Molecular regulation of virulence in Legionella pneumophila.

Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

Structural characterization of the Sel1-like repeat protein LceB from Legionella pneumophila.

Legionella are freshwater Gram-negative bacteria that in their normal environment infect protozoa. However, this adaptation also allows Legionella to infect human alveolar macrophages and cause pneumonia. Central to Legionella pathogenesis are more than 330 secreted effectors, of which there are nine core effectors that are conserved in all pathogenic species. Despite their importance, the biochemical function of several core effectors remains unclear. To address this, we have taken a structural approach to characterize the core effector of unknown function LceB, or Lpg1356, from Legionella pneumophila. Here, we solve an X-ray crystal structure of LceB using an AlphaFold model for molecular replacement. The experimental structure shows that LceB adopts a Sel1-like repeat (SLR) fold as predicted. However, the crystal structure captured multiple conformations of LceB, all of which differed from the AlphaFold model. A comparison of the predicted model and the experimental models suggests that LceB is highly flexible in solution. Additionally, the molecular analysis of LceB using its close structural homologs reveals sequence and structural motifs of known biochemical function. Specifically, LceB harbors a repeated KAAEQG motif that both stabilizes the SLR fold and is known to participate in protein-protein interactions with eukaryotic host proteins. We also observe that LceB forms several higher-order oligomers in solution. Overall, our results have revealed that LceB has conformational flexibility, self-associates, and contains a molecular surface for binding a target host-cell protein. Additionally, our data provides structural insights into the SLR family of proteins that remain poorly studied.

The Virulence of S. marcescens Strains Isolated From Contaminated Blood Products Is Divergent in the C. elegans Infection Model.

Bacterial contamination of platelet concentrates (PCs) can occur during blood donation or PC processing, necessitating routine screening to identify contaminated products in efforts to prevent adverse transfusion reactions in recipient patients. Serratia marcescens is a common bacterial contaminant, and its resilient nature coupled with genetic promiscuity imbue this environmental bacterium with resistance to disinfectants and antibiotics enhancing bacterial virulence. In this study, we aim to understand adaptive survival mechanisms through genetic characterization of two S. marcescens strains, CBS11 and CBS12, isolated from PCs by Canadian Blood Services. Genomic analyses of the two strains indicated that CBS11 has one chromosome and one plasmid (pAM01), whereas CBS12 has no plasmids. Phylogenetic analyses show that CBS11 and CBS12 are non-clonal strains, with CBS11 clustering closely with clinical strain CAV1492 and less so with environmental strain PWN146, and CBS12 clustering with a clinical strain AR_0027. Interestingly, pAM01 was most closely related to PWN146p1, a plasmid found in S. marcescens PWN146 strain associated with pinewood nematode Bursaphelenchus xylophilus. Lastly, the genomic diversity of CBS11 and CBS12 was not reflected in the antibiotic resistance profiles as they were remarkably similar to one another, but was reflected in the virulence phenotypes assessed in the Caenorhabditis elegans nematode infection model, with CBS11 being more virulent then CBS12. Taken together, we suggest that S. marcescens environmental isolates that feature evolutionary diverse genomics are better equipped to adapt and thrive in varied environments, such as that of PCs, and therefore is as much of a concern as multi-drug resistance for human infection potential.

The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Autorepressor PsrA is required for optimal Legionella pneumophila growth in Acanthamoeba castellanii protozoa.

Legionella pneumophila possesses a unique intracellular lifecycle featuring distinct morphological stages that include replicative forms and transmissive cyst forms. Expression of genes associated with virulence traits and cyst morphogenesis is concomitant, and governed by a complex stringent response based-regulatory network and the stationary phase sigma factor RpoS. In Pseudomonas spp., rpoS expression is controlled by the autorepressor PsrA, and orthologs of PsrA and RpoS are required for cyst formation in Azotobacter. Here we report that the L. pneumophila psrA ortholog, expressed as a leaderless monocistronic transcript, is also an autorepressor, but is not a regulator of rpoS expression. Further, the binding site sequence recognized by L. pneumophila PsrA is different from that of Pseudomonas PsrA, suggesting a repertoire of target genes unique to L. pneumophila. While PsrA was dispensable for growth in human U937-derived macrophages, lack of PsrA affected bacterial intracellular growth in Acanthamoeba castellanii protozoa, but also increased the quantity of poly-3-hydroxybutyrate (PHB) inclusions in matured transmissive cysts. Interestingly, overexpression of PsrA increased the size and bacterial load of the replicative vacuole in both host cell types. Taken together, we report that PsrA is a host-specific requirement for optimal temporal progression of L. pneumophila intracellular lifecycle in A. castellanii.

Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection.

