Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended‐spectrum β‐lactamases

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended‐spectrum β‐lactamase (ESBL)‐positive Klebsiella pneumoniae producing CTX‐M‐15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, β‐lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital‐associated materials was analysed. CTX‐M‐15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY‐2, DHA‐1). Independent of plasmid‐mediated beta‐lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC‐producing isolates. In conclusion, ESBL‐producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL‐producing E. coli strains.     

Evaluation of phytochemicals from medicinal plants of Myrtaceae family on virulence factor production by Pseudomonas aeruginosa

Virulence factors regulated by quorum sensing (QS) play a critical role in the pathogenesis of an opportunistic human pathogen, Pseudomonas aeruginosa in causing infections to the host. Hence, in the present work, the anti‐virulence potential of the medicinal plant extracts and their derived phytochemicals from Myrtaceae family was evaluated against P. aeruginosa. In the preliminary screening of the tested medicinal plant extracts, Syzygium jambos and Syzygium antisepticum demonstrated a maximum inhibition in QS‐dependent violacein pigment production by Chromobacterium violaceum DMST 21761. These extracts demonstrated an inhibitory activity over a virulence factor, pyoverdin, production by P. aeruginosa ATCC 27853. Gas chromatography–mass spectrometric (GC‐MS) analysis revealed the presence of 23 and 12 phytochemicals from the extracts of S. jambos and S. antisepticum respectively. Three top‐ranking phytochemicals, including phytol, ethyl linoleate and methyl linolenate, selected on the basis of docking score in molecular docking studies lowered virulence factors such as pyoverdin production, protease and haemolytic activities of P. aeruginosa to a significant level. In addition, the phytochemicals reduced rhamnolipid production by the organism. The work demonstrated an importance of plant‐derived compounds as anti‐virulence drugs to conquer P. aeruginosa virulence towards the host.     

Detection and characterization of a bacteriocin,putadicin T01, produced by Pseudomonas putida isolated from hot spring water

Pseudomonas strains isolated from hot spring water were tested for bacteriocin‐like substance (BLS) production using a target panel of closely related microorganisms and other Gram‐positive and Gram‐negative bacteria. Molecular identification was carried out through specific PCR and 16S RNA sequence analysis. Isolates were identified as Brevundimonas diminuta and Pseudomonas putida, the latter exhibited antimicrobial activity. Pseudomonas putida strains produce an inhibitory substance against other Pseudomonas strains and other species including food‐borne pathogens. The BLS was sensitive to the proteolytic action of proteinase K, pronase E and trypsin but resistant to α‐amylase, RNase and lipase C, reflecting its proteinaceous nature. The BLS was stable at 100 °C and also after thermal treatment at 121 °C for 15 min. Additionally, it was stable within a wide range of pH (2–10). The substance from P. putida T01 strain was bactericidal to Escherichia coli. SDS‐PAGE analysis of the partial purified supernatant of strain T01 revealed a BLS with an approximate molecular mass of 8 kDa. Therefore, the results of this study show that P. putida strain T01 produces a BLS with a higher activity spectrum, which may find application in human medicine and in minimally processed food preservation.     

A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires’ disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin‐containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow‐derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow‐derived macrophages other than the Fla mutant. Less legA9 mutant‐containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila‐containing vacuole for autophagy uptake.     

Wingless signaling regulates winner/loser status in Minute cell competition

Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild‐type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, we established two assay systems for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3‐RNAi‐expressing cells) system in which RpS3‐RNAi‐expressing wing pouch cells are eliminated from wild‐type wing disc and (ii) the SURE (supercompetition of RpS3‐expressing clones in RpS3/+ tissue) system in which RpS3‐over‐expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over‐expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down‐regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, we found that down‐regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. Our observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition.     

Brucella,nitrogen and virulence

Abstract

The brucellae are α-Proteobacteria causing brucellosis, an important zoonosis. Although multiplying in endoplasmic reticulum-derived vacuoles, they cause no cell death, suggesting subtle but efficient use of host resources. Brucellae are amino-acid prototrophs able to grow with ammonium or use glutamate as the sole carbon–nitrogen source in vitro. They contain more than twice amino acid/peptide/polyamine uptake genes than the amino-acid auxotroph Legionella pneumophila, which multiplies in a similar vacuole, suggesting a different nutritional strategy. During these two last decades, many mutants of key actors in nitrogen metabolism (transporters, enzymes, regulators, etc.) have been described to be essential for full virulence of brucellae. Here, we review the genomic and experimental data on Brucella nitrogen metabolism and its connection with virulence. An analysis of various aspects of this metabolism (transport, assimilation, biosynthesis, catabolism, respiration and regulation) has highlighted differences and similarities in nitrogen metabolism with other α-Proteobacteria. Together, these data suggest that, during their intracellular life cycle, the brucellae use various nitrogen sources for biosynthesis, catabolism and respiration following a strategy that requires prototrophy and a tight regulation of nitrogen use.     

The YvqE two‐component system controls biofilm formation and acid production in Streptococcus pyogenes

In Streptococcus pyogenes, proteins involved in determining virulence are controlled by stand‐alone response regulators and by two‐component regulatory systems. Previous studies reported that, compared to the parental strain, the yvqE sensor knockout strain showed significantly reduced growth and lower virulence. To determine the function of YvqE, we performed biofilm analysis and pH assays on yvqE mutants, and site‐directed mutagenesis of YvqE. The yvqE deletion mutant showed a slower acid production rate, indicating that YvqE regulates acid production from sugar fermentation. The mutant strain, in which the Asp²⁶ residue in YvqE was replaced with Asn, affected biofilm formation, suggesting that this amino acid senses hydrogen ions produced by fermentative sugar metabolism. Signals received by YvqE were directly or indirectly responsible for inducing pilus expression. This study shows that at low environmental pH, biofilm formation in S. pyogenes is mediated by YvqE and suggests that regulation of pilus expression by environmental acidification could be directly under the control of YvqE.     

Evidence of interspecies O antigen gene cluster transfer between Shigella boydii 15 and Escherichia fergusonii

An environmental bacterial isolate, Iso10, previously found to show serological cross‐reactivity with type‐specific Shigella boydii 15 antisera was subjected to further molecular and serological analyses that revealed interspecies transfer of the O antigen gene cluster. Western blot analysis of Iso10 cell surface extracts and purified lipopolysaccharides demonstrated strong cross‐reactivity with S. boydii 15‐specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. boydii 15. Biochemical and phylogenetic analyses identified the Iso10 isolate as Escherichia fergusonii. O antigen gene cluster analyses of Iso10, carried out by restriction fragment length analysis of the amplified ~10‐kb O antigen‐encoding gene cluster, revealed a profile highly similar to that of S. boydii 15, confirming the presence of the S. boydii 15 somatic antigen in Iso10. To the best of our knowledge, this is the first report of interspecies transfer of O antigen‐encoding genes between S. boydii and E. fergusonii, and it has implications for our understanding of the role of lateral gene transfer in the emergence of novel Shigella serotypes.     

Molecular characteristics of Staphylococcus aureus associated with chronic rhinosinusitis

The anterior nares have been regarded as the major carriage site of Staphylococcus aureus. From here, the organism can spread to other parts of the body where it might act as harmless commensal or cause mild to severe infections. Nasal sinuses are normally sterile, but in patients with chronic rhinosinusitis (CRS), the finding of S. aureus in maxillary sinus cultures is common. Isolates were obtained from the nares and maxillary sinus of patients with CRS and the nares of healthy controls. A significantly higher frequency of S. aureus was found in nares samples from patients (24/42) compared to controls (16/57) (p = 0.004). There is no consensus regarding whether S. aureus is a relevant pathogen in CRS. A DNA microarray was used to investigate the prevalence of S. aureus virulence genes with focus on staphylococcal enterotoxins, toxic shock syndrome toxin‐1, agr types, and cell wall‐associated proteins. The genotyping of S. aureus isolates revealed only small and non‐significant differences in gene prevalence between isolates collected from patients with CRS and those collected from healthy nasal carriers. This study provides an increased knowledge of the genetic pattern of virulence genes among S. aureus collected in CRS.     

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.     

Pathogenic potential and genetic diversity of environmental and clinical isolates of Pseudomonas aeruginosa

The aim of this study was to investigate the occurrence of virulence genes among clinical and environmental isolates of Pseudomonas aeruginosa and to establish their genetic relationships by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC‐PCR). A total of 60 P. aeruginosa isolates from environmental and clinical sources were studied. Of these, 20 bacterial isolates were from soil, 20 from water, and 20 from patients with cystic fibrosis. Analysis of ERIC‐PCR demonstrated that the isolates of P. aeruginosa showed a considerable genetic variability, regardless of their habitat. Numerous virulence genes were detected in both clinical and environmental isolates, reinforcing the possible pathogenic potential of soil and water isolates. The results showed that the environmental P. aeruginosa has all the apparatus needed to cause disease in humans and animals.     

Surface proteins of Staphylococcus aureus play an important role in experimental skin infection

Staphylococcus aureus is the most common cause of skin infections that range from mild diseases up to life‐threatening conditions. Mechanisms of S. aureus virulence in those infections remain poorly studied. To investigate the impact of S. aureus surface proteins on skin infection, we used mouse models of skin abscess formation and skin necrosis, induced by a subcutaneous injection of bacteria. In the skin abscess model, a sortase‐deficient S. aureus strain lacking all of its cell‐wall anchored proteins was less virulent than its wild‐type strain. Also, strains specifically lacking protein A, fibronecting binding proteins, clumping factor A or surface protein SasF were impaired in their virulence. When a model of dermonecrosis was studied, the S. aureus surface proteins could not be shown to be involved. In summary, surface proteins play an important role in virulence of S. aureus skin abscess infections, but not in formation of skin necrosis.     

The influence of a glucosyltransferase,encoded by gtfP,on biofilm formation by Streptococcus sanguinis in a dual‐species model

Streptococcus sanguinis is an early colonizer of tooth surfaces and forms biofilms with other species of microorganisms. In vitro, S. sanguinis produces water‐soluble glucans from sucrose and releases them into the culture supernatant; however, the role played by these glucans in biofilm formation is unclear. The present study examined both the effect of glucans on biofilm formation by S. sanguinis and the proportion of this bacterial species within the biofilms. Inactivation of the gtfP gene, annotated as glucosyltransferase in the S. sanguinis genome database, caused a marked reduction in the amount of water‐soluble glucans in the culture supernatant, but not in the amount of water‐insoluble glucans expressed on the bacterial cell surface. Scanning electron microscopy revealed that wild‐type S. sanguinis, but not the gtfP‐deficient mutant, produced large amounts of sticky material in the presence of 1% (w/v) sucrose. In addition, biofilm production by wild‐type bacteria was greater than that by the mutant strain. By contrast, co‐culture of mutant bacteria with Streptococcus mutans, S. sobrinus, S. oralis, S. gordonii, S. anginosus, or S. salivarius showed that inactivating the gtfP gene had little effect on the amount of biofilm produced. Furthermore, inactivating the gtfP gene did not greatly alter the proportion of S. sanguinis in the biofilms formed by the co‐cultures. Thus, despite the role of S. sanguinis glucosyltransferase in formation of water‐soluble glucans and biofilms in monoculture, the functional gene contributed little to biofilms in co‐culture experiments.     

