In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.

Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.

Improving phage therapy by evasion of phage resistance mechanisms.

Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.

Single-cell analysis reveals that cryptic prophage protease LfgB protects Escherichia coli during oxidative stress by cleaving antitoxin MqsA.

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role of MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.IMPORTANCEThe roles of toxin/antitoxin systems in cell physiology are few and include phage inhibition and stabilization of genetic elements; yet, to date, there are no single-transcriptome studies for toxin/antitoxin systems and few insights for prokaryotes from this novel technique. Therefore, our results with this technique are important since we discover and characterize a cryptic prophage protease that is regulated by the MqsR/MqsA toxin/antitoxin system in order to regulate the host response to oxidative stress.

Antimicrobial and antibiofilm activity of fungal metabolites on methicillin-resistant Staphylococcus aureus (ATCC 43300) mediated by SarA and AgrA.

Methicillin-resistant Staphylococcus aureus (MRSA) increases its antibiotic resistance by forming biofilms. Natural products (NP) or specialized metabolites have demonstrated their ability to decrease the virulence and pathogenesis of MRSA infections by inhibiting biofilm formation. The present study evaluated the antimicrobial and antibiofilm potential against MRSA of a small library of fungal NP isolated from Mexican biodiversity. The most potent antibacterial activity was observed for myrotecisin B, epiequisetin, equisetin, stachybotrolide acetate, monorden A, zearalenone, fuscin, and fusarubin. On the other hand, epifiscalin C, fiscalin C, dimethylglyotoxin, aspernolide B, and butyrolactones I and IV inhibited the biofilm formation without decreasing bacterial growth. To determine the putative mechanism of action of these compounds, docking analyses were performed against SarA and AgrA proteins, targets known to regulate biofilm production in MRSA. Overall, the results demonstrate that fungal NP may act as potential antibiofilm agents for treating MRSA infections.

Theoretical study of ArcB and its dimerization, interaction with anaerobic metabolites, and activation of ArcA.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Complete genome sequence of Paenibacillus sp. VCA1 isolated from crater lake of the El Chichón Volcano.

We report the complete genome of Paenibacillus sp. strain VCA1, which was isolated from sediment from El Chichón Volcano. This genome consists of 6,690,819 bp and 6,312 coding sequences, with 51.8% G+C content. Whole-genome sequencing was performed to explore the strain's biocontrol and plant growth promotion properties.

To cheat or not to cheat: cheatable and non-cheatable virulence factors in Pseudomonas aeruginosa.

Important bacterial pathogens such as Pseudomonas aeruginosa produce several exoproducts such as siderophores, degradative enzymes, biosurfactants, and exopolysaccharides that are used extracellularly, benefiting all members of the population, hence being public goods. Since the production of public goods is a cooperative trait, it is in principle susceptible to cheating by individuals in the population who do not invest in their production, but use their benefits, hence increasing their fitness at the expense of the cooperators' fitness. Among the most studied virulence factors susceptible to cheating are siderophores and exoproteases, with several studies in vitro and some in animal infection models. In addition to these two well-known examples, cheating with other virulence factors such as exopolysaccharides, biosurfactants, eDNA production, secretion systems, and biofilm formation has also been studied. In this review, we discuss the evidence of the susceptibility of each of those virulence factors to cheating, as well as the mechanisms that counteract this behavior and the possible consequences for bacterial virulence.

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Exoprotease exploitation and social cheating in a Pseudomonas aeruginosa environmental lysogenic strain with a noncanonical quorum sensing system.

Social cheating is the exploitation of public goods that are costly metabolites, like exoproteases. Exoprotease exploitation in Pseudomonas aeruginosa has been studied in reference strains. Experimental evolution with reference strains during continuous growth in casein has demonstrated that nonexoprotease producers that are lasR mutants are selected while they behave as social cheaters. However, noncanonical quorum-sensing systems exist in P. aeruginosa strains, which are diverse. In this work, the exploitation of exoproteases in the environmental strain ID4365 was evaluated; ID4365 has a nonsense mutation that precludes expression of LasR. ID4365 produces exoproteases under the control of RhlR, and harbors an inducible prophage. As expected, rhlR mutants of ID4365 behave as social cheaters, and exoprotease-deficient individuals accumulate upon continuous growth in casein. Moreover, in all continuous cultures, population collapses occur. However, this also sometimes happens before cheaters dominate. Interestingly, during growth in casein, ID4565's native prophage is induced, suggesting that the metabolic costs imposed by social cheating may increase its induction, promoting population collapses. Accordingly, lysogenization of the PAO1 lasR mutant with this prophage accelerated its collapse. These findings highlight the influence of temperate phages in social cheating.

Myriad applications of bacteriophages beyond phage therapy.

Bacteriophages are the most abundant biological entity on the planet, having pivotal roles in bacterial ecology, animal and plant health, and in the biogeochemical cycles. Although, in principle, phages are simple entities that replicate at the expense of their bacterial hosts, due the importance of bacteria in all aspects of nature, they have the potential to influence and modify diverse processes, either in subtle or profound ways. Traditionally, the main application of bacteriophages is phage therapy, which is their utilization to combat and help to clear bacterial infections, from enteric diseases, to skin infections, chronic infections, sepsis, etc. Nevertheless, phages can also be potentially used for several other tasks, including food preservation, disinfection of surfaces, treatment of several dysbioses, and modulation of microbiomes. Phages may also be used as tools for the treatment of non-bacterial infections and pest control in agriculture; moreover, they can be used to decrease bacterial virulence and antibiotic resistance and even to combat global warming. In this review manuscript we discuss these possible applications and promote their implementation.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

Efecto del extracto hexánico de Tagetes lucida sobre la inhibición del crecimiento de enterobacterias Shigella flexneri y Salmonella typhi / Effect of the hexanic extract of Tagetes lucida on the inhibition of the growth of enterobacteria Shigella flexneri and Salmonella typhi

La resistencia a los antibióticos aumenta la búsqueda de nuevas estrategias para combatir las enfermedades que causan, y el uso de plantas medicinales representa una estrategia altamente efectiva y valiosa, como el uso de Tagetes lucida con diferentes bacterias gram positivas y gram negativas.Objetivo: Evaluar la actividad biológica que tiene el extracto hexanico de la planta Tagetes lucida a diferentes concentraciones sobre la inhibición del crecimiento en placa y tubo de dos enterobacterias, Shigella flexneri y Salmonella typhiMétodos: En el siguiente trabajo, se evaluó un extracto de hexano de Tagetes lucida sobre la inhibición del crecimiento de dos enterobacterias, Shigella flexneri y Salmonella typhi utilizando diferentes concentraciones de vehículo para evaluar si afectaba el crecimiento bacteriano y también diferentes concentraciones de extracto para evaluar la actividad.Resultados: Realizados los estudios por triplicado se logró concretar que a partir de 75µl/µg de extracto se logra una inhibición casi total del crecimiento de ambas bacterias, tanto en método de placa, como en método de tubo. Y a partir de 100 µl/µg se logra una inhibición total.Conclusiones: Los resultados favorables obtenidos con 75 µl/µg, permiten confirmar que los extractos de plantas medicinales son una estrategia importante para combatir infecciones bacterianas multi-resistentes. Por otro lado permite dar paso a un estudio para evaluar los metabolitos más activos del extracto, así como, el mecanismo de acción sobre la inhibición del crecimiento de las bacterias en estudio. (AU)

Antibiotic resistance increases the search for new strategies to combat the diseases they cause, and the use of medicinal plants represents a highly effective and valuable strategy, such as the use of Tagetes lucida with different gram positive and gram negative bacteria.Objective: To evaluate the biological activity of the hexane extract of the Tagetes lucida plant at different concentrations on the inhibition of growth in plaque and tube of two enterobacteriaceae, Shigella flexneri and Salmonella typhiMethods: In the following work, a hexane extract from Tagetes lucida was evaluated on the growth inhibition of two enterobacteriaceae, Shigella flexneri and Salmonella typhi using different concentrations of vehicle to evaluate if it affected bacterial growth and also different concentrations of extract to evaluate activity.Results: Once the studies were carried out in triplicate, it was possible to specify that from 75µl/µg of extract, almost total inhibition of the growth of both bacteria was achieved, both in the plate method and in the tube method. And from 100 µl/µg total inhibition is achieved.Conclusions: The favorable results obtained with 75 µl/ µg, confirm that medicinal plant extracts are an important strategy to combat multi-drug resistant bacterial infections. On the other hand, it allows a study to be carried out to evaluate the most active metabolites of the extract, as well as the mechanism of action on the inhibition of the growth of the bacteria under study. (AU)

Cultivation of gastrointestinal microbiota in a new growth system revealed dysbiosis and metabolic disruptions in carcinoma-bearing rats.

A challenge in the study of gastrointestinal microbiota (GITm) is the validation of the genomic data with metabolic studies of the microbial communities to understand how the microbial networks work during health and sickness. To gain insights into the metabolism of the GITm, feces from healthy and sick rats with cancer were inoculated in a defined synthetic medium directed for anaerobic prokaryote growth (INC-07 medium). Significant differences between cultures of healthy and sick individuals were found: 1) the consumption of the carbon source and the enzyme activity involved in their catabolism (e.g., sucrase, lactase, lipases, aminotransferases, and dehydrogenases); 2) higher excretion of acetic, propionic, isobutyric, butyric, valeric, and isovaleric acids; 3) methane production; 4) ability to form biofilms; and 5) up to 500 amplicon sequencing variants (ASVs) identified showed different diversity and abundance. Moreover, the bowel inflammation induced by cancer triggered oxidative stress, which correlated with deficient antioxidant machinery (e.g., NADPH-producing enzymes) determined in the GITm cultures from sick individuals in comparison with those from control individuals. Altogether, the data suggested that to preserve the microbial network between bacteria and methanogenic archaea, a complete oxidation of the carbon source may be essential for healthy microbiota. The correlation of 16S rRNA gene metabarcoding between cultures and feces, as well as metabolomic data found in cultures, suggest that INC-07 medium may be a useful tool to understand the metabolism of microbiota under gut conditions.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Corrigendum: The Immune Response Against Acinetobacter baumannii, an Emerging Pathogen in Nosocomial Infections.

[This corrects the article DOI: 10.3389/fimmu.2017.00441.].