Legionnaires' disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

Complete Genome Sequences of Two Environmental Legionella Isolates Obtained from Potable Water Sourced in a First Nation Community.

Here, we report the complete genome sequences of two distinct isolates of Legionella that were obtained from potable water sourced from cistern-bearing homes within a First Nation community in Manitoba, Canada.

Complete Genome Sequences of Two Distinct Strains of Serratia marcescens Isolated from Contaminated Platelet Concentrates from Canadian Donors.

In this report, we present the genome sequences of two Serratia marcescens strains isolated as contaminants from platelet concentrates by Canadian Blood Services and designated CBS2010/11 (CBS11) and CBS2010/12 (CBS12). Genomic sequence analyses showed that CBS11 has one chromosome and one plasmid (pAM01), whereas CBS12 has no plasmids.

A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism.

Acinetobacter baumannii, a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in A. baumannii ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (AmiR and NasR transcription anti-termination regulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in A. baumannii. RNA-Seq analysis revealed that avnR deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the avnR deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in avnR deletion mutant. Biolog phenotypic array showed that deletion of avnR hampered A. baumannii ATCC17978's ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in A. baumannii, affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

The Caenorhabditis elegans Model of Legionella Infection.

Caenorhabditis elegans can serve as a simple genetic host to study interactions between Legionellaceae and their hosts and to examine the contribution of specific gene products to virulence and immunity. C. elegans nematodes have several appealing attributes as a host organism; they are inexpensive, have robust genetic analysis tools, have a simple anatomy yet display a wide range of complex behaviors, and, as invertebrates, do not require animal ethics protocols. Use of C. elegans as a host model complements cell-based models, providing additional support and consistency of the experimental data obtained from multiple models. The C. elegans innate immune system functions similarly to that of the alveolar macrophage including the apoptosis [a.k.a. programmed cell death (PCD)] pathway located within the germline. The digestive tract of C. elegans is a primary interface between the innate immune system and bacterial pathogens. Thus, the C. elegans host model provides an alternative approach to investigate L. pneumophila immunopathogenesis, particularly in the view of the recent discovery of Legionella-containing vacuoles within the gonadal tissues of Legionella-colonized nematodes supporting the plausible evolutionary origin of the strategies employed by L. pneumophila to counteract macrophage cellular responses.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System.

Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyRLp) is an ortholog of Escherichia coli OxyR; however, OxyRLp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila Lack of OxyRLp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyRLp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyRLp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyRLp directly bound to these promoter regions, suggesting that OxyRLp is a direct repressor. Interestingly, a strain overexpressing OxyRLp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyRLp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein.IMPORTANCELegionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyRLp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyRLp expression is regulated remain elusive, this study shows that OxyRLp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

Complete Genome Sequence of a Rhodococcus Species Isolated from the Winter Skate Leucoraja ocellata.

We report here a genome sequence for Rhodococcus sp. isolate UM008 isolated from the renal/interrenal tissue of the winter skate Leucoraja ocellata Genome sequence analysis suggests that Rhodococcus bacteria may act in a novel mutualistic relationship with their elasmobranch host, serving as biocatalysts in the steroidogenic pathway of 1α-hydroxycorticosterone.

Pseudomonas brassicacearum strain DF41 kills Caenorhabditis elegans through biofilm-dependent and biofilm-independent mechanisms.

Pseudomonas brassicacearum DF41 is a biocontrol agent that suppresses disease caused by the fungal pathogen Sclerotinia sclerotiorum A number of exometabolites are produced by DF41 including the lipopeptide sclerosin, hydrogen cyanide (HCN) and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional level by quorum sensing (QS) and the Gac-two component regulatory system. In order to be successful, a biocontrol agent must persist in the environment at levels sufficient for pathogen control. Bacterivorous predators, including nematodes, represent a challenge to the establishment of introduced microorganisms. In the current study, DF41 was investigated for its ability to resist predation by Caenorhabditis elegans. We discovered that this bacterium is capable of killing C. elegans through two different mechanisms: the first involves exposure to toxic metabolites; and the second entails biofilm formation on the nematode head blocking the buccal cavity. Biofilm formation on nematodes, which has only been reported for Yersinia spp. and Xenorhabdus nematophila, is dependent upon the Gac system. Biofilms were not observed when bacteria were grown on NaCl-containing media, and on C. elegans biofilm-resistant mutants. Co-culturing with nematodes lead to increased expression of the pdfRI-rfiA QS genes and hcnA which is under QS control. HCN was the most nematicidal of the exometabolites, suggesting that this bacterium can respond to predator cues and upregulate expression of toxins accordingly. In summary, DF41 is able to respond to the presence of C. elegans and through two distinct mechanisms it can escape predation. IMPORTANCE: Pseudomonas brassicacearum DF41 can suppress fungal pathogens through a process known as biocontrol. To be successful, a biocontrol agent must be able to persist in the environment at levels sufficient for pathogen control. Predators including the nematode Caenorhabditis elegans represent a threat to persistence. The aim of the current study was to investigate the DF41-C. elegans interaction. We discovered that DF41 is able to escape predation through two distinct mechanisms. The first involves exposure to toxic bacterial metabolites and the second entails formation of a sticky coating on the nematode head, called a biofilm, which blocks feeding and causes starvation. This is the first report of a pseudomonad forming biofilms on the C. elegans surface. When grown with C. elegans, DF41 exhibits altered gene expression and metabolite production indicating that this bacterium can sense the presence of these predators and adjust its physiology accordingly.

