Staphylococcal trafficking and infection-from 'nose to gut' and back

Staphylococcus aureus is an opportunistic human pathogen, which is a leading cause of infections worldwide. The challenge in treating S. aureus infection is linked to the development of multidrug-resistant strains and the mechanisms employed by this pathogen to evade the human immune defenses. In addition, S. aureus can hide asymptomatically in particular ‘protective’ niches of the human body for prolonged periods of time. In the present review, we highlight recently gained insights in the role of the human gut as an endogenous S. aureus reservoir next to the nasopharynx and oral cavity. In addition, we address the contribution of these ecological niches to staphylococcal transmission, including the roles of particular triggers as modulators of the bacterial dissemination. In this context, we present recent advances concerning the interactions between S. aureus and immune cells to understand their possible roles as vehicles of dissemination from the gut to other body sites. Lastly, we discuss the factors that contribute to the switch from colonization to infection. Altogether, we conclude that an important key to uncovering the pathogenesis of S. aureus infection lies hidden in the endogenous staphylococcal reservoirs, the trafficking of this bacterium through the human body and the subsequent immune responses

Time-resolved analysis of Staphylococcus aureus invading the endothelial barrier

Staphylococcus aureus is a leading cause of infections world-wide. Once this pathogen has reached the bloodstream, it can invade different parts of the human body by crossing the endothelial barrier. Infected endothelial cells may be lysed by bacterial products, but the bacteria may also persist intracellularly, where they are difficult to eradicate with antibiotics and cause relapses of infection. Our present study was aimed at investigating the fate of methicillin resistant S. aureus (MRSA) isolates of the USA300 lineage with different epidemiological origin inside endothelial cells. To this end, we established two in vitro infection models based on primary human umbilical vein endothelial cells (HUVEC), which mimic conditions of the endothelium when infection occurs. For comparison, the laboratory strain S. aureus HG001 was used. As shown by flow cytometry and fluorescence- or electron microscopy, differentiation of HUVEC into a cell barrier with cell-cell junctions sets limits to the rates of bacterial internalization, the numbers of internalized bacteria, the percentage of infected cells, and long-term intracellular bacterial survival. Clear strain-specific differences were observed with the HG001 strain infecting the highest numbers of HUVEC and displaying the longest intracellular persistence, whereas the MRSA strains reproduced faster intracellularly. Nonetheless, all internalized bacteria remained confined in membrane-enclosed LAMP-1-positive lysosomal or vacuolar compartments. Once internalized, the bacteria had a higher propensity to persist within the differentiated endothelial cell barrier, probably because internalization of lower numbers of bacteria was less toxic. Altogether, our findings imply that intact endothelial barriers are more likely to sustain persistent intracellular infection

Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis

Background: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. Results: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. Conclusions: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains

Fighting Acinetobacter baumannii infections with the acylase PvdQ

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections

Molecular typing and antimicrobial resistance profiling of 33 mastitis-related Staphylococcus aureus isolates from cows in the Comarca Lagunera region of Mexico

Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015-2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureus lineages, susceptible to most antimicrobials, were responsible for causing mastitis at the time of sampling. Lastly, many isolates carried the same small plasmid, designated pSAM1. The high prevalence of pSAM1 amongst the antimicrobial-susceptible isolates suggests an association with bovine colonization or mastitis rather than antimicrobial resistance

The effect of calcium palmitate on bacteria associated with infant gut microbiota

