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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.

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We report here a draft genome assembly of Lacticaseibacillus rhamnosus CRL 2244, recovered from wastewater in Argentina. The genome has a size of 2,898,100 bp, with G + C content of 46.73%. Comparative analysis reveals that its closest relative is L. rhamnosus 1.0320 (GCF_006151905.1), with an average nucleotide identity of 97.46%.

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Carbapenem-resistant Acinetobacter baumannii (CRAB) is a recognized nosocomial pathogen with limited antibiotic treatment options. Lactic acid bacteria (LAB) constitute a promising therapeutic alternative. Here we studied the antibacterial properties of a collection of LAB strains using phenotypic and transcriptomic analysis against A. baumannii clinical strains. One strain, Lacticaseibacillus rhamnosus CRL 2244, demonstrated a potent inhibitory capacity on A. baumannii with a significant killing activity. Scanning electron microscopy images showed changes in the morphology of A. baumannii with an increased formation of outer membrane vesicles. Significant changes in the expression levels of a wide variety of genes were also observed. Interestingly, most of the modified genes were involved in a metabolic pathway known to be associated with the survival of A. baumannii. The paa operon, Hut system, and fatty acid degradation were some of the pathways that were induced. The analysis reveals the impact of Lcb. rhamnosus CRL 2244 on A. baumannii response, resulting in bacterial stress and subsequent cell death. These findings highlight the antibacterial properties of Lcb. rhamnosus CRL 2244 and its potential as an alternative or complementary strategy for treating infections. Further exploration and development of LAB as a treatment option could provide valuable alternatives for combating CRAB infections.

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The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.

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Between 2015 and 2019, we hosted an International Phage Course at Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. The 2-week full-time course was hands-on and included lectures from renowned phage biologists. Participating students were able to meet and discuss with recognized experts from around the world in a familiar setting, facilitating the establishment of scientific collaborations and the expansion of their networks. Eighty-four students from 14 Latin American countries have participated in the course, which included isolation, characterization, genome sequencing, and annotation of novel phages. We have successfully created a coursework that enabled the acquisition of new knowledge and expertise in bacteriophage biology and strengthened ties among Latin American colleagues.

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Introduction: Because of the clinical relevance of Mycobacteria, and from a therapeutic perspective, there is an increasing interest to study phages that infect bacteria belonging to this genus. Materials and Methods: A phage was isolated from a soil sample, using Mycobacterium smegmatis as host. Its characterization included sequencing, annotation, and analysis of the genome, host range determination, and electron microscopy imaging. Results: Mycobacterium phage vB_MsmS_Celfi is a temperate phage able to infect Mycobacterium tuberculosis with high efficiency. From electron microscopy images, Celfi belongs to the Siphoviridae family. Genome analysis classified phage Celfi into cluster L, subcluster L2 of Actinobacteriophage clusters. Mycobacterium phage Celfi exhibits a Lysin B distant to those present in other members of the subcluster and other mycobacteriophages. Conclusions: The discovery of new phages that infect M. tuberculosis could contribute to the development of novel tools for detection systems and future treatment of the disease.

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Introduction: Only a few Lactobacillus casei phages have so far been characterized. As several L. casei strains are part of probiotic formulations, bacteriophage outbreaks targeting these strains can lead to critical losses within the dairy industry. Materials and Methods: A new L. casei phage was isolated from raw milk obtained from a milking yard from the province of Buenos Aires. The phage genome was sequenced, annotated, and analyzed. Morphology was determined by electron microscopy and the host range was established. Results: Lactobacillus phage vB_LcaM_Lbab1 is a member of the Herelleviridae family and features a host range including L. casei/Lactobacillus paracasei and Lactobacillus kefiri strains. We further analyzed the baseplate proteins in silico and found putative carbohydrate binding modules that are responsible for host recognition in other Lactobacillus phages. Conclusions: A new Lactobacillus phage was isolated and characterized. The focus was made on its host recognition mechanism, pointing toward the development of future strategies to avoid deleterious infections in the dairy industry.

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The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points •F. tropaeoli CRL 2034 harbors three ldh genes in its genome. •ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. •Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. •Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.

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We report the draft genome sequence of Fructobacillus tropaeoli CRL 2034, a strain isolated from ripe fig in Tucumán province, Argentina. The interest in studying the genome of this fructophilic lactic acid bacterium strain was motivated by its ability to produce high levels of mannitol from fructose. This polyol has multiple industrial applications; however, it is mainly used as low calorie sugar in the food industry. The assembled genome of this strain consists of a 1.66-Mbp circular chromosome with 1465 coding sequences and a G+C content of 44.6%. The analysis of this genome supports the one step reaction of fructose reduction to mannitol by the mannitol 2-dehydrogenase enzyme, which together with a fructose permease, were identified as involved in mannitol synthesis. In addition, a phylogenetic analysis was performed including other Leuconostocaceae members to which the Fructobacillus genus belongs to; according to the 16S rRNA gene sequences, the strain CRL 2034 was located in the Fructobacillus clade. The present genome sequence could be useful to further elucidate regulatory processes of mannitol and other bioactive metabolites and to highlight the biotechnological potential of this fruit-origin Fructobacillus strain.

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