The CpxRA two-component system contributes to Legionella pneumophila virulence.

The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation.

Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

Legionella pneumophila, a causative agent of Legionnaires' disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila.

Pyrrolnitrin and Hydrogen Cyanide Production by Pseudomonas chlororaphis Strain PA23 Exhibits Nematicidal and Repellent Activity against Caenorhabditis elegans.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1.

The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW(-1) day(-1)) and very little urea (0.21±0.004 µmol gFW(-1) day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H(+)-ATPase (subunit A) and Na(+)/K(+)-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H(+)-ATPase, carbonic anhydrase, Na(+)/K(+)-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l(-1) NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na(+)/K(+)-ATPase also increased significantly in response to 1 mmol l(-1) NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins.

The lack of OmpF, but not OmpC, contributes to increased antibiotic resistance in Serratia marcescens.

The environmental organism Serratia marcescens is one of the primary causes of numerous nosocomial outbreaks and opportunistic infections. Multi-drug resistance is now a common feature among S. marcescens clinical isolates, complicating the efficacy of treatment. Recent reports have attributed antibiotic resistance to altered porin expression as well as perturbation of the intrinsic AmpC beta-lactamase production pathway. In this study, we aimed to genetically correlate the absence of OmpF and OmpC classical porins with increased antibiotic resistance. In generating isogenic porin mutant strains, we avoided incorporating additional resistance through the use of antibiotic cassettes in gene replacement and adopted an alternative strategy in creating clean unmarked mutant strains. We found that lack of OmpF, but not OmpC, significantly increased antibiotic MIC values to the beta-lactam drugs such as ampicillin and cefoxitin as well as to nitrofurantoin. Furthermore, we found that cefoxitin did not induce intrinsic AmpC beta-lactamase production, indicating that the increased MIC values were a result of reduced permeability of cefoxitin due to the lack of OmpF. Genetic deletion of both ompF and ompC did not compromise the integrity of the bacterial cell envelope in optimal growth conditions, suggesting that other outer-membrane porins may function in a compensatory role to facilitate nutrient uptake and cell envelope integrity. Taken together, to our knowledge this is the first study that genetically correlates increased antibiotic resistance with altered porin expression in S. marcescens.

Enhanced pathogenicity of biofilm-negative Staphylococcus epidermidis isolated from platelet preparations.

BACKGROUND: The platelet (PLT) storage environment triggers the formation of surface-attached aggregates known as biofilms by the common PLT contaminant Staphylococcus epidermidis. The biofilm matrix is largely composed of polysaccharide intercellular adhesin (PIA) mediated by the icaADBC operon. However, PIA-negative S. epidermidis has been reported to form biofilms in PLT concentrates (PCs). Since biofilm formation is associated with increased virulence, this study was aimed at determining if PIA-negative S. epidermidis grown in PCs presents enhanced virulence using the nematode Caenorhabditis elegans as a host model for bacterial pathogenesis. STUDY DESIGN AND METHODS: Biofilm-positive S. epidermidis ATCC 35984 and 9142, which carry the icaADBC operon, and biofilm-negative S. epidermidis ATCC 12228 and 9142 ΔicaA were grown in regular media and in PCs and biofilm formation was quantified using a crystal violet assay. The virulence of these strains after passage through PCs was tested using nematode killing assays. Nematode survival was calculated using the Kaplan-Meier method and statistical differences were determined by log-rank analysis. RESULTS: All S. epidermidis strains were able to form biofilms in PCs. Although persistence of a biofilm-positive phenotype in the biofilm-negative strains grown in PCs was not observed after passage in regular medium, the virulence of all strains was significantly increased as demonstrated by shortened life spans of the nematodes in C. elegans killing assays. CONCLUSION: Our findings highlight the potential of an increased risk of nosocomial infections caused by S. epidermidis in transfusion recipients since PC storage conditions promote biofilm formation, and possibly pathogenicity, of strains traditionally known to be attenuated for virulence.