FlhF Is Required for Swarming Motility and Full Pathogenicity of Bacillus cereus

Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g., B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g., flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae. Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Functional analysis of bc5181 in Bacillus cereus

Previous studies on the systematic examination of the global transcriptional response of Bacillus cereus to disinfectant treatments showed a common up-regulation of stress-related genes. In this study, we focused our attention on one of these genes (bc5181) that encodes a protein belonging to the YbjQ-like superfamily. Since bc5181 expression was shown to be induced by mild or lethal concentrations of peracetic-acid (PAA), we examined its role under PAA treatment by evaluating the phenotypic response of B. cereus strains lacking or overexpressing this gene. A bc5181 null mutant (MP23) and a strain carrying an additional IPTG-inducible copy of this gene (MP25) were constructed from B. cereus ATCC 14579 and comparatively assayed. Treatment of mid-exponential phase cells with two PAA concentrations revealed that the lack or overexpression of bc5181 have no significant effect on bacterial survival to the mild PAA concentration. However, when the lethal concentration was used, a high resistance to PAA was observed in the strain MP25 compared to the wild-type, more pronouncedly following IPTG induction. Unexpectedly, a lower increase in the resistance to the lethal PAA concentration was also observed in the bc5181 null mutant MP23 compared to the wild type. To evaluate whether deprivation of bc5181 or increase in its expression could have other effects on B. cereus physiology, bacterial growth, carbohydrate utilization, antibiotic resistance, motility, chemotactic response, as well as virulence and pathogenicity of all strains were evaluated. Moreover, the effect of heterologous expression of bc5181 in B. subtilis, a microorganism naturally lacking a bc5181 homologue, was investigated. While no significant effect of bc5181 on bacterial growth, biochemistry and antibiotic resistance was evidenced, bc5181 overexpression was shown to cause a defect in swimming motility, chemotactic response toward the chemo-attractant L-glutamine, and swarming motility. In addition, increase in bc5181 expression level caused a significant reduction in B. cereus pathogenicity in an in vivo animal model of infection using the lepidoteran Galleria mellonella and in the production of some relevant B. cereus virulence factors, such as phosphatidylcholine-preferring phospholipase C (PC-PLC), proteases and general haemolysins. Heterologous expression of bc5181 in B. subtilis was shown to increase the production of proteases and the propensity to develop biofilm communities by this organism. In conclusion, bc5181 appears to be a non-essential stress-related gene whose overexpression increases resistance to PAA treatment, causes alterations in the motility and chemotactic behaviors, and leads to a reduction in B. cereus pathogenicity and virulence. The observation that heterologous bc5181 expression in a microorganism lacking ybjQ-like genes causes an increase in protease secretion and biofilm formation is also relevant, and further support a role of this gene in exerting a pleiotropic effect on bacterial cells

Allestimento di un mutante nel gene ybjQ di Bacillus cereus e sua caratterizzazione fenotipica

Bacillus cereus è un microrganismo Gram-positivo, sporigeno che si comporta da patogeno/patogeno opportunista per l’uomo. In quanto dotato di flagelli disposti su tutta la superficie batterica, questo microrganismo è capace di muoversi in mezzo liquido mediante motilità swimming. Inoltre, quando propagato su superfici con una appropriata concentrazione di nutrienti e viscosità, B. cereus è in grado di muoversi mediante swarming. Lo swarming rappresenta un particolare tipo di motilità cooperativa che prevede un profondo processo differenziativo cellulare che porta alla produzione di cellule swarm, più lunghe e dotate di un maggior numero di flagelli rispetto alle forme planctoniche. Le cellule swarm di B. cereus sono caratterizzate dalla capacità di sintetizzare/secernere una maggiore quantità di fattori di virulenza e contribuiscono alla patogenicità di questo microrganismo. Nonostante si ritenga che lo swarming rappresenti un fenomeno complesso che richiede l’alterata espressione di molti geni implicati in numerose funzioni cellulari, i meccanismi che portano alla produzione di cellule swarm in B. cereus rimangono quasi del tutto sconosciuti. In B. subtilis, la specie più studiata del genere Bacillus, è stato dimostrato che la proteina codificata dal gene swrA ha un ruolo importante nella regolazione del numero di flagelli ed è assolutamente necessario per il differenziamento swarming di questa specie batterica. La presente tesi si è prefissa lo scopo di valutare se in B. cereus esistesse un gene omologo a swrA di B. subtilis e se tale gene, eventualmente presente, fosse coinvolto nel differenziamento swarming e/o nella motilità in mezzo liquido di questo microrganismo. Mediante un’approfondita analisi in silico è stata identificata una ORF, denominata ybjQ, mostrante un certo grado di omologia con swrA di B. subtilis. Al fine di generare un mutante mancante di ybjQ, sono stati disegnati dei primer per amplificare due regioni genomiche fiancheggianti il gene ybjQ e comprendenti una porzione ybjQ ciascuna. Gli amplificati di PCR sono stati clonati in opportuni vettori plasmidici e, successivamente, in un vettore shuttle capace di replicarsi sia nei microrganismi Gram-negativi che Gram-positivi. Nella miscela di ligazione è stato incluso anche un frammento di DNA, preparato mediante digestione di uno specifico plasmide con opportuni enzimi di restrizione, portante una “cassetta genica” capace di conferire resistenza alla kanamicina. Con il prodotto di ligazione sono state trasformate cellule di E. coli, dalle quali il plasmide ottenuto è stato purificato ed impiegato per trasformare B. cereus. I ceppi trasformanti di B. cereus sono stati curati dal plasmide facendo crescere i microrganismi a temperatura non permissiva per la replicazione del plasmide e selezionando con aggiunta di kanamicina al terreno di coltura. Ciò favoriva la selezione di ceppi di B. cereus in cui fosse avvenuta ricombinazione tra la porzione genomica portante il gene ybjQ integro e la stessa porzione presente nel plasmide portante il gene ybjQ interrotto dalla “cassetta genica”. Mediante questi esperimenti è stato ottenuto un ceppo di B. cereus mutante nel gene ybjQ. E’ stata, quindi, effettuata una indagine comparativa per valutare quali difetti fenotipici potessero essere presenti nel ceppo mutante rispetto al ceppo parentale. In particolare è stato valutato: la velocità di crescita in diversi terreni, la motilità cellulare in mezzo liquido, la quantità di flagelli prodotti sia in mezzo liquido che su terreno solido, la lunghezza cellulare e il profilo delle proteine totali di secrezione e di quelle citoplasmatiche. I risultati finora ottenuti indicano che vi sono profonde differenze tra le funzioni che i geni ybjQ e swrA esercitano rispettivamente in B. cereus e in B. subtilis e che il gene ybjQ non sembra avere un ruolo significativo nella motilità e nel differenziamento swarming di B. cereus

Antimicrobial Activity of a New Preservative for Multiuse Ophthalmic Solutions

Purpose: The aim of this study was to examine the antimicrobial activity and the preservative efficacy of a novel preservative solution containing sodium hydroxymethyl glycinate (SHMG) and edetate disodium (EDTA), which is used for preservation of some commercial ophthalmic formulations. Methods: In vitro susceptibility assays were performed against several gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus cereus) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria representative of the microbial flora of epithelial surfaces or colonizing the conjunctiva, as well as against Candida albicans and Aspergillus niger. Using different concentrations of SHMG alone or in combination with EDTA, the minimal inhibitory and microbicidal concentrations against these organisms were assessed. In addition, 8 brands of multidose eye drops containing 0.002% SHMG and 0.1% EDTA as preservative were tested for antimicrobial activity using the antimicrobial effectiveness test recommended by the international pharmacopoeias. Results: The minimal inhibitory and bactericidal/fungicidal concentration values of SHMG ranged from 0.0025% to 0.0125% for bacteria and from 0.125% to 0.50% for mold and yeast. Susceptibility testing demonstrated that the addition of EDTA substantially increased the SHMG activity against all bacterial and fungal strains. The preservative effectiveness test was applied to commercial eye drops. All the drop solutions met the criteria reported by the U.S. Pharmacopeia for parenteral and ophthalmic preparations. All products also satisfied the major acceptance criteria of the European Pharmacopeia with respect to the antifungal activity. With regard to the antibacterial activity, the less-stringent criteria of the European Pharmacopeia were fulfilled. Conclusions: The present study demonstrates the efficacy of a novel preservative for ophthalmic solutions (SHMG/EDTA) and its activity in protecting selected commercial artificial tears against microbial contamination

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance

Frequency of isolation of <i>Bacillus</i> and <i>Paenibacillus</i> species from deep, usually sterile body sites or superficial and/or non-sterile body sites.

<p>Frequency of isolation of <i>Bacillus</i> and <i>Paenibacillus</i> species from deep, usually sterile body sites or superficial and/or non-sterile body sites.</p

Percentage of bacteria correctly identified to the species level by API 50 CHB and MALDI-TOF MS.

<p>Percentage of bacteria correctly identified to the species level by API 50 CHB and MALDI-TOF MS.</p

Percentages of strains among clinical <i>Bacillus</i> and <i>Paenibacillus</i> isolates showing virulence-related behaviors.

<p>Percentages of strains among clinical <i>Bacillus</i> and <i>Paenibacillus</i> isolates showing virulence-related behaviors.</p

Percentages of strains among clinical <i>Bacillus</i> and <i>Paenibacillus</i> isolates producing extracellular toxins/enzymes or possessing virulence genes.

<p>Percentages of strains among clinical <i>Bacillus</i> and <i>Paenibacillus</i> isolates producing extracellular toxins/enzymes or possessing virulence genes.</p