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Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo . We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro , ex vivo , and in vivo . To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections. Synopsis A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus . The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo . Mycoplasma pneumoniae offers unique features that might be of interest for a bacterial‐based therapeutic vector. Here, an attenuated version of this bacterium is generated after studying the role of different pathogenic factors. Using strong synthetic promoters and native secretion signals, a genetic platform is designed coding for antibiofilm and bactericidal enzymes. In vitro , ex vivo and in vivo studies confirmed the ability of the engineered M. pneumoniae strain to efficiently dissolve S. aureus biofilms. Graphical Abstract A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus . The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo .

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B . abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×10 9 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

Abortion and reproductive failures induced by Brucella are the main symptoms of animal brucellosis. Laboratory animal models are essential tools of research to study the Brucella pathogenesis before experimentation in natural hosts. To extend the existing knowledge, we studied B. melitensis 16M (virulent) and Rev1 (attenuated) as well as B. suis bv2 infections in pregnant mice. Here, we report new information about kinetics of infection (in spleens, blood, placentas, vaginal shedding, and foetuses), serum cytokine profiles, and histopathological features in placentas and the litter throughout mice pregnancy. Both B. melitensis strains showed a marked placental tropism and reduced viability of pups (mainly in 16M infections), which was preceded by an intense Th1-immune response during placental development. In contrast, B. suis bv2 displayed lower placental tropism, mild proinflammatory immune response, and scarce bacterial transmission to the litter, thus allowing foetal viability. Overall, our studies revealed three different smooth Brucella patterns of placental and foetal pathogenesis in mice, providing a useful animal model for experimental brucellosis.

The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

The brucellae are α-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-α release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

Extensive pig systems are gaining importance as quality production systems and as the standard for sustainable rural development and animal welfare. However, the effects of natural foods on Salmonella epidemiology remain unknown. Herein, we assessed the presence of Salmonella and the composition of the gut microbiota in pigs from both Salmonella-free and high Salmonella prevalence farms. In addition, risk factors associated with the presence of Salmonella were investigated. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), and exhibited a multi-drug resistance profile (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos’ cleaning/disinfection as the main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative populations, showing the former with increasing Proteobacteria and decreasing Bacteroides populations. Butyrate and propionate producers including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactobacillus were more abundant in the Salmonella-negative group, whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs is directly related to the natural vegetation accessible, determining the composition of the intestinal microbiota.

Antimicrobial resistance is a major global health threat, potentially causing 8.22 million deaths annually by 2050. Polymicrobial biofilms significantly contribute to this crisis, leading to treatment failure, especially in chronic airway infections caused by Staphylococcus aureus and Pseudomonas aeruginosa , common in ventilator-associated pneumonia and cystic fibrosis. To address this, we engineered an attenuated Mycoplasma pneumoniae strain, CV8_HAD, to secrete biofilm-disrupting enzymes (PelAh, PslGh, A1-II’ and Dispersin B). CV8_HAD showed strong in vitro activity against single and mixed-species biofilms of S. aureus and P. aeruginosa , and demonstrated in vivo efficacy against S. aureus biofilms in mice and mixed infections in Galleria mellonella larvae. This study establishes engineered M. pneumoniae as a promising therapeutic strategy for tackling microbial biofilms and highlights the potential of G. mellonella larvae models as an alternative to mouse models in advancing research on biofilm-targeting interventions.