HOTAIR but not ANRIL long non‐coding RNA contributes to the pathogenesis of multiple sclerosis

Studies have revealed that dysregulation in gene expression is one of the main aspects of multiple sclerosis (MS) pathogenesis. Although the molecular pathways underlying the immunomodulatory role of vitamin D (VD) in MS is not completely elucidated, VD has more recently become a topic of interest in immune regulation and is widely administered to patients with MS as an immunomodulatory supplement. Long non‐coding RNAs (lncRNAs) are known to play important roles in regulation of gene expression via different mechanisms. Given that VD‐related genes are regulated by epigenetic mechanisms, here we aimed to evaluate the role of VD in combination with HOTAIR and ANRIL lncRNAs using in vivo, in vitro and in silico experiments in MS pathogenesis. Our data revealed that HOTAIR but not ANRIL lncRNA is probably involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis through an unclear mechanism and it seems that by affecting the expression, inflammation and VD can influence HOTAIR‐related mechanisms, which require further study.     

Genotypic diversity and virulence markers of Yersinia enterocolitica biotype 1A strains isolated from clinical and non‐clinical origins

Yersinia enterocolitica biotype 1A (B1A) strains are considered as non‐pathogenic; however, some reports have identified some strains as the causal agents of infection. In South America, few studies molecularly characterized the strains of this biotype. This work typed 51 B1A strains isolated from clinical and non‐clinical sources from Brazil and Chile by Enterobacterial Repetitive Intergenic Consensus‐PCR (ERIC‐PCR) to elucidate their genotypic diversity, and verify the distribution of 11 virulence markers by PCR. The strains were divided into two groups, ERIC‐A and ERIC‐B, clustered independently of their clinical or non‐clinical origin. No differences were observed in the frequencies of the virulence markers between clinical and non‐clinical strains. However, the genes ystB, hreP and myfA occurred exclusively in the strains of the group ERIC‐A. Some clinical and non‐clinical strains were clustered in the same genetic group and presented the same number of virulence markers, which might suggest the role of the environment and food as a potential source of infection for humans and animals. The results corroborate with the hypothesis that B1A strains are divided into two main clusters that differ in the frequency of some virulence markers, a fact observed for the first time in South American strains.     

Refinement and delineation of the breakpoint regions of a chromosome 1;22 translocation in a patient with Costello syndrome*

Altered plasma levels of lipids and lipoproteins, obesity, hypertension, and diabetes are major risk factors for atherosclerotic cardiovascular disease. To identify genes that affect these traits and disorders, we looked for association between markers in candidate genes (apolipoprotein AII (apo AII), apolipoprotein AI‐CIII‐AIV gene cluster (apo AI‐CIII‐AIV), apolipoprotein E (apo E), cholesteryl ester transfer protein (CETP), cholesterol 7α‐hydroxylase (CYP7a), hepatic lipase (HL), and microsomal triglyceride transfer protein (MTP)) and known risk factors (triglycerides (Tg), total cholesterol (TC), apolipoprotein AI (apo AI), apolipoprotein AII (apo AII), apolipoprotein B (apo B), body mass index (BMI), blood pressure (BP), leptin, and fasting blood sugar (FBS) levels.) A total of 1,102 individuals from the Pacific island of Kosrae were genotyped for the following markers: Apo AII/MspI, Apo CIII/SstI, Apo AI/XmnI, Apo E/HhaI, CETP/TaqIB, CYP7a/BsaI, HL/DraI, and MTP/HhpI. After testing for population stratification, family‐based association analysis was carried out. Novel associations found were: 1) the apo AII/MspI with apo AI and BP levels, 2) the CYP7a/BsaI with apo AI and BMI levels. We also confirmed the following associations: 1) the apo AII/MspI with Tg level; 2) the apo CIII/SstI with Tg, TC, and apo B levels; 3) the Apo E/HhaI E2, E3, and E4 alleles with TC, apo AI, and apo B levels; and 4) the CETP/TaqIB with apo AI level. We further confirmed the connection between the apo AII gene and Tg level by a nonparametric linkage analysis. We therefore conclude that many of these candidate genes may play a significant role in susceptibility to heart disease. © 2002 Wiley‐Liss, Inc.