Iron Acquisition Systems Research Articles

Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico

One hundred and five uropathogenic Escherichia coli (UPEC) strains from patients with community-acquired urinary tract infections were characterized according to phylogenetic group, virulence factors, serogroup, antibiotic resistance, and genotype. The pathogenic phylogenetic groups (B2, D, and F) were found in 71.4% of the tested strains. Among them, the main uropathogenic serogroups were O8, O25, and O75, in which 97.1% of the strains had a multidrug-resistant profile. Sixteen virulence genes were analysed using a combination of polymerase chain reaction (PCR) assays, with the fimH, irp-2, iutA, aer, iucC, PAI, sat, iroN, usp, and cnf1 genes being mainly found in pathogenic phylogroups. The E. coli O25b-ST131 clone was identified in 32% of the strains assigned to the pathogenic phylogroup B2. These findings demonstrate that virulence genes encoding adhesin components, iron-acquisition systems, toxins, and pathogenicity-associated islands were highly prevalent among the pathogenic phylogroup of UPEC strains.

Urinary Tract Infections Caused by Uropathogenic Escherichia coli Strains-New Strategies for an Old Pathogen.

Urinary tract infections (UTIs) are among the most common infections worldwide. Uropathogenic Escherichia coli (UPECs) are the main causative agent of UTIs. UPECs initially colonize the human host adhering to the bladder epithelium. Adhesion is followed by the bacterial invasion of urothelial epithelial cells where they can replicate to form compact aggregates of intracellular bacteria with biofilm-like properties. UPEC strains may persist within epithelial urothelial cells, thus acting as quiescent intracellular bacterial reservoirs (QIRs). It has been proposed that host cell invasion may facilitate both the establishment and persistence of UPECs within the human urinary tract. UPEC strains express a variety of virulence factors including fimbrial and afimbrial adhesins, invasins, iron-acquisition systems, and toxins, which cooperate to the establishment of long lasting infections. An increasing resistance rate relative to the antibiotics recommended by current guidelines for the treatment of UTIs and an increasing number of multidrug resistant UPEC isolates were observed. In order to ameliorate the cure rate and improve the outcomes of patients, appropriate therapy founded on new strategies, as alternative to antibiotics, needs to be explored. Here, we take a snapshot of the current knowledge of coordinated efforts to develop innovative anti-infective strategies to control the diffusion of UPECs.

Iron availability enhances the cellular energetics of aerobic Escherichia coli cultures while upregulating anaerobic respiratory chains

Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 μM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems.

To kill or to be killed: pangenome analysis of Escherichia coli strains reveals a tailocin specific for pandemic ST131

BackgroundEscherichia coli (E. coli) has been one of the most studied model organisms in the history of life sciences. Initially thought just to be commensal bacteria, E. coli has shown wide phenotypic diversity including pathogenic isolates with great relevance to public health. Though pangenome analysis has been attempted several times, there is no systematic functional characterization of the E. coli subgroups according to the gene profile.ResultsSystematically scanning for optimal parametrization, we have built the E. coli pangenome from 1324 complete genomes. The pangenome size is estimated to be ~25,000 gene families (GFs). Whereas the core genome diminishes as more genomes are added, the softcore genome (≥95% of strains) is stable with ~3000 GFs regardless of the total number of genomes. Apparently, the softcore genome (with a 92% or 95% generation threshold) can define the genome of a bacterial species listing the critically relevant, evolutionarily most conserved or important classes of GFs. Unsupervised clustering of common E. coli sequence types using the presence/absence GF matrix reveals distinct characteristics of E. coli phylogroups B1, B2, and E. We highlight the bi-lineage nature of B1, the variation of the secretion and of the iron acquisition systems in ST11 (E), and the incorporation of a highly conserved prophage into the genome of ST131 (B2). The tail structure of the prophage is evolutionarily related to R2-pyocin (a tailocin) from Pseudomonas aeruginosa PAO1. We hypothesize that this molecular machinery is highly likely to play an important role in protecting its own colonies; thus, contributing towards the rapid rise of pandemic E. coli ST131.ConclusionsThis study has explored the optimized pangenome development in E. coli. We provide complete GF lists and the pangenome matrix as supplementary data for further studies. We identified biological characteristics of different E. coli subtypes, specifically for phylogroups B1, B2, and E. We found an operon-like genome region coding for a tailocin specific for ST131 strains. The latter is a potential killer weapon providing pandemic E. coli ST131 with an advantage in inter-bacterial competition and, suggestively, explains their dominance as human pathogen among E. coli strains.