Abstract Gut microbiota development in formula‐fed and breast‐fed infants is known to differ. This could relate to the usage of unmodified vegetable oil instead of mammalian fat in infant formula (IF), causing the enhanced formation of the poorly soluble soap calcium palmitate (CP) in the infant's gut. Here we investigate in vitro the possible influence of CP on the infant gut bacteria. The growth of several bacterial species dominant in the infant's gut was analyzed by culturing in media with CP. Faecalibacterium prausnitzii as a sensitive representative was analyzed in detail by scanning transmission electron microscopy, membrane staining, gas chromatography, and microbial fuel cell experiments. Of all bacteria tested, the growth of several bifidobacteria and F. prausnitzii was reduced at 0.01 mg/ml CP, Bifidobacterium infantis stopped growing completely. CP reduced the cell envelope thickness of F. prausnitzii, disturbed the cell membrane fatty acids and function of membrane proteins involved in electron transport. CP inhibited the growth of bifidobacteria and faecalibacteria. This suggests that modification of fat in IF may benefit the development of the gut microbiota in formula‐fed infants by supporting the colonization of important beneficial bacteria in early life. Future clinical studies are needed to confirm this

An Efficient UV-C Disinfection Approach and Biological Assessment Strategy for Microphones

Featured Application Disinfection of microphones by using UV-C to be applied in the entertainment industry to prevent infections as a consequence of microphone sharing between individuals. Hygiene is a basic necessity to prevent infections, and though it is regarded as vital in general, its importance has been stressed again during the pandemic. Microbes may spread through touch and aerosols and thereby find their way from host to host. Cleaning and disinfection of possibly contaminated surfaces prevents microbial spread, thus reducing potential illnesses. One item that is used by several people in a way that promotes close contact by touch and aerosol formation is the microphone. A microphone is a complex piece of equipment with respect to shape and various materials used to fabricate it and, hence, its disinfection is challenging. A new device has been developed to efficiently sterilize microphones by using UV-C and a biological assessment has been done to identify its efficacy and translatability. For this investigation, a contamination procedure was developed by using M13 bacteriophage as a model to illustrate the effectiveness of the disinfection. The susceptibility to UV-C irradiation of M13 in solution was compared to that of the PR8 H1N1 influenza virus, which has a similar UV-C susceptibility as SARS-CoV-2. It was found that 10 min of UV-C treatment reduced the percentage of infectious M13 by 99.3% based on whole microphone inoculation and disinfection. UV-C susceptibility of M13 and influenza in suspension were found to be very similar, indicating that the microphone sterilization method and device function are highly useful and broadly applicable

Polymorphism, genetic exchange and intragenic recombination of the aureolysin gene among Staphylococcus aureus strains

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of <it>S. aureus</it>, relatively little information was so far available concerning the <it>aur </it>gene diversity and mobility within and between the major subdivisions of the <it>S. aureus </it>population. Therefore, an epidemiologically and genetically diverse collection of <it>S. aureus </it>strains was used to determine the range of aureolysin (<it>aur</it>) gene polymorphism.</p> <p>Results</p> <p>Sequence analyses support the conclusion that the <it>aur </it>gene occurs in two distinct types of related sequences. The <it>aur </it>gene was much more polymorphic but, at the same time, showed higher purifying selection than genes utilized for multilocus sequence typing (MLST). Gene trees constructed from <it>aur </it>and concatenated MLST genes revealed several putative assortative recombination events (<it>i.e</it>. entire <it>aur </it>gene exchanges) between divergent lineages of <it>S. aureus</it>. Evidence for intragenic recombination events (<it>i.e</it>. exchanges of internal <it>aur </it>segments) across <it>aur </it>genes was also found. The biochemical properties and substrate specificity of the two types of aureolysin purified to homogeneity were studied, revealing minor differences in their affinity to low molecular weight synthetic substrates.</p> <p>Conclusion</p> <p>Although numerous nucleotide differences were identified between the <it>aur </it>alleles studied, our findings showed that a strong purifying selection is acting on the <it>aur </it>gene. Moreover, our study distinguishes between homologous exchanges of the entire <it>aur </it>gene (assortative recombination) between divergent <it>S. aureus </it>lineages and recombination events within <it>aur </it>genes.</p

Microfluidic-Chip-Based Multiple-Locus Variable-Number Tandem-Repeat Fingerprinting with New Primer Sets for Methicillin-Resistant Staphylococcus aureus

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory