae biogroup aegyptius.

Brazilian purpuric fever is a severe vascular disease caused by an invasive clone of Haemophilus influenzae biogroup aegyptius, which normally causes self-limiting eye infections. A previous genome subtraction procedure resulted in the isolation of a DNA fragment, which encodes a putative IgA1 protease, specific to the F3031 Brazilian purpuric fever type strain. Cloning and sequencing of the entire F3031 iga1 gene showed that the subtracted DNA fragment encompasses the iga1 region encoding the active site and the cleavage specificity determinant of the protein, which are different from the cognate regions of the proteases produced by other H. influenzae strains. Western and IgA cleavage assays together with clustering analysis showed that the F3031 IgA1 protease is most similar to the type 2 proteases produced by H. influenzae type c and e strains. Analysis of the promoter region of the F3031 iga1 gene revealed the presence of Fur binding sites. However, real-time PCR analysis and transcriptional fusion assays showed that the expression of iga1 is not regulated by iron or hemin under the conditions tested.

Cloning and sequencing of a genomic island found in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

A genomic island was identified in the Haemophilus influenzae biogroup aegyptius Brazilian purpuric fever (BPF) strain F3031. This island, which was also found in other BPF isolates, could not be detected in non-BPF biogroup aegyptius strains or in nontypeable or typeable H. influenzae strains, with the exception of a region present in the type b Eagan strain. This 34,378-bp island is inserted, in reference to H. influenzae Rd KW20, within a choline transport gene and contains a mosaic structure of Mu-like prophage genes, several hypothetical genes, and genes potentially encoding an Erwinia carotovora carotovoricin Er-like bacteriocin. The product of the tail fiber ORF in the bacteriocin-like region shows a hybrid structure where the C terminus is similar to an H. influenzae phage HP1 tail protein implicating this open reading frame in altering host specificity for a putative bacteriocin. Significant synteny is seen in the entire genomic island with genomic regions from Salmonella enterica subsp. enterica serovar Typhi CT18, Photorhabdus luminescens subsp. laumondii TT01, Chromobacterium violaceum, and to a lesser extent Haemophilus ducreyi 35000HP. In a previous work, we isolated several BPF-specific DNA fragments through a genome subtraction procedure, and we have found that a majority of these fragments map to this locus. In addition, several subtracted fragments generated from an independent laboratory by using different but related strains also map to this island. These findings underscore the importance of this BPF-specific chromosomal region in explaining some of the genomic differences between highly invasive BPF strains and non-BPF isolates of biogroup aegyptius.

Genomic analysis of the F3031 Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius by PCR-based subtractive hybridization.

PCR-based subtractive genome hybridization produced clones harboring inserts present in Brazilian purpuric fever (BPF) prototype strain F3031 but absent in noninvasive Haemophilus influenzae biogroup aegyptius isolate F1947. Some of these inserts have no matches in the GenBank database, while others are similar to genes encoding either known or hypothetical proteins. One insert represents a 2.3-kb locus with similarity to a Thermotoga maritima hypothetical protein, while another is part of a 7.6-kb locus that contains predicted genes encoding hypothetical, phage-related, and carotovoricin Er-like proteins. The presence of DNA related to these loci is variable among BPF isolates and nontypeable H. influenzae strains, while neither of them was detected in strains of types a to f. The data indicate that BPF-causing strain F3031 harbors unique chromosomal regions, most of which appear to be acquired from unrelated microbial sources.

Propiedades de superficie y adherencia de cepas de Acinetobacter baumannii / Surface characteristics and strains adherence of the Acetinetobacter baumannii

En este trabajo se estudió la capacidad que presentaban 14 cepas de Acinetobacter baumannii (A.b.) para adherirse a células epiteliales bucales (CEB), sustratos no biológicos y su correlación con el mecanismo de interacción hidrofóbica. Se estimó la adherencia de A.b. a CEB, mediante recuento microscópico en 40 CEB, y la hidrofobicidad de la superficie celular mediante la acción de agentes fisicoquímicos y la capacidad de unión a compuestos hidrofóbicos. El 92% de las cepas se unió a CEB (p<0,001). El 35% lo hizo marcadamente frente al cloruro de polivinilo (PVC) y xileno; un mayor porcentaje se unió al poliestireno (PE), pero con intensidad variable entre las distintas cepas. Una sola cepa autoagregó formando, en cultivos estáticos, película cohesiva en la interfase aire-líquido; esta misma cepa presentó el mayor valor de agregación después de calentar 1h a temperatura de ebullición. Los fenotipos hidrofóbicos agregaban a menor concentración de sulfato de amonio, excepto una cepa mucosa de A.b.. Hubo correlación en el comportamiento hidrofóbico e hidrofílico entre las distintas cepas. Solo una cepa se comportó como no adherente frente a los sustratos biológicos y no biológicos ensayados. Las diferentes características de superficie de las cepas estudiadas podría ser explicado por la heterogeneidad de la superficie celular, según se observa en los perfiles de membrana externa. Los datos indicarían que si bien la hidrofobicidad sola no podría ser considerada un factor relevante en la adherencia a CEB, es posible que tenga un efecto importante cuando se considera junto a otros factores, tales como carga de superficie, concentración iónica del medio y tensión interfasial

Propiedades de superficie y adherencia de cepas de Acinetobacter baumannii / Surface characteristics and strains adherence of the Acetinetobacter baumannii

En este trabajo se estudió la capacidad que presentaban 14 cepas de Acinetobacter baumannii (A.b.) para adherirse a células epiteliales bucales (CEB), sustratos no biológicos y su correlación con el mecanismo de interacción hidrofóbica. Se estimó la adherencia de A.b. a CEB, mediante recuento microscópico en 40 CEB, y la hidrofobicidad de la superficie celular mediante la acción de agentes fisicoquímicos y la capacidad de unión a compuestos hidrofóbicos. El 92% de las cepas se unió a CEB (p<0,001). El 35% lo hizo marcadamente frente al cloruro de polivinilo (PVC) y xileno; un mayor porcentaje se unió al poliestireno (PE), pero con intensidad variable entre las distintas cepas. Una sola cepa autoagregó formando, en cultivos estáticos, película cohesiva en la interfase aire-líquido; esta misma cepa presentó el mayor valor de agregación después de calentar 1h a temperatura de ebullición. Los fenotipos hidrofóbicos agregaban a menor concentración de sulfato de amonio, excepto una cepa mucosa de A.b.. Hubo correlación en el comportamiento hidrofóbico e hidrofílico entre las distintas cepas. Solo una cepa se comportó como no adherente frente a los sustratos biológicos y no biológicos ensayados. Las diferentes características de superficie de las cepas estudiadas podría ser explicado por la heterogeneidad de la superficie celular, según se observa en los perfiles de membrana externa. Los datos indicarían que si bien la hidrofobicidad sola no podría ser considerada un factor relevante en la adherencia a CEB, es posible que tenga un efecto importante cuando se considera junto a otros factores, tales como carga de superficie, concentración iónica del medio y tensión interfasial