Bioinformatic Analysis for Iron-Uptake System of Campylobacter spp. in humans and chickens

Asymptomatic Campylobacter jejuni colonisation is highly prevalent in chickens, however, it’s a major cause of bacterial foodborne disease in humans. Iron is an essential co-factor in many physiological processes and Campylobacter strains employ several non-redundant iron acquisition systems for survival and colonization. The outer membrane receptors CfrA and CfrB are involved in iron acquisition fromcatecholamine siderophores and are required for colonisation of the chicken GI tract. In contrast, ChuAB are part of an uptake system, enabling the utilization of iron in haem and is not required for colonisation. This study aims to compare differences in the iron-uptake systems present in Campylobacter strains isolated from chickens or humans. Analysis of Campylobacter jejuni/coli genome sequences in PubMLST (https://pubmlst.org/organisms/campylobacter-jejunicoli [https://pubmlst.org/organisms/campylobacter-jejunicoli]) added from the UK in 2018 reveals differences in sequence type (ST) and chuA, chuB, cfrA and cfrB between isolates from chicken (CI) or human (HI) sources. ST828 was the most common ST for the C. coliCI (45%) and ST827 (32%) for the HI. C. jejuniST5136 was the most common of the HI (10%) and CI (13%), ST50 (8%) for the HI and ST21 (7%) for the CI. >95% of isolates contained chuAand chuB, we also found the Chu system to be highly conserved. For cfrA81% of HI and 74% of CI contained the gene, and 92% of HI and 96% of CI harboured cfrB.<3% of all isolates had neither cfrAor cfrB, cfrBmore associated with CI than HI. This data analysis reveals potential gene targets towards vaccine development against Campylobacter strains.

The Regulatory Protein ChuP Connects Heme and Siderophore-Mediated Iron Acquisition Systems Required for Chromobacterium violaceum Virulence.

Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of chuR and vbuA, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.

Whole-Genome Sequencing of a Colistin-Resistant Acinetobacter baumannii Strain Isolated at a Tertiary Health Facility in Pretoria, South Africa.

Background: Acinetobacter baumannii’s (A. baumannii) growing resistance to all available antibiotics is of concern. The study describes a colistin-resistant A. baumannii isolated at a clinical facility from a tracheal aspirate sample. Furthermore, it determines the isolates’ niche establishment ability within the tertiary health facility. Methods: An antimicrobial susceptibility test, conventional PCR, quantitative real-time PCR, phenotypic evaluation of the efflux pump, and whole-genome sequencing and analysis were performed on the isolate. Results: The antimicrobial susceptibility pattern revealed a resistance to piperacillin/tazobactam, ceftazidime, cefepime, cefotaxime/ceftriaxone, imipenem, meropenem, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tigecycline, and colistin. A broth microdilution test confirmed the colistin resistance. Conventional PCR and quantitative real-time PCR investigations revealed the presence of adeB, adeR, and adeS, while mcr-1 was not detected. A MIC of 0.38 µg/mL and 0.25 µg/mL was recorded before and after exposure to an AdeABC efflux pump inhibitor. The whole-genome sequence analysis of antimicrobial resistance-associated genes detected beta-lactam: blaOXA-66; blaOXA-23; blaADC-25; blaADC-73; blaA1; blaA2, and blaMBL; aminoglycoside: aph(6)-Id; aph(3″)-Ib; ant(3″)-IIa and armA) and a colistin resistance-associated gene lpsB. The whole-genome sequence virulence analysis revealed a biofilm formation system and cell–cell adhesion-associated genes: bap, bfmR, bfmS, csuA, csuA/B, csuB, csuC, csuD, csuE, pgaA, pgaB, pgaC, and pgaD; and quorum sensing-associated genes: abaI and abaR and iron acquisition system associated genes: barA, barB, basA, basB, basC, basD, basF, basG, basH, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, and entE. A sequence type classification based on the Pasteur scheme revealed that the isolate belongs to sequence type ST2. Conclusions: The mosaic of the virulence factors coupled with the resistance-associated genes and the phenotypic resistance profile highlights the risk that this strain is at this South African tertiary health facility.

The fciTABC and feoABI systems contribute to ferric citrate acquisition in Stenotrophomonas maltophilia

BackgroundStenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia.MethodsCandidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2′-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein–protein interaction.ResultsSmlt2858 and Smlt2356, the homologues of FecA and FecC/D of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein–protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate.ConclusionsThe FciTABC and FeoABI systems contribute to ferric citrate acquisition in S. maltophilia.

Molecular Detection of Virulence genes (rmpA2, iuc & iroB) of Hypervirulent Klebsiella pneumoniae in Clinical Isolates from Patients in Khartoum State, Sudan

Background: Iron acquisition systems and regulator mucoid phenotype plays an important role in the pathogenesis and virulence of Klebsiella pneumoniae. This cross-sectional study aimed to investigate the prevalence of virulence genes iuc, iroB and rmpA2 of hypervirulent Klebsiella pneumoniae (hykp) in clinical isolates from patients in Khartoum.
 Methods: A total of 400 samples were collected from 250 (62.5%) males and 150 (37.5%) females. All samples were cultivated in agar plates to determine the presence of Klebsiella pneumoniae isolates. These isolates were screened for hypermucoviscosity phenotypically by string test. Antibiotic susceptibility tests were performed for all isolates to identify their resistance pattern, iuc, rmpA2, iroB genes were investigated by polymerase chain reaction.
 Results: Our results demonstrate that the majority of participants 150 (37.5%) are aged 31 – 40 years. Out of 400 samples cultivated, 313 (78.75%) were yield significant pathogenic growth of which 95 (23.75%) are K.pneumoniae isolates, and 220 (55.0%) are other pathogenic organisms while the rest of samples 87 (17.25%) were showed no pathogenic growth isolated. The frequency of iuc, iroB, rmpA2 were 74.7%, 57.9%, 2.1% respectively among the clinical isolates. String tests were positive among 30 (31.6%) isolates and the remaining 65 (68.4%) isolates gave negative results.
 Conclusion: There was a high prevalence of iuC virulence gene of hvkp, high frequency iron acquisition systems coding gene iuC gene, iroB gene compared to regulator mucoid phenotype A2 (rmpA2 gene) among clinical isolates.

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection indicating this bacterium overcomes host nutritional immunity. Despite this, there exists a significant knowledge gap regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis can usurp hydroxamate siderophores and staphyloferrin A and B from Staphylococcus aureus. These transport activities all required a functional FhuC ATPase. Moreover, we show that the acquisition of catechol siderophores and catecholamine stress hormones by S. lugdunensis required the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitated the ferrous iron transport system encoded by feoAB. Heme iron was acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice, we demonstrated that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci (isd, fhuC, sst-1, and feo), we demonstrate that only a strain deficient for all of these systems was attenuated in its ability to proliferate to high numbers in the murine kidney. We propose the concerted action of heme and non-heme iron acquisition systems also enable S. lugdunensis to cause human infection.

High Affinity Iron Acquisition Systems Facilitate but Are Not Essential for Colonization of Chickens by Salmonella Enteritidis.

The roles of TonB mediated Fe3+ (ferric iron) uptake via enterobactin (involving biosynthesis genes entABCDEF) and Fe2+ (ferrous iron) uptake through the FeoABC transporter are poorly defined in the context of chicken-Salmonella interactions. Both uptake systems are believed to be the major contributors of iron supply in the Salmonella life cycle. Current evidence suggests that these iron uptake systems play a major role in pathogenesis in mammals and as such, they represent promising antibacterial targets with therapeutic potential. We investigated the role of these iron uptake mechanisms regarding the ability of Salmonella Enteritidis (SEn) strains to colonize in a chicken infection model. Further we constructed a bioluminescent reporter to sense iron limitation during gastrointestinal colonization of Salmonella in chicken via ex vivo imaging. Our data indicated that there is some redundancy between the ferric and ferrous iron uptake mechanisms regarding iron acquisition during SEn pathogenesis in chicken. We believe that this redundancy of iron acquisition in the host reservoir may be the consequence of adaptation to unique avian environments, and thus warrants further investigation. To our knowledge, this the first report providing direct evidence that both enterobactin synthesis and FeoABC mediated iron uptake contribute to the virulence of SEn in chickens.

Genetic Diversity of Antimicrobial Resistance and Key Virulence Features in Two Extensively Drug-Resistant Acinetobacter baumannii Isolates.

In recent decades, Acinetobacter baumannii emerged as a major infective menace in healthcare settings due to scarce therapeutic options to treat infections. Therefore, undertaking genome comparison analyses of multi-resistant A. baumannii strains could aid the identification of key bacterial determinants to develop innovative anti-virulence approaches. Following genome sequencing, we performed a molecular characterization of key genes and genomic comparison of two A. baumannii strains, #36 and #150, with selected reference genomes. Despite a different antibiotic resistance gene content, the analyzed strains showed a very similar antibiogram profile. Interestingly, the lack of some important virulence determinants (i.e., bap, ata and omp33–36) did not abrogate their adhesive abilities to abiotic and biotic surfaces, as reported before; indeed, strains retained these capacities, although to a different extent, suggesting the presence of distinct vicarious genes. Conversely, secretion systems, lipopolysaccharide (LPS), capsule and iron acquisition systems were highly similar to A. baumannii reference strains. Overall, our analyses increased our knowledge on A. baumannii genomic content and organization as well as the genomic events occurring in nosocomial isolates to better fit into changing healthcare environments.

Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.

P44 Nosocomial neurosurgical meningitis due to XDR Pseudomonas aeruginosa: clinical experience with cefiderocol

BackgroundCarbapenem resistance due to MBL production is relatively uncommon in Pseudomonas aeruginosa infections in the UK. We report a case of post-operative meningitis caused by a New-Delhi MBL-producing (NDM-1) strain, outlining the microbiological and management challenges.Patient caseThe patient was admitted to intensive care after craniotomy and evacuation for an intracerebral haemorrhage. Three weeks later she was febrile due to nosocomial meningitis. Purulent material evacuated from extradural and subdural spaces cultured P. aeruginosa, along with blood cultures and CSF. Susceptibility testing as per EUCAST methodology indicated the isolate was resistant to all β-lactams, aminoglycosides and quinolones but susceptible to colistin. Susceptibility to cefiderocol by disc diffusion was in the area of technical uncertainty at 21 mm (S≥22 mm). Immunochromatographic flow testing (CARBA5 NG Biotech) detected an NDM carbapenemase. WGS (Illumina) confirmed the presence of blaNDM-1 and a plethora of AMR genes responsible for the XDR phenotype {cephalosporins (blaPDC-10, blaOXA-488), aminoglycosides [aph(3')-VIa], quinolones (crpP), macrolides [mph(E), msr(E)], phenicols (catB7) and fosfomycin (fosA)}. No mutations in PBPs associated with resistance to β-lactams or iron acquisition systems involved in uptake of siderophore conjugated antibiotics were identified. The isolate belonged to ST1047, serotype 07, previously identified as a high-risk epidemic clone originating in Myanmar.ResultsTreatment was commenced with IV and intrathecal colistin as well as IV cefiderocol (Figure 1); a novel siderophore cephalosporin licensed for Gram-negative infections with limited treatment options. No synergy between the two drugs could be demonstrated in chequerboard assays. Although CSF sterility was achieved on three subsequent cultures with clinical response, the patient sadly died due to the underlying intracerebral injury. Four other patients subsequently became colonized with this strain and infection control measures (twice weekly screening of all patients, environmental cleaning, water testing, isolation of cases) were instituted, bringing the outbreak under control. Variable number tandem repeat analysis linked these strains to that of another patient discharged from the unit 9 months prior, but not previously reported in the UK.Figure 1.Clinical timeline, culture results, antimicrobial regimens and cefiderocol levels. BC, blood culture; Resp, respiratory specimen; Swab, craniotomy swab; Pus, subdural and extradural pus; Cro, ceftriaxone; Mero, meropenem; Cefid, cefiderocol; Vanc, vancomycin; IT, intrathecal; MU, million units; bd, two times a day; od, once daily.ConclusionsThe first problem encountered was a lack of evidence for the CNS penetration of cefiderocol and efficacy in the treatment of meningitis. The external ventricular drain required to administer intrathecal colistin enabled us to monitor CSF cefiderocol levels. Five CSF samples were shipped at −80°C for analysis in Germany by mass spectrometry. CSF cefiderocol levels of 1.22 μg/mL, 1.34 μg/mL, 2.39 μg/mL, 3.53 μg/mL and 3.90 μg/mL were disappointingly close to the pharmacodynamic breakpoint of 2 μg/mL and only available after treatment withdrawal. Dosing of cefiderocol was initially cautious starting at 1 g/8 h due to a creatinine clearance of 53 mL/min but later increased to 1.5 g/8 h. Further dose increases guided by serum and CSF monitoring could feasibly lead to therapeutic CSF cefiderocol levels. Clinical and pharmacological studies are needed in this area. To our knowledge this is the first attempt to treat a case of XDR neurosurgical meningitis using cefiderocol with precision treatment and achieving microbiological cure.

Preliminary Investigation of Iron Acquisition in Hypervirulent Klebsiella pneumoniae Mediated by Outer Membrane Vesicles.

ObjectiveTo investigate the role of outer membrane vesicles (OMVs) and related proteins in iron acquisition of hypervirulent Klebsiella pneumoniae (HVKP) and classic Klebsiella pneumoniae (cKP).MethodsThe OMVs of HVKP and cKP under iron-deficient and iron-sufficient media were extracted and purified by ultracentrifugation. Transmission electron microscopy (TEM) was used to identify OMVs. The quantitative proteomics were performed based on mass spectrometry.ResultsFour OMVs samples secreted by HVKP and cKP under iron-deficient and iron-sufficient environment were isolated and collected (HVKP OMVs under iron-deficient environment (A1), HVKP OMVs under iron-sufficient environment (A2), cKP OMVs under iron-deficient environment (B1), cKP OMVs under iron-sufficient environment (B2)). The amount of OMVs released by HVKP in iron-deficient medium was significantly larger than that in iron-sufficient medium (P < 0.05). HVKP secreted more OMVs than cKP in iron-deficient medium (P < 0.05). A total of 1074 kinds of proteins were identified in four samples. A comparison between the iron-deficient vs iron-sufficient environment showed that 61 proteins in HVKP OMVs were identified with a significant change in abundance under iron-deficient environment. Among them, 17 proteins were related to iron acquisition and transportation systems. While in cKP OMVs, 62 proteins significantly changed under iron-deficient environment in which 5 proteins were related to iron acquisition and transportation systems. Upon comparison of the HVKP vs cKP OMVs under iron deficiency, 81 proteins were detected with a significant change in which 8 proteins were related to iron acquisition and transportation systems.ConclusionAbove all, the results of this study suggest a potential role for OMVs in iron acquisition of HVKP and provide evidence of potential connections between OMVs and strong iron-acquisition ability of HVKP during iron limitation.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats.

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

Iron Homeostasis in Pseudomonas aeruginosa: Targeting Iron Acquisition and Storage as an Antimicrobial Strategy.

Pseudomonas aeruginosa causes a wide array of life-threatening acute and chronic infections in humans. This opportunistic pathogen is metabolically highly versatile and harbors multiple virulence factors that allow infection of essentially any organ of the human body. The high capacity of this bacterium to acquire iron facilitates its versatility and is considered one of the P. aeruginosa virulence hallmarks. Iron functions as a redox cofactor of enzymes required for vital biological processes and is thus essential for all living organisms. However, in aerobic environments, iron is mainly present in its ferric form, which is insoluble and poorly bioavailable. This problem increases in the human body because, as a reaction to the infection, the host induces a "nutritional immunity" response aiming to reduce the amount of iron available for invading microorganisms. P. aeruginosa contains several mechanisms for iron acquisition including (1) production of siderophores pyoverdine and pyochelin; (2) use of xenosiderophores produced by other microorganisms; (3) direct transport of ferrous ions; and (4) utilization of host iron carriers (e.g., heme). However, although essential, iron results toxic when present in excess because it facilitates the production of reactive oxygen species (ROS) that damage bacterial cells. P. aeruginosa contains ferritins and efflux systems for iron withdrawal to avoid excess of this metal. Production of iron acquisition and removal systems is highly regulated to ensure sufficient iron for metabolic needs while preventing its toxicity. This chapter covers the different mechanisms used by P. aeruginosa to maintain iron homeostasis, which is vital for this pathogen to grow and proliferate in the host. We also highlight current strategies to block P. aeruginosa infections by disrupting iron homeostasis.

Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli.

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli . Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius.

The thermophilic bacterium Parageobacillus thermoglucosidasius has recently gained interest due to its ability to catalyze the water gas shift reaction, where the oxidation of carbon monoxide (CO) is linked to the evolution of hydrogen (H2) gas. This phenotype is largely predictable based on the presence of a genomic region coding for a carbon monoxide dehydrogenase (CODH—Coo) and hydrogen evolving hydrogenase (Phc). In this work, seven previously uncharacterized strains were cultivated under 50% CO and 50% air atmosphere. Despite the presence of the coo—phc genes in all seven strains, only one strain, Kp1013, oxidizes CO and yields H2. The genomes of the H2 producing strains contain unique genomic regions that code for proteins involved in nickel transport and the detoxification of catechol, a by-product of a siderophore-mediated iron acquisition system. Combined, the presence of these genomic regions could potentially drive biological water gas shift (WGS) reaction in P. thermoglucosidasius.

Expression of Genes Located on the Incompatibility Group FIB Plasmids at Transcription and Protein Levels in Iron-Modified Growth Conditions.

Salmonella enterica strains often harbor plasmids representing several incompatibility groups (Inc) including IncFIB, which have been previously associated with carrying antimicrobial resistance and virulence associated genes. To better understand the distribution of virulence genes on IncFIB plasmids, we analyzed 37 complete whole genome and plasmid sequences of different S. enterica isolates from multiple serovars. Many of the sequences analyzed carried multiple virulence-associated genes, including those associated with iron acquisition systems; thus we aimed to determine how iron-rich (IR) and various iron-depleted (ID) conditions affected the transcription of iron acquisition and virulence genes including sitA, iutA, iucA, and enolase at different time intervals. sitA, iutA, and enolase from S. enterica that were grown in Luria-Bertani broth (LB) ID (LBID) conditions were substantially upregulated when compared to LBIR conditions. For both S. enterica strains that were grown at various LBID conditions, addition of 200 μM bipyridyl in the growth medium yielded the highest transcription for all four genes, followed by the 100 μM concentration. An antibody using a peptide targeting aerobactin receptor gene iutA encoded by IncFIB was generated and used to examine the protein expression in the wild-type, recipient, and transconjugant strain in LB, LBID, and LBIR growth conditions using Western blot analyses. A 70 KDa protein band was detected in the wild-type and transconjugant that carried the IncFIB plasmid, while this band was not detected in the recipient strain that lacked this plasmid.